STIM1-dependent store-operated calcium entry mediates sex differences in macrophage chemotaxis and monocyte recruitment.

Infiltration of monocyte-derived cells to sites of infection and injury is greater in males than in females, due in part, to increased chemotaxis, the process of directed cell movement toward a chemical signal. The mechanisms governing sexual dimorphism in chemotaxis are not known. We hypothesized a role for the store-operated calcium entry (SOCE) pathway in regulating chemotaxis by modulating leading and trailing edge membrane dynamics. We measured the chemotactic response of bone marrow-derived macrophages migrating toward complement component 5a (C5a). Chemotactic ability was dependent on sex and inflammatory phenotype (M0, M1, and M2), and correlated with SOCE. Notably, females exhibited a significantly lower magnitude of SOCE than males. When we knocked out the SOCE gene, stromal interaction molecule 1 (STIM1), it eliminated SOCE and equalized chemotaxis across both sexes. Analysis of membrane dynamics at the leading and trailing edges showed that STIM1 influences chemotaxis by facilitating retraction of the trailing edge. Using BTP2 to pharmacologically inhibit SOCE mirrored the effects of STIM1 knockout, demonstrating a central role of STIM/Orai-mediated calcium signaling. Importantly, by monitoring the recruitment of adoptively transferred monocytes in an in vivo model of peritonitis, we show that increased infiltration of male monocytes during infection is dependent on STIM1. These data support a model in which STIM1-dependent SOCE is necessary and sufficient for mediating the sex difference in monocyte recruitment and macrophage chemotactic ability by regulating trailing edge dynamics.

The last days of Aporia crataegi (L.) in Britain: Evaluating genomic erosion in an extirpated butterfly.

Current rates of habitat degradation and climate change are causing unprecedented declines in global biodiversity. Studies on vertebrates highlight how conservation genomics can be effective in identifying and managing threatened populations, but it is unclear how vertebrate-derived metrics of genomic erosion translate to invertebrates, with their markedly different population sizes and life histories. The Black-veined White butterfly (Aporia crataegi) was extirpated from Britain in the 1920s. Here, we sequenced historical DNA from 17 specimens collected between 1854 and 1924 to reconstruct demography and compare levels of genomic erosion between extirpated British and extant European mainland populations. We contrast these results using modern samples of the Common Blue butterfly (Polyommatus icarus); a species with relatively stable demographic trends in Great Britain. We provide evidence for bottlenecks in both these species around the period of post-glacial colonization of the British Isles. Our results reveal different demographic histories and Ne for both species, consistent with their fates in Britain, likely driven by differences in life history, ecology and genome size. Despite a difference, by an order of magnitude, in historical effective population sizes (Ne), reduction in genome-wide heterozygosity in A. crataegi was comparable to that in P. icarus. Symptomatic of A. crataegi's disappearance were marked increases in runs-of-homozygosity (RoH), potentially indicative of recent inbreeding, and accumulation of putatively mildly and weakly deleterious variants. Our results provide a rare glimpse of genomic erosion in a regionally extinct insect and support the potential use of genomic erosion metrics in identifying invertebrate populations or species in decline.

TASK inhibition by mild acidosis increases Ca2+ oscillations to mediate pH sensing in rat carotid body chemoreceptor cells.

