Focal laser stimulation of fly nociceptors activates distinct axonal and dendritic Ca2+ signals.

Drosophila class IV neurons are polymodal nociceptors that detect noxious mechanical, thermal, optical, and chemical stimuli. Escape behaviors in response to attacks by parasitoid wasps are dependent on class IV cells, whose highly branched dendritic arbors form a fine meshwork that is thought to enable detection of the wasp's needle-like ovipositor barb. To understand how mechanical stimuli trigger cellular responses, we used a focused 405-nm laser to create highly localized lesions to probe the precise position needed to evoke responses. By imaging calcium signals in dendrites, axons, and soma in response to stimuli of varying positions, intensities, and spatial profiles, we discovered that there are two distinct nociceptive pathways. Direct stimulation to dendrites (the contact pathway) produces calcium responses in axons, dendrites, and the cell body, whereas stimulation adjacent to the dendrite (the noncontact pathway) produces calcium responses in the axons only. We interpret the noncontact pathway as damage to adjacent cells releasing diffusible molecules that act on the dendrites. Axonal responses have higher sensitivities and shorter latencies. In contrast, dendritic responses have lower sensitivities and longer latencies. Stimulation of finer, distal dendrites leads to smaller responses than stimulation of coarser, proximal dendrites, as expected if the contact response depends on the geometric overlap of the laser profile and the dendrite diameter. Because the axon signals to the central nervous system to trigger escape behaviors, we propose that the density of the dendritic meshwork is high not only to enable direct contact with the ovipositor but also to enable neuronal activation via diffusing signals from damaged surrounding cells. Dendritic contact evokes responses throughout the dendritic arbor, even to regions distant and distal from the stimulus. These dendrite-wide calcium signals may facilitate hyperalgesia or cellular morphological changes after dendritic damage.

Protospacer Adjacent Motif-Induced Allostery Activates CRISPR-Cas9.

CRISPR-Cas9 is a genome editing technology with major impact in life sciences. In this system, the endonuclease Cas9 generates double strand breaks in DNA upon RNA-guided recognition of a complementary DNA sequence, which strictly requires the presence of a protospacer adjacent motif (PAM) next to the target site. Although PAM recognition is essential for cleavage, it is unknown whether and how PAM binding activates Cas9 for DNA cleavage at spatially distant sites. Here, we find evidence of a PAM-induced allosteric mechanism revealed by microsecond molecular dynamics simulations. PAM acts as an allosteric effector and triggers the interdependent conformational dynamics of the Cas9 catalytic domains (HNH and RuvC), responsible for concerted cleavage of the two DNA strands. Targeting such an allosteric mechanism should enable control of CRISPR-Cas9 functionality.

Alda-1 Protects Against Acrolein-Induced Acute Lung Injury and Endothelial Barrier Dysfunction.

Inhalation of acrolein, a highly reactive aldehyde, causes lung edema. The underlying mechanism is poorly understood and there is no effective treatment. In this study, we demonstrated that acrolein not only dose-dependently induced lung edema but also promoted LPS-induced acute lung injury. Importantly, acrolein-induced lung injury was prevented and rescued by Alda-1, an activator of mitochondrial aldehyde dehydrogenase 2. Acrolein also dose-dependently increased monolayer permeability, disrupted adherens junctions and focal adhesion complexes, and caused intercellular gap formation in primary cultured lung microvascular endothelial cells (LMVECs). These effects were attenuated by Alda-1 and the antioxidant N-acetylcysteine, but not by the NADPH inhibitor apocynin. Furthermore, acrolein inhibited AMP-activated protein kinase (AMPK) and increased mitochondrial reactive oxygen species levels in LMVECs-effects that were associated with impaired mitochondrial respiration. AMPK total protein levels were also reduced in lung tissue of mice and LMVECs exposed to acrolein. Activation of AMPK with 5-aminoimidazole-4-carboxamide-1-ß-4-ribofuranoside blunted an acrolein-induced increase in endothelial monolayer permeability, but not mitochondrial oxidative stress or inhibition of mitochondrial respiration. Our results suggest that acrolein-induced mitochondrial dysfunction may not contribute to endothelial barrier dysfunction. We speculate that detoxification of acrolein by Alda-1 and activation of AMPK may be novel approaches to prevent and treat acrolein-associated acute lung injury, which may occur after smoke inhalation.

