Frequency, kinetics and determinants of viable SARS-CoV-2 in bioaerosols from ambulatory COVID-19 patients infected with the Beta, Delta or Omicron variants.

Airborne transmission of SARS-CoV-2 aerosol remains contentious. Importantly, whether cough or breath-generated bioaerosols can harbor viable and replicating virus remains largely unclarified. We performed size-fractionated aerosol sampling (Andersen cascade impactor) and evaluated viral culturability in human cell lines (infectiousness), viral genetics, and host immunity in ambulatory participants with COVID-19. Sixty-one percent (27/44) and 50% (22/44) of participants emitted variant-specific culture-positive aerosols <10µm and <5µm, respectively, for up to 9 days after symptom onset. Aerosol culturability is significantly associated with lower neutralizing antibody titers, and suppression of transcriptomic pathways related to innate immunity and the humoral response. A nasopharyngeal Ct <17 rules-in ~40% of aerosol culture-positives and identifies those who are probably highly infectious. A parsimonious three transcript blood-based biosignature is highly predictive of infectious aerosol generation (PPV > 95%). There is considerable heterogeneity in potential infectiousness i.e., only 29% of participants were probably highly infectious (produced culture-positive aerosols <5µm at ~6 days after symptom onset). These data, which comprehensively confirm variant-specific culturable SARS-CoV-2 in aerosol, inform the targeting of transmission-related interventions and public health containment strategies emphasizing improved ventilation.

Selected questions and controversies about bedaquiline: a view from the field.

Although there has been a slow decline in tuberculosis (TB) incidence worldwide, the prevalence of drug-resistant TB in most high-burden countries has increased. Drug-resistant TB is associated with high mortality, is a threat to health care workers in TB-endemic countries and is prohibitively costly, which diverts resources away from drug-susceptible cases. Amplification of resistance means that there is an increasing proportion of patients with multidrug-resistant TB who have extensively drug-resistant TB (XDR-TB) or are programmatically untreatable. Thus, new treatment options are urgently needed. Bedaquiline (BDQ) is the first new drug specifically developed for TB to be licensed for use in almost 40 years. BDQ has sterilising activity and also shows promise as a component of new treatment-shortening regimens for drug-susceptible TB. Here we review insights from the field into the use of BDQ, issues relevant to the practising clinician, implications for the selection for antiretroviral therapy, pharmacokinetic issues relevant to clinical practice and implications for combination therapy. Given the increasing prevalence of resistance beyond XDR-TB, we also discuss how the development of resistance to BDQ can be minimised.

The canine Purkinje fiber: an in vitro model system for acquired long QT syndrome and drug-induced arrhythmogenesis.

Torsade de pointes is a rare but potentially fatal ventricular arrhythmia associated with drug-induced delayed repolarization and prolongation of the QT interval. To determine if the arrhythmogenic potential of noncardiac drugs can be assessed in vitro, we evaluated the effects of 12 drugs on the action potential duration (APD) of cardiac Purkinje fibers and compared results with clinical observations. APD changes in canine and porcine fibers were evaluated under physiologic conditions (37 degrees C, [K+]0 = 4 mM) using standard microelectrode techniques. Six of seven drugs associated with QT prolongation or torsade de pointes in man (cisapride, erythromycin, grepafloxacin, moxifloxacin, sertindole, and sotalol) affected concentration-dependent prolongation of the APD in canine fibers during slow stimulation (2-s basic cycle length), attaining greater than 15% prolongation at high concentrations (> or = 10-fold clinically encountered plasma levels). Each of five drugs not linked clinically to QT prolongation and torsade de pointes (azithromycin, enalaprilat, fluoxetine, indomethacin, and pinacidil) failed to attain 15% prolongation, with fluoxetine, indomethacin, and pinacidil abbreviating the APD. Drugs eliciting the greatest prolongation also demonstrated prominent reverse rate-dependent effects. The antihistamine terfenadine (linked to dose-dependent QT prolongation and torsade de pointes clinically) only minimally prolonged the APD in canine and porcine fibers (and exerted no effect on midmyocardial fibers from left ventricular free wall) at supratherapeutic concentrations. On the basis of concentration-dependent APD prolongation and reverse rate-dependent effects, this Purkinje fiber model detects six of seven drugs linked clinically to acquired long QT syndrome and torsade de pointes, and clears each of five drugs not associated with repolarization abnormalities (overall 92% accuracy), validating the utility of this Purkinje fiber model in the preclinical evaluation of QT prolongation and proarrhythmic risk by noncardiac drugs.

Intrinsic lidocaine affinity for Na channels expressed in Xenopus oocytes depends on alpha (hH1 vs. rSkM1) and beta 1 subunits.