Severe levels of acidosis (pH < 6.8) have been shown to cause a sustained rise in cytosolic Ca2+ concentration in carotid body Type 1 (glomus) cells. To understand how physiologically relevant levels of acidosis regulate Ca2+ signaling in glomus cells, we studied the effects of small changes in extracellular pH (pHo) on the kinetics of Ca2+ oscillations. A decrease in pHo from 7.4 to 7.3 (designated mild) and 7.2 (designated moderate) acidosis produced significant increases in the frequency and amplitude of Ca2+ oscillations. These effects of acidosis on Ca2+ oscillations were not blocked by NS383 and amiloride [acid-sensing ion channel (ASIC) inhibitors]. Mild and moderate levels of acidosis, however, caused a small but significant inhibition of two-pore domain acid-sensing K+ channels (TASK) (TASK-1- and TASK-3-like channels) and depolarized the cell by 6-13 mV. Acidosis-induced increase in Ca2+ oscillations was inhibited by nifedipine (1 µM; L-type Cav inhibitor) and by TTA-P2 (20 µM; T-type Cav inhibitor). Mild inhibition of TASK activity by N-[(2,4-difluorophenyl)methyl]-2'-[[[2-(4methoxyphenyl)acetyl]amino]methyl][1,1'-biphenyl]-2-carboxamide (A1899) (0.3 µM) and 1-[1-[6-[[1,1'-biphenyl]-4-ylcarbonyl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine-4-yl]-4-piperidinyl]-1-butanon (PK-THPP) (0.1 µM) increased Ca2+ oscillation frequency to levels similar to those observed with mild-moderate acidosis. Mild acidosis (pHo 7.3) and mild hypoxia (∼5%O2) produced similar levels of changes in the kinetics of Ca2+ oscillations. Block of tetraethylammonium (TEA)-sensitive Kv channels did not affect acid-induced increase in Ca2+ oscillations. Our study shows that mild and moderate levels of acidosis increase the frequency and amplitude of Ca2+ oscillations primarily by inhibition of TASK without involving ASICs, and suggests a major role of TASK for signal transduction in response to a physiological change in pHo.

Signatures of increasing environmental stress in bumblebee wings over the past century: Insights from museum specimens.

Determining when animal populations have experienced stress in the past is fundamental to understanding how risk factors drive contemporary and future species' responses to environmental change. For insects, quantifying stress and associating it with environmental factors has been challenging due to a paucity of time-series data and because detectable population-level responses can show varying lag effects. One solution is to leverage historic entomological specimens to detect morphological proxies of stress experienced at the time stressors emerged, allowing us to more accurately determine population responses. Here we studied specimens of four bumblebee species, an invaluable group of insect pollinators, from five museums collected across Britain over the 20th century. We calculated the degree of fluctuating asymmetry (FA; random deviations from bilateral symmetry) between the right and left forewings as a potential proxy of developmental stress. We: (a) investigated whether baseline FA levels vary between species, and how this compares between the first and second half of the century; (b) determined the extent of FA change over the century in the four bumblebee species, and whether this followed a linear or nonlinear trend; (c) tested which annual climatic conditions correlated with increased FA in bumblebees. Species differed in their baseline FA, with FA being higher in the two species that have recently expanded their ranges in Britain. Overall, FA significantly increased over the century but followed a nonlinear trend, with the increase starting c. 1925. We found relatively warm and wet years were associated with higher FA. Collectively our findings show that FA in bumblebees increased over the 20th century and under weather conditions that will likely increase in frequency with climate change. By plotting FA trends and quantifying the contribution of annual climate conditions on past populations, we provide an important step towards improving our understanding of how environmental factors could impact future populations of wild beneficial insects.

The Mechanical Characterization and Comparions of Male and Female Calvaria Under Four-Point Bending Impacts.

The circumstances in which we mechanically test and critically assess human calvarium tissue would find relevance under conditions encompassing real-world head impacts. These conditions include, among other variables, impact velocities, and strain rates. Compared to quasi-static loading on calvaria, there is less reporting on the impact loading of the calvaria and consequently, there are relatively fewer mechanical properties on calvaria at relevant impact loading rates available in the literature. The purpose of this work was to report on the mechanical response of 23 human calvarium specimens subjected to dynamic four-point bending impacts. Impacts were performed using a custom-built four-point impact apparatus at impact velocities of 0.86-0.89 m/s resulting in surface strain rates of 2-3/s-representative of strain rates observed in vehicle collisions and blunt impacts. The study revealed comparable effective bending moduli (11-15 GPa) to the limited work reported on the impact mechanics of calvaria in the literature, however, fracture bending stress (10-47 MPa) was relatively less. As expected, surface strains at fracture (0.21-0.25%) were less compared to studies that performed quasi-static bending. Moreover, the study revealed no significant differences in mechanical response between male and female calvaria. The findings presented in this work are relevant to many areas including validating surrogate skull fracture models in silico or laboratory during impact and optimizing protective devices used by civilians to reduce the risk of a serious head injury.

