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The prevalence of human and animal helminth infections remains staggeringly high, thus urging the need for concerted efforts towards this area of research. GABA receptors, encoded by the unc-49 gene, mediate body muscle inhibition in Caenorhabditis elegans and parasitic nematodes and are targets of anthelmintic drugs. Thus, the characterization of nematode GABA receptors provides a foundation for rational anti-parasitic drug design. We therefore explored UNC-49 channels from C. elegans muscle cultured cells of the first larval stage at the electrophysiological and behavioral levels. Whole-cell recordings reveal that GABA, muscimol and the anthelmintic piperazine elicit macroscopic currents from UNC-49 receptors that decay in their sustained presence, indicating full desensitization. Single-channel recordings show that all drugs elicit openings of ∼2.5 pA (+100 mV), which appear either as brief isolated events or in short bursts. The comparison of the lowest concentration required for detectable channel opening, the frequency of openings and the amplitude of macroscopic currents suggest that piperazine is the least efficacious of the three drugs. Macroscopic and single-channel GABA-activated currents are profoundly and apparently irreversibly inhibited by ivermectin. To gain further insight into ivermectin action at C. elegans muscle, we analyzed its effect on single-channel activity of the levamisol-sensitive nicotinic receptor (L-AChR), the excitatory receptor involved in neuromuscular transmission. Ivermectin produces a profound inhibition of the frequency of channel opening without significant changes in channel properties. By revealing that ivermectin inhibits C. elegans muscle GABA and L-AChR receptors, our study adds two receptors to the already known ivermectin targets, thus contributing to the elucidation of its pleiotropic effects. Behavioral assays in worms show that ivermectin potentiates piperazine-induced paralysis, thus suggesting that their combination is a good strategy to overcome the increasing resistance of parasites, an issue of global concern for human and animal health.
Citation: Hernando G, Bouzat C (2014) Caenorhabditis elegans Neuromuscular Junction: GABA Receptors and Ivermectin Action. PLoS ONE 9(4): e95072. https://doi.org/10.1371/journal.pone.0095072
Editor: William Phillips, University of Sydney, Australia
Received: December 20, 2013; Accepted: March 23, 2014; Published: April 17, 2014
Copyright: © 2014 Hernando, Bouzat. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work has been supported by grants from the Universidad Nacional del Sur (UNS), Agencia Nacional de Promoción Científica y Tecnológica (ANPCYT), and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) from Argentina. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
GABA-gated chloride channels, members of the Cys-loop receptor family, play an important inhibitory role in the nervous system of vertebrates  and invertebrates . Defective GABA neurotransmission causes neurological disorders and GABAA receptors are targets for therapeutically important drugs, including benzodiazepines, barbiturates, and anesthetics , , . In invertebrates, GABA receptors are important targets for insecticides and nematicides . GABA receptors are homo- or hetero-pentameric proteins composed of an extracellular domain that carries the agonist binding sites and a transmembrane region that forms the ion pore –. The presence of a large number of GABA subunits results in the formation of a wide variety of functionally and pharmacologically different receptor subtypes .
In Caenorhabditis elegans, body wall muscles receive innervations from both cholinergic (excitatory) and GABAergic (inhibitory) motor neurons. Acetylcholine released from motor neurons stimulates muscle contraction on one side of the body, and simultaneously activates an inhibitory motor neuron that projects to the opposite side of the body to release GABA. Thus, GABA elicits muscle relaxation, facilitating the smooth body bend necessary for coordinated locomotion . Muscle cells contain one GABA and two types of acetylcholine receptors (AChR), the levamisole-sensitive (L-AChR) and the nicotine-sensitive AChR (N-AChR). The C. elegans muscle GABA receptor is encoded by the unc-49 gene, which is translated into three subunits: UNC-49A, UNC-49B, and UNC-49C. In adult C. elegans, the GABA receptor has been shown to be composed of UNC-49B and C subunits . The UNC-49B subunit confers synaptic localization and allows channel activation whereas UNC-49C is a non essential modulatory subunit that co-assembles with UNC-49B. The unc-49 null mutant exhibits the “shrinker” phenotype, owing to hypercontraction of the body wall muscles on both sides of the body. The worms become resistant to muscimol, which is a full agonist of vertebrate GABAA receptors. This drug relaxes all body wall muscles and causes lengthening of adult worms , .
The unc-49 gene is also present in parasitic nematodes, including Haemonchus contortus , Brugia malayi and Trichinella spiralis , Ascaris suum and Loa loa , and GABA receptors are targets of widely used anthelmintic agents. Within these agents, piperazine (PZE) is the active constituent of over-the-counter remedies for threadworm infection in children. Its mode of action has been primarily studied in A. suum, where its GABA-mimetic action causes a flaccid, reversible paralysis of body wall muscle .
Ivermectin (IVM) has been used successfully to treat billions of humans, livestock and pets. It activates glutamate-activated chloride channels (GluClR) in nematodes and also activates and modulates a great variety of vertebrate and invertebrate Cys-loop receptors, including GABA receptors . Understanding the molecular basis of IVM wide spectrum of action may be important for the development of novel anthelmintic agents.
UNC-49 receptors share significant structural and pharmacological overlap with mammalian GABAA receptors in some aspects and differ greatly in others. These differences could be exploited in parasitic drug design. However, information about functional properties of nematode GABA receptors is still scarce. We therefore here explored macroscopic and single-channel properties of GABA receptors from C. elegans L1 muscle cells, determined how they are activated and modulated by anthelmintic agents, and evaluated the behavioral effects of these agents in the worm.
The characterization of GABA receptors in a genetically tractable organism and model of parasitic nematodes provides new avenues of exploration for selective drugs as well as for defining structural determinants of activation and modulation in the Cys-loop receptor family.
