Integrating molecular and radiological screening tools during community-based active case-finding for tuberculosis and COVID-19 in southern Africa.

Objectives: To evaluate diagnostic yield and feasibility of integrating testing for TB and COVID-19 using molecular and radiological screening tools during community-based active case-finding (ACF).

Methods: Community-based participants with presumed TB and/or COVID-19 were recruited using a mobile clinic. Participants underwent simultaneous point-of-care (POC) testing for TB (sputum; Xpert Ultra) and COVID-19 (nasopharyngeal swabs; Xpert SARS-CoV-2).

Reactive oxygen species production induced by pore opening in cardiac mitochondria: The role of complex II.

Succinate-driven reverse electron transport (RET) through complex I is hypothesized to be a major source of reactive oxygen species (ROS) that induces permeability transition pore (PTP) opening and damages the heart during ischemia/reperfusion. Because RET can only generate ROS when mitochondria are fully polarized, this mechanism is self-limiting once PTP opens during reperfusion. In the accompanying article (Korge, P.

Reactive oxygen species production induced by pore opening in cardiac mitochondria: The role of complex III.

Recent evidence has implicated succinate-driven reverse electron transport (RET) through complex I as a major source of damaging reactive oxygen species (ROS) underlying reperfusion injury after prolonged cardiac ischemia. However, this explanation may be incomplete, because RET on reperfusion is self-limiting and therefore transient. RET can only generate ROS when mitochondria are well polarized, and it ceases when permeability transition pores (PTP) open during reperfusion.

Hexokinase-mitochondrial interactions regulate glucose metabolism differentially in adult and neonatal cardiac myocytes.

In mammalian tumor cell lines, localization of hexokinase (HK) isoforms to the cytoplasm or mitochondria has been shown to control their anabolic (glycogen synthesis) and catabolic (glycolysis) activities. In this study, we examined whether HK isoform differences could explain the markedly different metabolic profiles between normal adult and neonatal cardiac tissue. We used a set of novel genetically encoded optical imaging tools to track, in real-time in isolated adult (ARVM) and neonatal (NRVM) rat ventricular myocytes, the subcellular distributions of HKI and HKII, and the functional consequences on glucose utilization.

The cardiac Na+-Ca2+ exchanger has two cytoplasmic ion permeation pathways.

The Na(+)-Ca(2+) exchanger (NCX) is a ubiquitously expressed plasma membrane protein. It plays a fundamental role in Ca(2+) homeostasis by moving Ca(2+) out of the cell using the electrochemical gradient of Na(+) as the driving force. Recent structural studies of a homologous archaebacterial exchanger, NCX_Mj, revealed its outward configuration with two potential ion permeation pathways exposed to the extracellular environment.

Ca2+-dependent structural rearrangements within Na+-Ca2+ exchanger dimers.

Cytoplasmic Ca(2+) is known to regulate Na(+)-Ca(2+) exchanger (NCX) activity by binding to two adjacent Ca(2+)-binding domains (CBD1 and CBD2) located in the large intracellular loop between transmembrane segments 5 and 6. We investigated Ca(2+)-dependent movements as changes in FRET between exchanger proteins tagged with CFP or YFP at position 266 within the large cytoplasmic loop. Data indicate that the exchanger assembles as a dimer in the plasma membrane.

Conformational changes of a Ca2+-binding domain of the Na+/Ca2+ exchanger monitored by FRET in transgenic zebrafish heart.

The Na(+)/Ca(2+) exchanger is the major Ca(2+) extrusion mechanism in cardiac myocytes. The activity of the cardiac Na(+)/Ca(2+) exchanger is dynamically regulated by intracellular Ca(2+). Previous studies indicate that Ca(2+) binding to a high-affinity Ca(2+)-binding domain (CBD1) in the large intracellular loop is involved in regulation.

Phosphatidylinositol-4,5-bisphosphate (PIP2) regulation of strong inward rectifier Kir2.1 channels: multilevel positive cooperativity.

Inwardly rectifying potassium (Kir) channels are gated by the interaction of their cytoplasmic regions with membrane-bound phosphatidylinositol-4,5-bisphosphate (PIP(2)). In the present study, we examined how PIP(2) interaction regulates channel availability and channel openings to various subconductance levels (sublevels) as well as the fully open state in the strong inward rectifier Kir2.1 channel.

Dynamic modulation of intracellular glucose imaged in single cells using a FRET-based glucose nanosensor.

To study intracellular glucose homeostasis, the glucose nanosensor FLIPglu-600 microM, which undergoes changes in fluorescence resonance energy transfer (FRET) upon interaction with glucose, was expressed in four mammalian cell lines: COS-7, CHO, HEK293, and C2C12. Upon addition of extracellular glucose, the intracellular FRET ratio decreased rapidly as intracellular glucose increased. The kinetics were fast (tau=5 to 15 s) in COS and C2C12 cells and slow (tau=20 to 40 s) in HEK and CHO cells.

On the Breslow-Holubkov estimator.

Breslow and Holubkov (J Roy Stat Soc B 59:447-461 1997a) developed semiparametric maximum likelihood estimation for two-phase studies with a case-control first phase under a logistic regression model and noted that, apart for the overall intercept term, it was the same as the semiparametric estimator for two-phase studies with a prospective first phase developed in Scott and Wild (Biometrica 84:57-71 1997). In this paper we extend the Breslow-Holubkov result to general binary regression models and show that it has a very simple relationship with its prospective first-phase counterpart. We also explore why the design of the first phase only affects the intercept of a logistic model, simplify the calculation of standard errors, establish the semiparametric efficiency of the Breslow-Holubkov estimator and derive its asymptotic distribution in the general case.