Mycorrhizal symbiosis pathway and edaphic fertility frame root economics space among tree species.

The root economics space (RES) is multidimensional and largely shaped by belowground biotic and abiotic influences. However, how root-fungal symbioses and edaphic fertility drive this complexity remains unclear. Here, we measured absorptive root traits of 112 tree species in temperate and subtropical forests of China, including traits linked to functional differences between arbuscular mycorrhizal (AM) and ectomycorrhizal (ECM) hosts. Our data, from known mycorrhizal tree species, revealed a 'fungal-symbiosis' dimension distinguishing AM from ECM species. This divergence likely resulted from the contrasting mycorrhizal evolutionary development of AM vs ECM associations. Increased root tissue cortical space facilitates AM symbiosis, whereas increased root branching favours ECM symbiosis. Irrespective of mycorrhizal type, a 'root-lifespan' dimension reflecting aspects of root construction cost and defence was controlled by variation in specific root length and root tissue density, which was fully independent of root nitrogen content. Within this function-based RES, we observed a substantial covariation of axes with soil phosphorus and nitrate levels, highlighting the role played by these two axes in nutrient acquisition and conservation. Overall, our findings demonstrate the importance of evolved mycorrhizal symbiosis pathway and edaphic fertility in framing the RES, and provide theoretical and mechanistic insights into the complexity of root economics.

3D Microstructure-Based Finite Element Simulation of Cold-Sprayed Al-Al2O3 Composite Coatings Under Quasi-Static Compression and Indentation Loading.

This study developed microstructure-based finite element (FE) models to investigate the behavior of cold-sprayed aluminum-alumina (Al-Al2O3) metal matrix composite (MMC) coatings subject to indentation and quasi-static compression loading. Based on microstructural features (i.e., particle weight fraction, particle size, and porosity) of the MMC coatings, 3D representative volume elements (RVEs) were generated by using Digimat software and then imported into ABAQUS/Explicit. State-of-the-art physics-based modeling approaches were incorporated into the model to account for particle cracking, interface debonding, and ductile failure of the matrix. This allowed for analysis and informing on the deformation and failure responses. The model was validated with experimental results for cold-sprayed Al-34 wt.% Al2O3 and Al-46 wt.% Al2O3 metal matrix composite coatings under quasi-static compression by comparing the stress versus strain histories and observed failure mechanisms (e.g., matrix ductile failure). The results showed that the computational framework is able to capture the response of this cold-sprayed material system under compression and indentation, both qualitatively and quantitatively. The outcomes of this work have implications for extending the model to materials design and for applications involving different types of loading in real-world application (e.g., erosion and fatigue).

Alignment-free inference of hierarchical and reticulate phylogenomic relationships.

We are amidst an ongoing flood of sequence data arising from the application of high-throughput technologies, and a concomitant fundamental revision in our understanding of how genomes evolve individually and within the biosphere. Workflows for phylogenomic inference must accommodate data that are not only much larger than before, but often more error prone and perhaps misassembled, or not assembled in the first place. Moreover, genomes of microbes, viruses and plasmids evolve not only by tree-like descent with modification but also by incorporating stretches of exogenous DNA. Thus, next-generation phylogenomics must address computational scalability while rethinking the nature of orthogroups, the alignment of multiple sequences and the inference and comparison of trees. New phylogenomic workflows have begun to take shape based on so-called alignment-free (AF) approaches. Here, we review the conceptual foundations of AF phylogenetics for the hierarchical (vertical) and reticulate (lateral) components of genome evolution, focusing on methods based on k-mers. We reflect on what seems to be successful, and on where further development is needed.

Ca2+ oscillations in rat carotid body type 1 cells in normoxia and hypoxia.