Materials and Methods
Caenorhabditis elegans strains
Nematode strains used: N2: Bristol wild-type; PD4251: ccIs4251;dpy-20(e1282) which produces GFP in all body wall muscles and vulval muscles; EG1653: oxIs22 [unc-49p::unc-49::GFP + lin-15(+)] psoralen integration of oxEx129 [unc-49Bp(long)::GFP + lin-15(+)], which expresses GFP associated to UNC-49R ; and the null mutants CB407: unc-49(e407) in which the UNC-49B essential subunit is disrupted ; DA1316: avr-14(ad1302);avr-15(ad1051);glc-1(pk54) in which a simultaneous mutation of three genes, avr-14, avr-15, and glc-1, encoding glutamate-gated chloride channel (GluCl) alpha-type subunits confers high-level resistance to the antiparasitic drug ivermectin . All nematodes strains were obtained from the Caenorhabditis Genetic Center, which is funded by the NIH National Center for Research Resources (NCRR). Nematode strains were maintained at 18–25°C using freshly prepared Nematode Growth Medium (NGM) petri dishes that have been spread with Escherichia coli (OP50) as a source of food , .
Isolation and culture of C. elegans muscle cells
Cells were isolated and cultured as described before –. Briefly, adult nematodes were exposed to an alkaline hypochlorite solution and eggs were treated with 1 unit/ml chitinase. The dissociated embryo cells were filtered and placed on glass coverslips coated with poly-O-Ornithine. Cultures were maintained at 22–24°C in a humidified incubator in L-15 medium containing 10% fetal bovine serum. Complete differentiation to the various cell types that comprise the newly hatched Larva 1 (L1) was observed within 24 h as described by Christensen et al. . Electrophysiological experiments were performed 1–5 days after cell isolation.
Single-channel currents were recorded in the cell-attached patch configuration at 20°C , . The bath and pipette solutions contained 140 mM NaCl, 3 mM CaCl2, 5 mM KCl, 5 mM MgCl2, 11 mM glucose, 5 mM HEPES (pH 7.4) for UNC-49 receptors and 142 mM KCl, 5.4 mM NaCl, 1.8 mM CaCl2, 1.7 mM MgCl2, and 10 mM HEPES (pH 7.4) for L-AChRs. Drugs were obtained from Sigma-Aldrich Co. The stock solution for ivermectin (IVM) was prepared in dimethyl sulphoxide (DMSO) and the final DMSO concentration used in all assays was lower than 0.1%. Single-channel currents were recorded using an Axopatch 200 B patch-clamp amplifier (Molecular Devices), digitized at 5 µs intervals, and detected by the half-amplitude threshold criterion using the TAC 4.0.10 program (Bruxton Corporation). Open- and closed-time histograms were plotted using a logarithmic abscissa and a square root ordinate and fitted to the sum of exponential functions by maximum likelihood using TACFit (Bruxton Corporation).
To recognize bursts and quantify their durations, a critical closed time (τcrit) was defined as the point of intersection between the briefest and the succeeding component, and openings separated by closings briefer than this time constitute a burst . Typically, τcrit ranged from 0.15 to 0.20 ms. Burst duration histograms were well described by the sum of two exponentials, with the briefest duration component corresponding to isolated events and the longest duration component, to bursts. The burst duration was taken from the duration of the slowest component of the burst duration histogram.
Macroscopic currents were recorded in the whole-cell configuration at 20°C at a holding potential of −70 mV . The pipette solution contained 134 mM KCl, 10 mM EGTA, 1 mM MgCl2, and 10 mM HEPES (pH 7.3). The extracellular solution (ECS) contained 140 mM NaCl, 3 mM CaCl2, 5 mM KCl, 5 mM MgCl2, 11 mM glucose, 5 mM HEPES (pH 7.4). The cell membrane capacitance (Cm) was determined using the software Windows Whole Cell Program (WinWCP; Strathclyde Institute of Pharmacy and Biomedical Sciences, UK) after obtaining the whole-cell configuration. All tested L1 muscle cells exhibited Cm values varying from 2 to 6 pF. The perfusion system consisted of solution reservoirs, manual switching valves, a four channel VC3 valve controller (ALA Scientific Instruments), a solenoid-driven pinch valve, and three tubes (0.3 mm inner diameter) inserted into the culture dish , . One tube contained the ECS alone and the others, ECS with drugs. The cell was positioned within several hundred microns of the three output tubes where streams coming from them would intersect. The solution exchange time was estimated by the open pipette method, by stepping the patch held at 0 mV in ECS and then applying a 2000-ms pulse of ECS diluted 1:1 with water. Typical exchange times ranged from 1 to 2 ms. Macroscopic currents were filtered at 5 kHz, digitized at 20 kHz, and analyzed using WinWCP. Each current represents the average from 2–4 individual traces obtained from the same cell, which were aligned with each other at the point where they reached 50% of maximum. Peak currents (Ipeak) correspond to the value obtained by extrapolation of the decay to this point. The rise time of the currents (t20–80) was determined by calculating the time in which the current increased from 20 to 80% of the peak amplitude. Decays were fitted by a double-exponential function:(Equation 1)Where Iss is the steady state current, and τf and τs, the decay time constants.
Cells were grown in cultured dishes from MatTek (glass bottom culture dishes Nº 0 MatTek Corporation, Ashland, MA, U.S.A). Fluorescence images were obtained by laser scanning confocal microscopy (Leica TCS SP2) with a 63X water objective. Images were collected and processed with LCS software (Leica) and Photoshop (Adobe Systems, San Jose, CA). The percentage of green cells was determined by counting cells in ten different fields for each dish (n = 3 dishes). The result is expressed as the mean ± SD from three different cell isolations. Quantitative analysis of GABA receptor distribution was conducted by measuring the intensity profile along the cell on the focal plane of the membrane by using LCS software (Leica). An arbitrary 7.5 µm line was traced through the cell starting and ending it in a background zone and a curve of fluorescence intensity was constructed for each point of the line.