OBJECTIVE: The affinity of lidocaine for the alpha-subunit of the Na channel has been reported to be greater for heart than for non-heart alpha-subunits, and also to be no different. Lidocaine block has a complex voltage dependence caused by a higher affinity for the inactivated state over the resting state. Inactivation kinetics, however, depend upon the alpha-subunit isoform and the presence of the auxiliary beta 1-subunit and will affect measures of block. METHODS: We studied the voltage dependence of lidocaine block of Na currents by a two microelectrode voltage clamp in oocytes injected with RNA for the Na channel alpha-subunits of human heart (hH1a) or a rat skeletal muscle (rSkM1) alone, or coexpressed with the beta 1-subunit. RESULTS: The midpoints of availability for a 25-s conditioning potential in control solutions were -65 mV for rSkM1, -50 for rSkM1 + beta 1, -78 mV for hH1a and -76 for hH1a + beta 1. The Kd of tonic lidocaine block was measured at -90, -100, -110, -120 and -130 mV in the same oocytes. The apparent Kd for both isoforms +/- beta 1 became greater with more negative holding potentials, but tended to reach different plateaus at -130 mV (Kd = 2128 microM for rSkM1, 1760 microM for rSkM1 + beta 1, 433 for hH1a, and 887 microM for hH1a + beta 1). Inactivated state affinities, assessed by fitting the shift in the Boltzmann midpoint of the availability relationship to the modulated receptor model, were 4 microM for rSkM1, 1 microM for rSkM1 + beta 1, 7 microM for hH1a and 9 microM for hH1a + beta 1. CONCLUSION: The heart Na channel alpha-subunits expressed in oocytes have an intrinsically higher rest state affinity for lidocaine compared to rSkM1 after the voltage- and state dependence of block are considered. Coexpression with beta 1 modestly increased the rest affinity of lidocaine for rSkM1, but had the opposite effect for hH1a.

Transitional changes in membrane potential and intracellular [Ca2+] in rat basophilic leukemia cells.

Using whole-cell current-clamp measurements we have found that thapsigargin-mediated activation of store-regulated Ca2+ entry in rat basophilic leukemia cells is accompanied by complex changes in membrane potential. These changes consisted of: (i) an initial slow, small depolarization, (ii) a transitional change in potential to a depolarized value and (iii) transitional changes between a hyperpolarized and a depolarized potential. These complex changes in potential can be explained by the interaction between the endogenous inwardly rectifying K+ conductance and the generation of a small inward current. To investigate the possible influence of these changes of potential on [Ca2+]i, single cell measurements of fura2 fluorescence were undertaken alone or in combination with current-clamp measurements. Thapsigargin-mediated activation of the store-regulated Ca2+ entry pathway was accompanied by a marked increase of [Ca2+]i. During this increase, transient, abrupt declines in [Ca2+]i were detected in approximately 60% of the cells investigated. These changes of [Ca2+]i are consistent with the observed changes of membrane potential recorded under current-clamp.

Coexpression of beta 1 with cardiac sodium channel alpha subunits in oocytes decreases lidocaine block.

Coexpression of the rat beta 1 subunit with rat brain and skeletal muscle sodium channel alpha subunits in Xenopus oocytes normalizes currents by accelerating sodium current decay kinetics, shifting steady state availability relationships, and accelerating recovery from inactivation. Unlike brain and skeletal muscle, the heart alpha subunit expressed without beta 1 has native-like decay kinetics in oocytes. Messenger RNA for beta 1 has been found in heart, but whether and how it affects cardiac sodium channel function are unclear. We studied coexpression of human heart alpha subunit with beta 1 in Xenopus oocytes using two microelectrode voltage-clamp and macropatch techniques. Coexpression with beta 1 caused a significant positive shift of 3-7 mV in the midpoint of the steady state inactivation relationship but did not affect single-channel conductance, activation, current decay, or recovery from inactivation. Sensitivity to lidocaine block, however, was decreased for both resting state block (Kd = 0.5-1.3 mM) and phasic block in response to pulse trains, but inactivated state block was not affected (Kd = approximately 10 microM). Coexpression with beta 1 increased the rate of recovery from lidocaine block, which accounted for the major part of the observed differences in tonic and phasic block. A beta 1 construct with the cytoplasmic tail removed also produced these effects, demonstrating that the beta 1 cytoplasmic tail was not involved in altering lidocaine block. We conclude that the beta 1 subunit is capable of affecting function of the cardiac sodium channel in oocytes by decreasing tonic and phasic lidocaine block with small effects on gating.

The saxitoxin/tetrodotoxin binding site on cloned rat brain IIa Na channels is in the transmembrane electric field.

The rat brain IIa (BrIIa) Na channel alpha-subunit and the brain beta 1 subunit were coexpressed in Xenopus oocytes, and peak whole-oocyte Na current (INa) was measured at a test potential of -10 mV. Hyperpolarization of the holding potential resulted in an increased affinity of STX and TTX rested-state block of BrIIa Na channels. The apparent half-block concentration (ED50) for STX of BrIIa current decreased with hyperpolarizing holding potentials (Vhold). At Vhold of -100 mV, the ED50 was 2.1 +/- 0.4 nM, and the affinity increased to a ED50 of 1.2 +/- 0.2 nM with Vhold of -140 mV. In the absence of toxin, the peak current amplitude was the same for all potentials negative to -90 mV, demonstrating that all of the channels were in a closed conformation and maximally available to open in this range of holding potentials. The Woodhull model (1973) was used to describe the increase of the STX ED50 as a function of holding potential. The equivalent electrical distance of block (delta) by STX was 0.18 from the extracellular milieu when the valence of STX was fixed to +2. Analysis of the holding potential dependence of TTX block yielded a similar delta when the valence of TTX was fixed to +1. We conclude that the guanidinium toxin site is located partially within the transmembrane electric field. Previous site-directed mutagenesis studies demonstrated that an isoform-specific phenylalanine in the BrIIa channel is critical for high affinity toxin block. Therefore, we propose that amino acids at positions corresponding to this Phe in the BrIIa channel, which lie in the outer vestibule of the channel adjacent to the pore entrance,are partially in the transmembrane potential drop.