We studied the mechanisms by which carotid body glomus (type 1) cells produce spontaneous Ca2+ oscillations in normoxia and hypoxia. In cells perfused with normoxic solution at 37°C, we observed relatively uniform, low-frequency Ca2+ oscillations in >60% of cells, with each cell showing its own intrinsic frequency and amplitude. The mean frequency and amplitude of Ca2+ oscillations were 0.6 ± 0.1 Hz and 180 ± 42 nM, respectively. The duration of each Ca2+ oscillation ranged from 14 to 26 s (mean of ∼20 s). Inhibition of inositol (1,4,5)-trisphosphate receptor and store-operated Ca2+ entry (SOCE) using 2-APB abolished Ca2+ oscillations. Inhibition of endoplasmic reticulum Ca2+-ATPase (SERCA) using thapsigargin abolished Ca2+ oscillations. ML-9, an inhibitor of STIM1 translocation, also strongly reduced Ca2+ oscillations. Inhibitors of L- and T-type Ca2+ channels (Cav; verapamil>nifedipine>TTA-P2) markedly reduced the frequency of Ca2+ oscillations. Thus, Ca2+ oscillations observed in normoxia were caused by cyclical Ca2+ fluxes at the ER, which was supported by Ca2+ influx via Ca2+ channels. Hypoxia (2-5% O2) increased the frequency and amplitude of Ca2+ oscillations, and Cav inhibitors (verapamil>nifedipine>>TTA-P2) reduced these effects of hypoxia. Our study shows that Ca2+ oscillations represent the basic Ca2+ signaling mechanism in normoxia and hypoxia in CB glomus cells.

Protective Headgear Attenuates Forces on the Inner Table and Pressure in the Brain Parenchyma During Blast and Impact: An Experimental Study Using a Simulant-Based Surrogate Model of the Human Head.

Military personnel sustain head and brain injuries as a result of ballistic, blast, and blunt impact threats. Combat helmets are meant to protect the heads of these personnel during injury events. Studies show peak kinematics and kinetics are attenuated using protective headgear during impacts; however, there is limited experimental biomechanical literature that examines whether or not helmets mitigate peak mechanics delivered to the head and brain during blast. While the mechanical links between blast and brain injury are not universally agreed upon, one hypothesis is that blast energy can be transmitted through the head and into the brain. These transmissions can lead to rapid skull flexure and elevated pressures in the cranial vault, and, therefore, may be relevant in determining injury likelihood. Therefore, it could be argued that assessing a helmet for the ability to mitigate mechanics may be an appropriate paradigm for assessing the potential protective benefits of helmets against blast. In this work, we use a surrogate model of the head and brain to assess whether or not helmets and eye protection can alter mechanical measures during both head-level face-on blast and high forehead blunt impact events. Measurements near the forehead suggest head protection can attenuate brain parenchyma pressures by as much as 49% during blast and 52% during impact, and forces on the inner table of the skull by as much as 80% during blast and 84% during impact, relative to an unprotected head.

Migratory flexibility in native Hawai'ian amphidromous fishes.

We assessed the prevalence of life history variation across four of the five native amphidromous Hawai'ian gobioids to determine whether some or all exhibit evidence of partial migration. Analysis of otolith Sr.: Ca concentrations affirmed that all are amphidromous and revealed evidence of partial migration in three of the four species. We found that 25% of Lentipes concolor (n = 8), 40% of Eleotris sandwicensis (n = 20) and 29% of Stenogobius hawaiiensis (n = 24) did not exhibit a migratory life-history. In contrast, all individuals of Sicyopterus stimpsoni (n = 55) included in the study went to sea as larvae. Lentipes concolor exhibited the shortest mean larval duration (LD) at 87 days, successively followed by E. sandwicensis (mean LD = 102 days), S. hawaiiensis (mean LD = 114 days) and S. stimpsoni (mean LD = 120 days). These findings offer a fresh perspective on migratory life histories that can help improve efforts to conserve and protect all of these and other at-risk amphidromous species that are subject to escalating anthropogenic pressures in both freshwater and marine environments.

Rapid analysis of metagenomic data using signature-based clustering.