Assays were performed with young adult hermaphrodites or L1 worms from synchronized plates. Measurement of drug sensitivity. Paralysis was determined on agar plates containing the tested drug at room temperature as described before . Body paralysis was followed by visual inspection at the indicated time (every 30–60 minutes) and was defined as the lack of complete body movement in response to prodding. We evaluated different types of nematode paralysis: flaccid that is mediated by inhibitory stimulation, in which worms appear lengthened; spastic paralysis that is mediated by AChR stimulation and worms appear shorter , , and stationary, in which worms do not show either flaccid or spastic paralysis. Stationary worms retain normal muscular rigidity and the capability of responding to prodding by contracting body wall muscle .
individual young adult C. elegans were placed in 200 µl of M9 buffer in the absence or presence of the drug in a 96-well microlitre plate. After 30 minutes in the solution at room temperature, the number of thrashes (bends of the body from one side to the other) were counted during 1 minute . The experiments were repeated 3 times for each condition (10 worms tested each time).
L1 muscle cells respond to GABAergic agonists
Cultured muscle cells corresponding to the Larva 1 stage (L1) can be identified by their spindle-shaped morphology (Fig.1; ). In the PD4251 strain, which produces GFP in all body wall and vulval muscles, muscle cells can be easily distinguished by fluorescence microscopy (Fig. 1A, right panel). Confocal microscopy observation of cultures obtained from the EG1653 strain, which expresses GFP associated with GABA receptors , shows that 78±10% of cultured cells with spindle-shaped morphology express GABA receptors, which appear to be homogeneously distributed on the cell membrane (Fig. 1B and C).
Confocal microscopy of L1 muscle cells in culture from worms expressing GFP in muscle (PD4251 strain, (A)) or associated to GABA receptors (EG1653 strain, (B)). Left panels correspond to DIC and Right panels correspond to fluorescence microscopy. Bar: 20 µm. (C) Fluorescence intensity profile at the membrane level of representative cells shown in panel B, two of which show GFP expression (marked by green and red lines) and the other corresponds to the basal fluorescence (blue line).
To verify that L1 muscle cells express functional GABA receptors, we measured macroscopic currents elicited by GABA in the whole-cell configuration at −70 mV pipette potential. In the control PD4251 strain, 80±12% of muscle (green) cells showed responses to 1 mM GABA application whereas no GABA-activated currents were detected from L1 muscle cells derived from unc-49(e407) mutant , which lacks UNC-49 receptors. 1 mM GABA-elicited currents reach a peak in less than ∼4 ms (t20-80 = 4±2 ms, n = 13; Fig. 2A). The mean peak current (Ipeak) is -21.7±11 pA/pF at −70 mV. Currents decay in the continuous presence of GABA to reach steady state current values (Iss) close to zero (Iss/Ipeak = 0.05±0.03), indicating full desensitization. Decays are fitted by a double exponential function (τfast = 19±9 ms and τslow = 85±40 ms). At 10 mM GABA, peak currents are between 4- and 5-fold larger than at 1 mM (−105.7±65 pA/pF, n = 9; Fig. 2A and B). Despite the variability in current amplitudes among different cells, the difference between 1 and 10 mM GABA is statistically significant (Fig. 2B). Muscimol (1 mM) also elicits GABA-activated currents from L1 muscle cells with a mean peak current of −47.8±13 pA/pF (n = 5), which is statistically different from that of 1 mM GABA-currents (Fig. 2A and B). 1 mM piperazine (PZE) does not elicit detectable currents. However, 10 mM PZE elicits currents in ∼76% of the cells, indicating that PZE acts as an agonist of C. elegans UNC-49 receptors. Peak currents are smaller and statistically different to those elicited by 10 mM GABA (Ipeak = −20±13 pA/pF, n = 5; Fig. 2B). Increasing PZE concentration to 25 mM does not produce larger currents than those elicited by 10 mM PZE (Ipeak = 21.7±6 pA/pF n = 3; p>0.30), suggesting that this drug does not behave as a full agonist. Unfortunately, an extended pharmacological study in the same cell is not possible because of the seal stability and the resealing of the whole cell. Nevertheless, the results suggest that PZE is less efficacious than GABA and muscimol at C. elegans UNC-49 receptors.
(A) Representative whole-cell currents from L1 cultured muscle cells (PD4251 strain) elicited by a 1 s-pulse of GABA, muscimol and piperazine. Pipette potential: −70 mV. Each current corresponds to a different cell and represents the average of at least 2–4 applications of the same agonist per cell. (B) Percentage of the peak current normalized to that elicited by 10 mM GABA. ANOVA with Bonfferroni multi comparison post test. (* p<0.05, ** p<0.01). (C) Whole-cell currents activated by 1 mM GABA or 10 mM PZE before (black line) and after pre-exposure to 30 µM IVM during 2 min (grey line).
Ivermectin (IVM) has been shown to activate and modulate a large number of vertebrate and invertebrate Cys-loop receptors. We therefore explored if it also affects GABA receptors from C. elegans. Currents are not elicited by application of IVM, indicating that it is not capable of activating GABA receptors (n = 6 cells). Pre-exposure of L1 cells to 30 µM IVM during 2 min fully inhibits 1 mM GABA-responses (n = 6 cells) and inhibits 90±3% of 10 mM PZE-responses (n = 8 cells, Fig. 2 C). Peak currents cannot be recovered after 5-min wash with buffer alone, suggesting apparent irreversibility. As a control, we verified that GABA-activated currents are not inhibited when cells are pre-exposed 2 minutes to 0.1% DMSO in the absence of IVM (n = 3, peak current >85% of the control peak). Thus, we conclude that IVM acts as an inhibitor of C. elegans UNC-49 receptors.