A novel role for connexin hemichannel in oxidative stress and smoking-induced cell injury.

Oxidative stress is linked to many pathological conditions, including ischemia, atherosclerosis and neurodegenerative disorders. The molecular mechanisms of oxidative stress induced pathophysiology and cell death are currently poorly understood. Our present work demonstrates that oxidative stress induced by reactive oxygen species and cigarette smoke extract depolarize the cell membrane and open connexin hemichannels.

Activation of inwardly rectifying potassium (Kir) channels by phosphatidylinosital-4,5-bisphosphate (PIP2): interaction with other regulatory ligands.

All members of the inwardly rectifying potassium channels (Kir1-7) are regulated by the membrane phospholipid, phosphatidylinosital-4,5-bisphosphate (PIP(2)). Some are also modulated by other regulatory factors or ligands such as ATP and G-proteins, which give them their common names, such as the ATP sensitive potassium (K(ATP)) channel and the G-protein gated potassium channel. Other more non-specific regulators include polyamines, kinases, pH and Na(+) ions.

ATP-sensitive K+ channels: regulation of bursting by the sulphonylurea receptor, PIP2 and regions of Kir6.2.

ATP-sensitive K+ channels composed of the pore-forming protein Kir6.2 and the sulphonylurea receptor SUR1 are inhibited by ATP and activated by Phosphatidylinositol Bisphosphate (PIP2). Residues involved in binding of these ligands to the Kir6.

Long polyamines act as cofactors in PIP2 activation of inward rectifier potassium (Kir2.1) channels.

Phosphatidylinosital-4,5-bisphosphate (PIP2) acts as an essential factor regulating the activity of all Kir channels. In most Kir members, the dependence on PIP2 is modulated by other factors, such as protein kinases (in Kir1), G(betagamma) (in Kir3), and the sulfonylurea receptor (in Kir6). So far, however, no regulator has been identified in Kir2 channels.

ATP sensitivity of ATP-sensitive K+ channels: role of the gamma phosphate group of ATP and the R50 residue of mouse Kir6.2.

ATP-sensitive K (K(ATP)) channels are composed of Kir6, the pore-forming protein, and the sulphonylurea receptor SUR, a regulatory protein. We and others have previously shown that positively charged residues in the C terminus of Kir6.2, including R201 and K185, interact with the alpha and beta phosphate groups of ATP, respectively, to induce channel closure.

Regulation of the ATP-sensitive K channel Kir6.2 by ATP and PIP(2).

ATP-sensitive K (K(ATP)) channels are blocked by ATP and activated by PIP(2). Both negatively-charged ligands are presumed to bind to positively-charged residues on the N-and C-termini of the channel's cytoplasmic domain. Evidence summarized here suggests that the channel's interaction with ATP and PIP(2) is regulated by separate groups of residues, involving both direct charge-charge interactions and allosteric effects.

Regulation of gating by negative charges in the cytoplasmic pore in the Kir2.1 channel.

Inward rectifier K(+) channels commonly exhibit long openings (slow gating) punctuated by rapid open-close transitions (fast gating), suggesting that two separate gates may control channel open-closed transitions. Previous studies have suggested possible gate locations at the selectivity filter and at the 'bundle crossing', where the two transmembrane segments (M1 and M2) cross near the cytoplasmic end of the pore. Wild-type Kir2.

Mechanosensitivity of GIRK channels is mediated by protein kinase C-dependent channel-phosphatidylinositol 4,5-bisphosphate interaction.

Gprotein-activated inwardly rectifying K+ channel (GIRK or Kir3) currents are inhibited by mechanical stretch of the cell membrane, but the underlying mechanisms are not understood. In Xenopus oocytes heterologously expressing GIRK channels, membrane stretch induced by 50% reduction of osmotic pressure caused a prompt reduction of GIRK1/4, GIRK1, and GIRK4 currents by 16.6-42.

Molecular mechanism for ATP-dependent closure of the K+ channel Kir6.2.

In the ATP-dependent K+ (KATP) channel pore-forming protein Kir6.2, mutation of three positively charged residues, R50, K185 and R201, impairs the ability of ATP to close the channel. The mutations do not change the channel open probability (Po) in the absence of ATP, supporting the involvement of these residues in ATP binding.

Inward rectification by polyamines in mouse Kir2.1 channels: synergy between blocking components.

We recently characterized two distinct mechanisms by which the polyamine spermine blocks Kir2.1 channels: (1) by reduction of negative surface charges in the cytoplasmic pore, thereby reducing single-channel conductance, and (2) by direct open channel transmembrane pore block. The extent to which the surface charge reduction component is mediated by passive surface charge screening versus binding of polyamines to these charges, as well as the extent to which the surface charge reduction and pore block mechanisms are synergistic, versus simply additive, was not established.

Molecular basis for Kir6.2 channel inhibition by adenine nucleotides.

K(ATP) channels are comprised of a pore-forming protein, Kir6.x, and the sulfonylurea receptor, SURx. Interaction of adenine nucleotides with Kir6.

Spermine block of the strong inward rectifier potassium channel Kir2.1: dual roles of surface charge screening and pore block.

Inward rectification in strong inward rectifiers such as Kir2.1 is attributed to voltage-dependent block by intracellular polyamines and Mg(2+). Block by the polyamine spermine has a complex voltage dependence with shallow and steep components and complex concentration dependence.