BACKGROUND: Sequencing highly-variable 16S regions is a common and often effective approach to the study of microbial communities, and next-generation sequencing (NGS) technologies provide abundant quantities of data for analysis. However, the speed of existing analysis pipelines may limit our ability to work with these quantities of data. Furthermore, the limited coverage of existing 16S databases may hamper our ability to characterise these communities, particularly in the context of complex or poorly studied environments. RESULTS: In this article we present the SigClust algorithm, a novel clustering method involving the transformation of sequence reads into binary signatures. When compared to other published methods, SigClust yields superior cluster coherence and separation of metagenomic read data, while operating within substantially reduced timeframes. We demonstrate its utility on published Illumina datasets and on a large collection of labelled wound reads sourced from patients in a wound clinic. The temporal analysis is based on tracking the dominant clusters of wound samples over time. The analysis can identify markers of both healing and non-healing wounds in response to treatment. Prominent clusters are found, corresponding to bacterial species known to be associated with unfavourable healing outcomes, including a number of strains of Staphylococcus aureus. CONCLUSIONS: SigClust identifies clusters rapidly and supports an improved understanding of the wound microbiome without reliance on a reference database. The results indicate a promising use for a SigClust-based pipeline in wound analysis and prediction, and a possible novel method for wound management and treatment.

Effects of hurricanes and climate oscillations on annual variation in reproduction in wet forest, Puerto Rico.

Interannual changes in global climate and weather disturbances may influence reproduction in tropical forests. Phenomena such as the El Niño Southern Oscillation (ENSO) are known to produce interannual variation in reproduction, as do severe storms such as hurricanes. Using stationary trap-based phenology data collected fortnightly from 1993 to 2014 from a hurricane-affected (1989 Hugo, 1998 Georges) subtropical wet forest in northeastern Puerto Rico, we conducted a time series analysis of flowering and seed production. We addressed (1) the degree to which interannual variation in flower and seed production was influenced by global climate drivers and time since hurricane disturbance, and (2) how long-term trends in reproduction varied with plant lifeform. The seasonally de-trended number of species in flower fluctuated over time while the number of species producing seed exhibited a declining trend, one that was particularly evident during the second half of the study period. Lagged El Niño indices and time series hurricane disturbance jointly influenced the trends in numbers of flowering and fruiting species, suggesting complex global influences on tropical forest reproduction with variable periodicities. Lag times affecting flowering tended to be longer than those affecting fruiting. Long-term patterns of reproduction in individual lifeforms paralleled the community-wide patterns, with most groups of lifeform exhibiting a long-term decline in seed but not flower production. Exceptions were found for hemiepiphytes, small trees, and lianas whose seed reproduction increased and then declined over time. There was no long-term increase in flower production as reported in other Neotropical sites.

TASK-1 (K2P3) and TASK-3 (K2P9) in Rabbit Carotid Body Glomus Cells.

Glomus cells isolated from rabbit and rat/mouse carotid bodies have been used for many years to study the role of ion channels in hypoxia sensing. Studies show that hypoxia inhibits the inactivating K+ channels (Kv4) in rabbits, but inhibits TASK in rats/mice to elicit the hypoxic response. Because the role of TASK in rabbit glomus cells is not known, we isolated glomus cells from rabbits and studied the expression of TASK mRNA in the whole carotid body (CB), changes in [Ca2+]i and TASK activity. RT-PCR showed that rabbit CB expressed mRNA for TASK-3 and several Kv (Kv2.1, Kv3.1 and Kv3.3). In rabbit glomus cells in which 20 mM KClo elevated [Ca2+], anoxia also elicited a strong rise in [Ca2+]. In cell-attached patches with 140 mM KCl in the pipette, basal openings of ion channels with single-channel conductance levels of 16-pS, 34-pS, and 42-pS were present. TREK-like channels were also observed. In inside-out patches with high [Ca2+]i, BK was activated. The 42-pS channel opened spontaneously and briefly. The 16-pS and 34-pS channels showed properties similar to those of TASK-1 and TASK-3, respectively. TASK activity in cell-attached patches was lower than that in rat glomus cells under identical recording conditions. Hypoxia (~0.5%O2) reduced TASK activity by ~52% and depolarized the cells by ~30 mV. Our results show that the O2-sensitive TASK contributes to the hypoxic response in rabbit glomus cells.