Single-channel currents of GABA receptors from L1 muscle cells
To gain further insight of how anthelmintic drugs act on GABA receptors we recorded single-channel currents from cell-attached patches in L1 muscle cells. Single-channel openings are detected at concentrations higher than 0.5 mM although they are very infrequent at this concentration. The proportion of patches that show detectable single-channel activity is very low at all GABA concentrations. At 5 mM GABA, only ∼15% of the patches show detectable openings. Channel openings exhibit single amplitudes of 2.5±0.6 pA at +100 mV pipette potential (Fig. 3A), 2.9±0.3 pA at +140 mV, and 3.5±0.2 pA at +160 mV (n = 6). By the slope of the current-voltage relationship, we estimated a value of 22±0.8 pS (n = 6) for channel conductance.
Single channels currents were recorded from muscle cultured cells in the presence of GABA (1, 5 or 10 mM) (A) and 1 mM muscimol or 10 mM piperazine (PZE) (B). Representative channel traces and open and closed time histograms are shown for each condition. (C) Representative traces of single-channel activity in the presence of IVM alone (top traces) or together with 1 mM or 5 mM GABA in the pipette solution (bottom). Channel openings are shown as upward deflections. Pipette potential: +100 mV. Filter: 9 kHz.
No opening events were detected in the absence of GABA (n = 30 cells) or in cell-attached patches from the unc-49(e407) mutant (n = 10 cells), thus revealing that the observed channel activity is originated from UNC-49 receptors.
At all GABA concentrations (1-10 mM), single-channel activity occurs as isolated opening events and short bursts composed of successive openings (Fig. 3A). Open-time distributions at all GABA concentrations are well described by a single exponential component with mean durations between ∼0.18 and 0.40 ms (Fig. 3A and Table 1). Bursts are readily detected at 5 mM GABA and show a mean duration of 1.1±0.25 ms (n = 5; Fig. 3A). Closed time distributions can be well fitted by three or four exponential components (Fig. 3A). The duration of the main closed component decreases with GABA concentration, indicating the increase in the frequency of openings with agonist concentration (Fig. 3A and Table 1).
Single UNC-49 channel activity is also detected in the presence of 1 mM muscimol and 10 mM PZE (Fig. 3B). As expected, the amplitude of opening events is similar to that of GABA-activated channels (2.4±0.8 pA and 2.5±0.6 pA for 1 mM muscimol and 10 mM PZE, respectively, p>0.8; Fig. 3B). Mean open times (Table 1) and burst durations (0.94±0.29 ms and 1.2±0.14 ms for 1 mM muscimol and 10 mM PZE, respectively) are not statistically different from those of 5 mM GABA-activated channels (p>0.4). The frequency of opening events from recordings in the presence of 10 mM PZE is systematically lower than that of 10 mM GABA (See representative traces in Fig. 3). The reduced frequency is clearly reflected in the closed time histograms by the displacement to longer durations of the main closed component (Fig. 3 and Table 1). This result is in agreement with the smaller macroscopic responses elicited by PZE, again suggesting that this drug is less efficacious than GABA.
Single-channel activity is not detected when IVM (20–40 µM) alone is present in the pipette solution (n = 12 and n = 14 for 20 and 40 µM IVM, respectively), indicating that it is not capable of activating C. elegans GABA receptors (Fig. 3). Also, GABA (1, 5 or 7 mM) does not elicit detectable single-channel activity if IVM is present in the pipette solution (n = 24 and n = 28 for 20 and 40 µM IVM, respectively; Fig. 3C). This result reveals that IVM produces fast and apparently irreversible inhibition of GABA receptors.
Effects of ivermectin on levamisole-sensitive AChRs
Since the L-AChR is the main Cys-loop receptor involved in muscle contraction, we evaluated whether it is, as the muscle GABA receptor, modulated by IVM. To this end, we took advantage of our previous characterization of single-channel activity of L-AChR on L1 cells , . As shown in Figure 4A, single channels of L-AChRs activated by 50 µM ACh are readily detected from L1 muscle cells. Channel activity shows openings of 3.5±0.3 pA (n = 5) at +100 mV pipette potential that appear isolated or in short bursts (Fig. 4A, top trace). As previously described , , , single-channel activity remains constant during the course of the 15-min recording. In contrast, when 50 µM IVM is present in the pipette solution together with 50 µM ACh, single-channel activity is detected at the beginning of the recording but it dramatically decreases after the first minute; only sporadic and infrequent activation episodes are observed thereafter (Fig. 4A, lower trace). As a control, we verified that 0.1% DMSO does not produce any change in single-channel activity (n = 5).
(A) Single-channels activated by 50 µM ACh were recorded in the absence (top) and presence of 50 µM IVM in the pipette solution (bottom). Three different traces are shown for each condition and correspond to the recording during the first, third and fifth minute. (B) Representative traces of single-channels activated by 50 µM ACh were recorded in the absence (top) and presence of 50 µM IVM in the pipette solution (bottom) from cells previously incubated with 0.1% DMSO (top) or 50 µM IVM in 0.1% DMSO (bottom). In brackets it is indicated the pre-incubation condition (0.1% DMSO for the control and 0.1% DMSO plus 50 µM IVM. (C) Comparison of the number of opening events in the first 30 seconds of recording as in (B). ** p<0.01 (Student t-Test). (D) Bar chart showing the percentage of opening events per minute during the first 5-min recording as in (B). The data correspond to five different recordings for each condition. (E) Representative traces and open and closed time histograms of channels recorded as explained in Panel B. Traces are shown at two temporal scales. Pipette potential: +100 mV. Filter: 9 kHz.