Antimalarial efficacy of MMV390048, an inhibitor of Plasmodium phosphatidylinositol 4-kinase.

As part of the global effort toward malaria eradication, phenotypic whole-cell screening revealed the 2-aminopyridine class of small molecules as a good starting point to develop new antimalarial drugs. Stemming from this series, we found that the derivative, MMV390048, lacked cross-resistance with current drugs used to treat malaria. This compound was efficacious against all Plasmodium life cycle stages, apart from late hypnozoites in the liver. Efficacy was shown in the humanized Plasmodium falciparum mouse model, and modest reductions in mouse-to-mouse transmission were achieved in the Plasmodium berghei mouse model. Experiments in monkeys revealed the ability of MMV390048 to be used for full chemoprotection. Although MMV390048 was not able to eliminate liver hypnozoites, it delayed relapse in a Plasmodium cynomolgi monkey model. Both genomic and chemoproteomic studies identified a kinase of the Plasmodium parasite, phosphatidylinositol 4-kinase, as the molecular target of MMV390048. The ability of MMV390048 to block all life cycle stages of the malaria parasite suggests that this compound should be further developed and may contribute to malaria control and eradication as part of a single-dose combination treatment.

A novel multiple-stage antimalarial agent that inhibits protein synthesis.

There is an urgent need for new drugs to treat malaria, with broad therapeutic potential and novel modes of action, to widen the scope of treatment and to overcome emerging drug resistance. Here we describe the discovery of DDD107498, a compound with a potent and novel spectrum of antimalarial activity against multiple life-cycle stages of the Plasmodium parasite, with good pharmacokinetic properties and an acceptable safety profile. DDD107498 demonstrates potential to address a variety of clinical needs, including single-dose treatment, transmission blocking and chemoprotection. DDD107498 was developed from a screening programme against blood-stage malaria parasites; its molecular target has been identified as translation elongation factor 2 (eEF2), which is responsible for the GTP-dependent translocation of the ribosome along messenger RNA, and is essential for protein synthesis. This discovery of eEF2 as a viable antimalarial drug target opens up new possibilities for drug discovery.

Focal Inflammation Causes Carbenoxolone-Sensitive Tactile Hypersensitivity in Mice.

A focal and transitory inflammation induced by injection of complete Freund's adjuvant (CFA) in the submandibular skin of mice elicits pain behavior that persists for several weeks after the initial inflammation has resolved. Chronic pain, assessed as tactile hypersensitivity to stimulation with von Frey filaments, was evident from 1-7 weeks following CFA injection, although inflammation at the injection site was resolved by 3-4 weeks. In contrast, there were no changes in tactile sensitivity in the paw (un-injected site for comparison), no alterations in open field behavior and no differences in a functional observation battery evident in CFA-treated mice compared to controls (saline-injected) or to baseline (before CFA injection). Neither strain (Balb/c vs. C57BL/6) nor sex differences in baseline tactile threshold were significant in the submandibular skin. CFA-induced tactile hypersensitivity was also not a function of strain or sex. A single intraperitoneal injection of the gap junction blocker carbenoxolone (CBX) restored normal tactile thresholds in CFA-treated mice when administered at the peak of inflammation (1 week), after significant resolution of inflammation (3 weeks) or after total resolution of inflammation (4 and 5 weeks) without altering the tactile threshold of control subjects, tactile threshold in the paw or open field behavior. Thus, in this novel model of post-inflammatory pain, transitory inflammation induced persistent sex- and strain-independent behavioral hypersensitivity that was reversed by the gap junction blocker CBX, suggesting neuronal and/or glial plasticity as a major component of the chronic pain.