The transcription factor GLI1 modulates the inflammatory response during pancreatic tissue remodeling.

Pancreatic cancer, one of the deadliest human malignancies, is almost uniformly associated with a mutant, constitutively active form of the oncogene Kras. Studies in genetically engineered mouse models have defined a requirement for oncogenic KRAS in both the formation of pancreatic intraepithelial neoplasias, the most common precursor lesions to pancreatic cancer, and in the maintenance and progression of these lesions. Previous work using an inducible model allowing tissue-specific and reversible expression of oncogenic Kras in the pancreas indicates that inactivation of this GTPase at the pancreatic intraepithelial neoplasia stage promotes pancreatic tissue repair. Here, we extend these findings to identify GLI1, a transcriptional effector of the Hedgehog pathway, as a central player in pancreatic tissue repair upon Kras inactivation. Deletion of a single allele of Gli1 results in improper stromal remodeling and perdurance of the inflammatory infiltrate characteristic of pancreatic tumorigenesis. Strikingly, this partial loss of Gli1 affects activated fibroblasts in the pancreas and the recruitment of immune cells that are vital for tissue recovery. Analysis of the mechanism using expression and chromatin immunoprecipitation assays identified a subset of cytokines, including IL-6, mIL-8, Mcp-1, and M-csf (Csf1), as direct GLI1 target genes potentially mediating this phenomenon. Finally, we demonstrate that canonical Hedgehog signaling, a known regulator of Gli1 activity, is required for pancreas recovery. Collectively, these data delineate a new pathway controlling tissue repair and highlight the importance of GLI1 in regulation of the pancreatic microenvironment during this cellular process.

VX-509 (decernotinib) is a potent and selective janus kinase 3 inhibitor that attenuates inflammation in animal models of autoimmune disease.

Cytokines, growth factors, and other chemical messengers rely on a class of intracellular nonreceptor tyrosine kinases known as Janus kinases (JAKs) to rapidly transduce intracellular signals. A number of these cytokines are critical for lymphocyte development and mediating immune responses. JAK3 is of particular interest due to its importance in immune function and its expression, which is largely confined to lymphocytes, thus limiting the potential impact of JAK3 inhibition on nonimmune physiology. The aim of this study was to evaluate the potency and selectivity of the investigational JAK3 inhibitor VX-509 (decernotinib) [(R)-2-((2-(1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-yl)amino)-2-methyl-N-(2,2,2-trifluoroethyl)butanamide] against JAK3 kinase activity and inhibition of JAK3-mediated signaling in vitro and JAK3-dependent physiologic processes in vivo. These results demonstrate that VX-509 potently inhibits JAK3 in enzyme assays (Ki = 2.5 nM + 0.7 nM) and cellular assays dependent on JAK3 activity (IC50 range, 50-170 nM), with limited or no measurable potency against other JAK isotypes or non-JAK kinases. VX-509 also showed activity in two animal models of aberrant immune function. VX-509 treatment resulted in dose-dependent reduction in ankle swelling and paw weight and improved paw histopathology scores in the rat collagen-induced arthritis model. In a mouse model of oxazolone-induced delayed-type hypersensitivity, VX-509 reduced the T cell-mediated inflammatory response in skin. These findings demonstrate that VX-509 is a selective and potent inhibitor of JAK3 in vitro and modulates proinflammatory response in models of immune-mediated diseases, such as collagen-induced arthritis and delayed-type hypersensitivity. The data support evaluation of VX-509 for treatment of patients with autoimmune and inflammatory diseases such as rheumatoid arthritis.

Increase in cytosolic Ca2+ produced by hypoxia and other depolarizing stimuli activates a non-selective cation channel in chemoreceptor cells of rat carotid body.