We also tested the effect of the pre-exposure of cells to 50 µM IVM on single-channel activity. To this end, we incubated the culture dishes for ∼15–20 min with 0.1% DMSO in the absence (Fig. 4B, top trace) or presence (Fig. 4B, lower trace) of 50 µM IVM and recorded ACh-activated single-channel currents as described above. In the first 30 seconds of recording, the number of opening events is significantly reduced in IVM-treated cells and it is statistically different from that of 0.1% DMSO-treated cells (n = 5, p<0.01; Fig. 4C). To quantify the decrease of single-channel activity as a function of time due to the presence of IVM, we measured the number of opening events per minute during the first 5 min for each condition (Figure 4D). In the control, the number of events remains constant during the course of the recording (Fig. 4D and ). However, in the presence of IVM, the frequency of events profoundly decreases after the first minute. This is clearly illustrated by the percentage of events detected in the first and second minute, which decreases from ∼80% to ∼10%, respectively.
The few opening events from IVM-treated cells were analyzed to determine if the drug affects single-channel properties. Open time histograms are fitted by the sum of two exponential components, which are identical to those detected in the control condition (Fig. 4E and Table 1). Closed-time distributions of L-AChRs from both conditions are well described by the sum of three or four components. The main closed component is displaced to longer durations in recordings from cells previously treated with IVM (p<0.01; Fig. 4E and Table 1), in agreement with the decreased frequency of opening events.
Taken together, our single-channel study reveals that muscle L-AChR and GABA receptors are profoundly inhibited by IVM.
Behavioral effects of anthelmintic drugs
PZE produces flaccid paralysis and lengthening of parasitic and C. elegans worms , . Since our results show that PZE is an agonist of C. elegans UNC-49 channels in L1 cells, we also explored drug sensitivity at this stage. 2 h-exposure of worms to 1-10 mM PZE does not affect significantly worm locomotion whereas 25 mM PZE significantly affects worm behavior producing flaccid paralysis. Under this condition, ∼40% adult and ∼20% L1 worms are paralyzed after 2 h (Fig. 5A). The reduced sensitivity of L1 stage may be due to the fact that at this stage GABA receptors are not present in dorsal muscle and are only present in ventral muscle . At 40 mM PZE ∼90% of L1 worms are paralyzed after 2 h on agar plates.
(A) Synchronized L1 and adult wild-type worms were placed on plates containing the specified drug [25 mM Piperazine (PZE), and/or 300 µM ivermectin (IVM)], and observed at the indicated times to determine the fraction of worms that respond to prodding, which were considered as “moving worms”. (B) Wild-type and triple mutant avr-14(ad1302);avr-15(ad1051);glc-1(pk54) (DA1316 strain), adult worms were placed on agar plates containing the specified drug [40 mM PZE, and/or 300 µM IVM]. Each point represents the average of 3-5 experiments, n = 30 worms, error bars = SD. (C) Effect of IVM on nematode swimming measured as thrashes per minute for wild type and the triple mutant. Measurements were performed after 30 min of incubation in M9 buffer or M9 buffer plus 10 µM IVM.
The features of IVM-induced paralysis have been well described , . After 2-h exposure on agar plates containing 0.3 mM IVM, worms are stationary but they still respond to prodding (Fig. 5A). The combination of 0.3 mM IVM with 25 mM PZE significantly enhances the flaccid paralysis produced by 25 mM PZE alone, resulting in ∼75% of adult and ∼80% of L1 worms that do not respond to prodding after 2 h (Fig. 5A). Thus, IVM causes a synergic effect on PZE-induced paralysis at both stages. As a control, we verified that no changes in worm behavior occur in the presence of 1% DMSO alone. The PZE and IVM doses used in both human and veterinary medicine appear to be high (e.g. 50–110 mg/kg host weight for PZE and 0.2 –200 mg/kg host weight for IVM –), in close agreement with the concentrations required to show effects in our assays.
To gain further insights into IVM targets, we used the triple mutant strain on GluClRs subunits, avr-14(ad1302);avr-15(ad1051);glc-1(pk54), which has been shown to exhibit high resistance to IVM . Non statistical differences in the sensitivity to 40 mM PZE on agar plates are detected between wild-type and triple mutant worms (Fig 5B). In contrast, PZE-induced paralysis is enhanced by the presence of 0.3 mM IVM in wild-type, as shown above, but not in triple mutant worms (Fig. 5B), thus suggesting that GluClRs are involved in the IVM potentiation of PZE paralysis.
Thrashing assays in the presence of 10 µM IVM show that 100% of wild-type worms are stationary. The triple mutant worms show a reduction of 50% in the thrashing rate (Fig. 5C), suggesting that other receptors besides GluClRs may contribute to the IVM effect, at the micromolar range, on worm locomotion .
The prevalence of human and animal helminth infections remains staggeringly high, thus urging the need for concerted efforts towards this area of research , . GABA receptors, encoded by the unc-49 gene, mediate body muscle inhibition during locomotion in C. elegans and parasitic nematodes and are therefore important targets of anthelmintic drugs , , , . Thus, the molecular and pharmacological characterization of these nematode receptors will have important implications for the development of novel anthelmintic drugs as well as for our understanding of GABA receptor pharmacology.
We here identified functional muscle GABA receptors composed of UNC-49 subunits in C. elegans L1 muscle cells. Not all L1 muscle cells do respond to GABA application, nor do they show GFP-label associated to GABA receptors, in agreement with the fact that at the L1 stage GABA receptors are expressed only in body ventral muscles and not in body dorsal muscles , . Thus, our study gives further evidence that the L1 cell culture reproduces the in vivo situation .
Overall responses of GABA receptors to anthelmintic drugs were evaluated by measuring macroscopic currents from L1 muscle cells in the whole-cell configuration. Unfortunately, these cultured muscle cells are not technically suitable for successive drug applications and they therefore do not allow complete pharmacological assays. This relatively limited information is nevertheless still valuable when complemented with single-channel recordings and behavioral assays.
