Two-pore channels as master regulators of membrane trafficking and endocytic well-being.

Two-pore channels (TPCs) are a ubiquitous class of Ca2+- and Na+-permeable ion channels expressed within the endo-lysosomal system. They have emerged as central regulators of a wide array of physiological processes intimately linked to information processing. In this short review, we highlight how molecular and chemical strategies have uncovered multiple roles for TPCs in regulating various aspects of endo-lysosomal trafficking associated with disease. We summarise advances in the identification of new small molecules to pharmacologically target TPCs for medical benefit. Lastly, we discuss possible underpinning molecular mechanism(s) that translate TPC-mediated ionic fluxes to function.

Polyhexamethylene Biguanide:Polyurethane Blend Nanofibrous Membranes for Wound Infection Control.

Polyhexamethylene biguanide (PHMB) is a broad-spectrum antiseptic which avoids many efficacy and toxicity problems associated with antimicrobials, in particular, it has a low risk of loss of susceptibility due to acquired antimicrobial resistance. Despite such advantages, PHMB is not widely used in wound care, suggesting more research is required to take full advantage of PHMB's properties. We hypothesised that a nanofibre morphology would provide a gradual release of PHMB, prolonging the antimicrobial effects within the therapeutic window. PHMB:polyurethane (PU) electrospun nanofibre membranes were prepared with increasing PHMB concentrations, and the effects on antimicrobial activities, mechanical properties and host cell toxicity were compared. Overall, PHMB:PU membranes displayed a burst release of PHMB during the first hour following PBS immersion (50.5-95.9% of total released), followed by a gradual release over 120 h (≤25 wt % PHMB). The membranes were hydrophilic (83.7-53.3°), gradually gaining hydrophobicity as PHMB was released. They displayed superior antimicrobial activity, which extended past the initial release period, retained PU hyperelasticity regardless of PHMB concentration (collective tensile modulus of 5-35% PHMB:PU membranes, 3.56 ± 0.97 MPa; ultimate strain, >200%) and displayed minimal human cell toxicity (<25 wt % PHMB). With further development, PHMB:PU electrospun membranes may provide improved wound dressings.

Mining of Ebola virus entry inhibitors identifies approved drugs as two-pore channel pore blockers.

Two-pore channels (TPCs) are Ca2+-permeable ion channels localised to the endo-lysosomal system where they regulate trafficking of various cargoes including viruses. As a result, TPCs are emerging as important drug targets. However, their pharmacology is ill-defined. There are no approved drugs to target them. And their mechanism of ligand activation is largely unknown. Here, we identify a number of FDA-approved drugs as TPC pore blockers. Using a model of the pore of human TPC2 based on recent structures of mammalian TPCs, we virtually screened a database of ~1500 approved drugs. Because TPCs have recently emerged as novel host factors for Ebola virus entry, we reasoned that Ebola virus entry inhibitors may exert their effects through inhibition of TPCs. Cross-referencing hits from the TPC virtual screen with two recent high throughput anti-Ebola screens yielded approved drugs targeting dopamine and estrogen receptors as common hits. These compounds inhibited endogenous NAADP-evoked Ca2+ release from sea urchin egg homogenates, NAADP-mediated channel activity of TPC2 re-routed to the plasma membrane, and PI(3,5)P2-mediated channel activity of TPC2 expressed in enlarged lysosomes. Mechanistically, single channel analyses showed that the drugs reduced mean open time consistent with a direct action on the pore. Functionally, drug potency in blocking TPC2 activity correlated with inhibition of Ebola virus-like particle entry. Our results expand TPC pharmacology through the identification of approved drugs as novel blockers, support a role for TPCs in Ebola virus entry, and provide insight into the mechanisms underlying channel regulation. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

Assembly of microbial communities in replicate nutrient-cycling model ecosystems follows divergent trajectories, leading to alternate stable states.

We studied in detail the reproducibility of community development in replicate nutrient-cycling microbial microcosms that were set up identically and allowed to develop under the same environmental conditions. Multiple replicate closed microcosms were constructed using pond sediment and water, enriched with cellulose and sulphate, and allowed to develop over several months under constant environmental conditions, after which their microbial communities were characterized using 16S rRNA gene sequencing. Our results show that initially similar microbial communities can follow alternative - yet stable - trajectories, diverging in time in a system size-dependent manner. The divergence between replicate communities increased in time and decreased with larger system size. In particular, notable differences emerged in the heterotrophic degrader communities in our microcosms; one group of steady state communities was enriched with Firmicutes, while the other was enriched with Bacteroidetes. The communities dominated by these two phyla also contained distinct populations of sulphate-reducing bacteria. This biomodality in community composition appeared to arise during recovery from a low-diversity state that followed initial cellulose degradation and sulphate reduction.