The current model of O2 sensing by carotid body chemoreceptor (glomus) cells is that hypoxia inhibits the outward K(+) current and causes cell depolarization, Ca(2+) influx via voltage-dependent Ca(2+) channels and a rise in intracellular [Ca(2+)] ([Ca(2+)]i). Here we show that hypoxia (<5% O2), in addition to inhibiting the two-pore domain K(+) channels TASK-1/3 (TASK), indirectly activates an ∼20 pS channel in isolated glomus cells. The 20 pS channel was permeable to K(+), Na(+) and Cs(+) but not to Cl(-) or Ca(2+). The 20 pS channel was not sensitive to voltage. Inhibition of TASK by external acid, depolarization of glomus cells with high external KCl (20 mm) or opening of the Ca(2+) channel with FPL64176 activated the 20 pS channel when 1 mm Ca(2+) was present in the external solution. Ca(2+) (10 µm) applied to the cytosolic side of inside-out patches activated the 20 pS channel. The threshold [Ca(2+)]i for activation of the 20 pS channel in cell-attached patches was ∼200 nm. The reversal potential of the 20 pS channel was estimated to be -28 mV. Our results reveal a sequential mechanism in which hypoxia (<5% O2) first inhibits the K(+) conductance and then activates a Na(+)-permeable, non-selective cation channel via depolarization-induced rise in [Ca(2+)]i. Our results suggest that inhibition of K(+) efflux and stimulation of Na(+) influx both contribute to the depolarization of glomus cells during moderate to severe hypoxia.

THIK-1 (K2P13.1) is a small-conductance background K(+) channel in rat trigeminal ganglion neurons.

The goal of this study was to determine the molecular identity of a small-conductance (~5-pS) background K(+) channel expressed in trigeminal ganglion (TG) neurons. We tested the hypothesis that the 5-pS channel is a K2P channel by comparing the pharmacological and single-channel properties of THIK-1 expressed in HEK293 cells. As reported earlier, whole-cell THIK-1 current was inhibited by halothane and activated by arachidonic acid. Among 25 additional modulators tested, bupivacaine (100 µM), quinidine (50 µM) and Ba(2+) (3 mM) and cold (10 °C) were most effective inhibitors of THIK-1 current (>50 % inhibition). In cell-attached patches with high KCl in the pipette and bath solutions, THIK-1 produced a small-conductance (~5 pS) channel with a weak inwardly rectifying current-voltage relationship. Halothane, bupivacaine and cold inhibited the single-channel activities of both THIK-1 and the 5-pS channel in TG neurons, whereas arachidonic acid augmented them. THIK-1 expressed in HEK293 cells and the 5-pS channels in TG neurons were insensitive to hypoxia. Reverse transcriptase-PCR, Western blot and immunocytochemical analyses suggested that THIK-1 mRNA and protein were expressed in TG neurons. These results show that THIK-1 is functionally expressed in TG neurons and contributes to the background K(+) conductance.

The marine cyanobacterial metabolite gallinamide A is a potent and selective inhibitor of human cathepsin L.

A number of marine natural products are potent inhibitors of proteases, an important drug target class in human diseases. Hence, marine cyanobacterial extracts were assessed for inhibitory activity to human cathepsin L. Herein, we have shown that gallinamide A potently and selectively inhibits the human cysteine protease cathepsin L. With 30 min of preincubation, gallinamide A displayed an IC50 of 5.0 nM, and kinetic analysis demonstrated an inhibition constant of ki = 9000 ± 260 M(-1) s(-1). Preincubation-dilution and activity-probe experiments revealed an irreversible mode of inhibition, and comparative IC50 values display a 28- to 320-fold greater selectivity toward cathepsin L than closely related human cysteine cathepsin V or B. Molecular docking and molecular dynamics simulations were used to determine the pose of gallinamide in the active site of cathepsin L. These data resulted in the identification of a pose characterized by high stability, a consistent hydrogen bond network, and the reactive Michael acceptor enamide of gallinamide A positioned near the active site cysteine of the protease, leading to a proposed mechanism of covalent inhibition. These data reveal and characterize the novel activity of gallinamide A as a potent inhibitor of human cathepsin L.