Macroscopic currents elicited by GABA show rapid onset as well as rapid and full decay under the sustained pulse of agonist, indicating full desensitization . At 1 mM, muscimol elicits larger currents than those elicited by 1 mM GABA, indicating that it is a full and potent agonist of C. elegans UNC-49 receptors as of vertebrate GABAA receptors. In contrast, muscimol has been shown to be less potent than GABA at Ascaris suum and Haemonchus contortus receptors , . PZE is used to treat Ascaris lumbricoides and Enterobius vermicularis infections in humans. Nematode paralysis has been shown to be mediated by activation of GABA-currents in A. suum . We here show that it is also an agonist of C. elegans muscle GABA receptors since it is capable of eliciting macroscopic currents, which are of reduced amplitude when compared to GABA-elicited currents.
C. elegans GABA currents have been recorded previously from oocytes expressing either UNC-49B/C or UNC-49B subunits . The GABA-activated currents recorded from our L1 cultured cells seem to be more similar to those corresponding to UNC-49B/C heteromers than to UNC-49B homomers, which show significantly slower desensitization .
In spite of the low number of active patches and the few opening events, we were able to describe the properties of single UNC-49 channels from L1 muscle cells. Channel openings activated by GABA, muscimol or PZE exhibit single amplitudes of ∼2.5 pA (+100 mV pipette potential) and brief open durations. The opening events are not detected in the absence of agonist or in patches from the unc-49 null mutants, thus revealing that the observed channel activity is originated from UNC-49 receptors. The lowest PZE concentration that allows detectable opening events as well as the frequency of opening events is reduced when compared to the other agonists. These observations together with the reduced macroscopic currents suggest that PZE is less efficacious than GABA. In agreement with this conclusion, PZE has been shown to act as a low-efficacy agonist of GABA receptors of the parasitic nematodes A. suum ,  and H. contortus .
Up to our knowledge the only -though limited- description of single C. elegans UNC-49 channel activity was made in HEK cells transfected with UNC-49B and C subunits . The estimated conductance was ∼37 pS for UNC-49B homomers and ∼30 pS for UNC-49B/C heteromers .This latter value is in close agreement with the conductance determined from our single-channel recordings from L1 cells (∼22 pS) as well as with the conductance determined for GABA receptors from A. suum (∼22 pS, ). Thus, the comparison of the desensitization rate of macroscopic currents and the single-channel conductance determined in our study with results from heterologously expressed UNC-49 receptors suggests that receptors in L1 muscle cells may be UNC49B/C heteromers, as previously reported for both C. elegans and parasitic nematodes , , .
Although IVM has revolutionized the treatment of nematode and arthropod parasites, much remains to be learned about how it works and how resistant to it is developed . The antiparasitic effects of IVM are known to be mediated by paralysis of the pharyngeal muscle through glutamate-gated chloride channel receptors (GluClR) , ,  and of the somatic muscle through GABA receptors . However, at the molecular level, IVM has been shown to activate and modulate a great number of Cys-loop receptors. IVM activates vertebrate GABA receptors, potentiates GABA receptors from H. contortus and inhibits GABA receptors from A. suum , , . Our study reveals that it inhibits UNC-49 receptors from C. elegans. IVM inhibition appears to be irreversible (currents cannot be recovered after 5-min wash) in agreement with its reported actions at other anionic Cys-loop receptors . IVM action at GABA receptors is more similar between C. elegans and the parasite A. suum than between the latter and H. contortus, in line with the fact that C. elegans is no more dissimilar to parasitic nematodes than each individual species of parasite is to another , .
Therefore, although GABA receptor subunit family appears conserved among nematodes there are variations in the properties of how individual channels respond to IVM among species. Understanding the molecular bases of these differences may lead to more rational and specific treatments.
With respect to AChRs, IVM has been shown to potentiate vertebrate α7 AChRs  and to inhibit C. elegans N-AChRs (ACR-16)  and α7-5HT3A chimeric receptors . Our study extends the information of IVM targets by showing that it inhibits C. elegans L-AChR, which is the main excitatory receptor involved in locomotion. Assuming that the few detected openings from IVM-treated cells correspond to IVM-treated receptors, our detailed single-channel study reveals that IVM fully inhibits channel activity without affecting open-channel lifetime This observation may be mechanistically explained by the stabilization of desensitized states, inhibition of channel opening, or irreversible channel block. In conclusion, our study reveals that IVM inhibits C. elegans muscle GABA and AChR receptors, thus contributing to the understanding of the wide spectrum of action of this complex drug.
The crystal structure of GluClR complexed with IVM shows that the drug binds at subunit interfaces on the periphery of transmembrane domains between M3 on the principal subunit (+) and M1 on the complementary subunit (-) and it makes important contacts with the M2 pore-lining α-helix and the M2–M3 linker . However, the relationship between binding and channel activation is not completely understood . Based on the structure of GluCl-IVM and on how different receptors are activated or modulated by IVM, several residues in M2, M3 and M1 domains have been pointed as involved in activation and potentiation . The comparison of these residues in UNC-49 subunits between C. elegans and H. contortus does not reveal a clear pattern of substitutions which may explain why their GABA receptors are oppositely modulated by IVM. Thus, this opposite IVM modulation could be due to different changes elicited by IVM binding rather than to IVM binding itself. In fact, mutations in residues from other loops outside the IVM-binding site affect the efficacy of IVM activation , . By molecular docking and mutagenesis studies it has been suggested that the site of action of IVM at AChRs may be located at an intrasubunit cavity (not intersubunit as in the GluCl receptor) in the transmembrane region , . Thus, the occupancy of different sites or/and the intrinsic capability of each receptor to respond to IVM binding may lead to the pleiotropic effects of this drug.
Understanding the mechanistic and structural bases underlying the final response of IVM may not only be important for the development of novel anthelmintic agents but also for drugs targeting human Cys-loop receptors associated to neurological diseases. In this respect, drugs that alter receptor responses to normal released neurotransmitter, such as positive allosteric modulators, are emerging as key therapeutic treatments for neurodegenerative and neurological diseases .
The combination of drugs is a strategy tending to fight against the increase in anthelmintic resistance. Despite being a low-efficacy GABAergic agonist, PZE is capable of inducing flaccid paralysis in C. elegans. We show that IVM can be used to potentiate the paralysis induced by PZE (at both L1 and adult stages) and that this potentiation may be mediated by GluClRs since it does not occur in the triple mutant strain. The explanation for the synergic effect seems to be not so straightforward since IVM inhibits UNC-49 receptors but potentiates PZE flaccid paralysis. However, IVM action is highly complex given its broad spectrum of action . Although understanding the neuronal circuit basis underlying the synergic IVM-PZE effect requires further investigation, our results suggest that the combination of the two drugs may result in a good alternative strategy to overcome the ever increasing resistance of parasites to these drugs, which is of global concern for human and cattle health.
Conceived and designed the experiments: GH CB. Performed the experiments: GH CB. Analyzed the data: GH CB. Contributed reagents/materials/analysis tools: GH CB. Wrote the paper: GH CB.
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14,000 Shares in Healthequity Inc (HQY) Purchased by Nomura Holdings Inc.
Nomura Holdings Inc. acquired a new position in shares of Healthequity Inc (NASDAQ:HQY) during the second quarter, according to the company in its most recent Form 13F filing with the Securities and Exchange Commission (SEC). The fund acquired 14,000 shares of the company’s stock, valued at approximately $1,051,000.
A number of other hedge funds and other institutional investors also recently added to or reduced their stakes in the business. BlackRock Inc. boosted its stake in Healthequity by 11.3% during the second quarter. BlackRock Inc. now owns 7,688,768 shares of the company’s stock worth $577,427,000 after buying an additional 777,915 shares during the last quarter. FMR LLC lifted its stake in shares of Healthequity by 26.5% in the 2nd quarter. FMR LLC now owns 3,410,542 shares of the company’s stock valued at $256,132,000 after purchasing an additional 715,441 shares during the last quarter. JPMorgan Chase & Co. lifted its stake in shares of Healthequity by 6.8% in the 1st quarter. JPMorgan Chase & Co. now owns 1,229,111 shares of the company’s stock valued at $74,411,000 after purchasing an additional 78,021 shares during the last quarter. Carillon Tower Advisers Inc. lifted its stake in shares of Healthequity by 751.9% in the 2nd quarter. Carillon Tower Advisers Inc. now owns 752,482 shares of the company’s stock valued at $56,511,000 after purchasing an additional 664,157 shares during the last quarter. Finally, Northern Trust Corp lifted its stake in shares of Healthequity by 1.5% in the 1st quarter. Northern Trust Corp now owns 664,085 shares of the company’s stock valued at $40,204,000 after purchasing an additional 9,575 shares during the last quarter. 96.94% of the stock is currently owned by institutional investors.
HQY stock opened at $92.46 on Monday. The firm has a market capitalization of $5.74 billion, a P/E ratio of 171.22, a PEG ratio of 3.21 and a beta of 1.46. Healthequity Inc has a 12-month low of $42.92 and a 12-month high of $99.99.
Several equities analysts have recently commented on HQY shares. Bank of America lowered Healthequity from a “buy” rating to a “neutral” rating and upped their price target for the company from $84.00 to $105.00 in a research note on Thursday, September 6th. They noted that the move was a valuation call. SunTrust Banks upped their price target on Healthequity to $105.00 and gave the company a “buy” rating in a research note on Wednesday, September 5th. Cantor Fitzgerald lowered Healthequity from an “overweight” rating to a “neutral” rating and set a $65.00 price target on the stock. in a research note on Wednesday, May 30th. Oppenheimer upped their price target on Healthequity from $68.00 to $85.00 and gave the company an “outperform” rating in a research note on Tuesday, June 5th. Finally, KeyCorp upped their price target on Healthequity from $72.00 to $82.00 and gave the company an “overweight” rating in a research note on Tuesday, June 5th. Four analysts have rated the stock with a hold rating, nine have issued a buy rating and two have given a strong buy rating to the stock. Healthequity currently has a consensus rating of “Buy” and a consensus price target of $93.08.
In related news, VP Darcy G. Mott sold 4,000 shares of the business’s stock in a transaction on Monday, August 20th. The stock was sold at an average price of $86.37, for a total value of $345,480.00. Following the sale, the vice president now owns 99,200 shares in the company, valued at approximately $8,567,904. The transaction was disclosed in a document filed with the Securities & Exchange Commission, which is available through this link. Also, Director Frank Medici sold 15,596 shares of the business’s stock in a transaction on Tuesday, July 10th. The shares were sold at an average price of $80.15, for a total value of $1,250,019.40. Following the completion of the sale, the director now owns 26,341 shares in the company, valued at approximately $2,111,231.15. The disclosure for this sale can be found here. Insiders sold a total of 45,500 shares of company stock worth $3,684,201 in the last quarter. Company insiders own 4.00% of the company’s stock.
Healthequity Company Profile
HealthEquity, Inc provides various solutions for managing health care accounts, health reimbursement arrangements, and flexible spending accounts for health plans, insurance companies, and third-party administrators in the United States. The company offers healthcare saving and spending platform, a cloud-based platform for individuals to make health saving and spending decisions, pay healthcare bills, compare treatment options and prices, receive personalized benefit and clinical information, earn wellness incentives, grow their savings, and make investment choices; and health savings accounts.
Recommended Story: Outstanding Shares and The Effect on Share Price
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The main difference between the Premier League and the Championship is the standard of attacking play, so any team with an average defence has little hope of staying up – just look at Norwich last season.
It leaves the newly promoted sides with a few different options of bolstering the defence when they come up, but every one carries a big risk.
They can rely on their existing players or take a chance on players from the lower divisions in the hope they can handle the step up, but there’s no way of knowing how good they are until it’s too late.
They can also take a chance on a player from abroad, but the Premier League is notoriously fast and physical so it’s common to see defenders struggle when they don’t get a free kick every time an opposing player has the audacity to put the slightest bit of pressure on them.
READ MORE: Mixed news at Chelsea as defender touted for imminent exit is expected to be included in their Premier League squad
The final option is to take a chance on a veteran player who has a lot of experience in the league, and it looks like West Brom are going to go down that route.
A report from Noticiasaominuto has quoted the Telegraph in saying that West Brom are in advanced talks with the former Chelsea and Zenit defender Branislav Ivanovic, and it looks like the move is going to happen.
On the plus side he’s played 261 times in the Premier League for Chelsea, he’s vastly experienced and he’s also handy for a few goals each season so you can understand why he could be seen as a good addition.
It’s absolutely worth pointing out that he’s 36 years old and you’ll almost be able to hear his legs creaking as the season goes on, so they won’t have any chance of playing a high pressing game.
He usually played at right back for Chelsea but you have to think he would play centrally for West Brom, but his age does suggest this is a gamble that might not pay off.


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On a lighter note, I”m going to take a week off the blog. Need to catch up on my reading and some family stuff. Also need to break in a new ham radio. See you in a week.
Sowell nails it, as usual.
One of the arguments for Medicare is that the elderly don’t want to be a burden to their children. Apparently it is all right to be a burden to other people’s children, who are paying taxes.
– Dr. Thomas Sowell, Random Thoughts, 07/25/2012
Generational Warfare – Reason.com: In a 1999 address to the National Education Association’s Women’s Equality Summit, then-First Lady Hillary Clinton was even more explicit in celebrating her own generation’s freedom from the burdens of traditional caretaking responsibilities. “Were it not for Social Security, many of us would be supporting our parents,” intoned the author of It Takes a Village. “We would take them in; we would do what we needed to do to try to provide the resources they required to stay above poverty, to live as comfortably as we could afford. And that would cause a lot of difficult decisions in our lives, wouldn’t it?”


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A lot or a little?
The parents' guide to what's in this movie.
What parents need to know
Parents need to know that Twist is a gritty British modern day retelling of Oliver Twist, with fast-paced action, gender-flipped characters, and some strong language and violence. Though the main characters are all thieves and hustlers, they mostly display good moral values and positive traits such as loyalty, bravery, and kindness. The value of teamwork is a key theme, but there's no escaping the fact that petty crime is being glorified. One particularly villainous character -- Sikes (Lena Headey) -- could bother younger viewers and the story briefly touches on the death of a mother and the idea of being alone in the world. There are several guns, two shootings, and some fist fights along with language that includes one "f--k." After a character is seen with a bloody lip and nose, it's suggested that she has been the victim of domestic violence. There is some smoking and drinking with one depiction of drunkenness. A lot of the characters initially put great importance on material goods, and some well-known brands are depicted. Despite this, the overall tone is one of a mischievous adventure, with cool characters and a rousing soundtrack.
- Parents say
- Kids say
What's the story?
A modern interpretation of Dickens' Oliver Twist, TWIST sees artist Oliver (Raff Law) get involved with a rag-tag group of young grifters, led by master thief Fagin (Michael Caine). Seeing the potential in this streetwise charmer, the gang immediately enlist Oliver in their latest hustle -- an audacious art heist to retrieve a valuable painting that was stolen from Fagin many years before. As they come up against a smarmy art dealer, two earnest police officers, and the psychopathic Sikes (Lena Headey), Oliver and his new friends prove themselves to be expert con artists -- but when the heat is on, do they really know who they can trust?
Is it any good?
With a free running chase across London rooftops and a murder in the first five minutes, this modern spin on Dickens' classic sets itself up as a high-octane crime caper right from the off. Reminiscent of any number of Guy Ritchie movies, Twist is a morally ambiguous tale. Baby-faced con artists justify their thieving ways (they only steal from criminals) and charm themselves into the audience's affections with their audacious rule-breaking and lovable rogue shtick. A sparkling cast of top British talent includes the legendary Caine as a modern-day Fagin, and newcomer Law (son of Jude Law and Sadie Frost) as Oliver. Uncannily like his real-life father, Law has swagger but also a certain sensitivity. Sophie Simnett as Red (aka Nancy) is coolheaded and natural, while Headey plays the psychopathic Sikes with a chilling malevolence.
A misjudged moment when Sikes kisses Red -- against her will -- is overly uncomfortable and sinister, but goes against the generally larky tone of the story. Much is changed from the original Oliver Twist -- not least the gender-flipped Dodge and Sikes characters and the core plot line surrounding an elaborate art heist. But the essence is still here -- orphans, misfits, and thieves with honor working together to stick it to the man. It's clever, funny, and full of energy. Edgy enough for teens to enjoy but compelling enough for their parents too.
Talk to your kids about ...
Discuss the language used in the movie. Did it seem necessary or excessive? What did it contribute to the movie?
Discuss the "gender flipping" in the movie -- when a traditionally male character is re-imagined as a female role. Did it work for you? How did these characters compare to how you might have seen them portrayed before? Can you think of any other movies that have used "gender flipping" for certain roles?
Do you think any of the characters in the movie make good role models? Fagin's gang are hustlers but do they also have strong morals? Which characters displayed strengths like empathy, teamwork, or integrity?
How did this retelling of Dickens' classic compare to the original story? Do you think this is a clever interpretation or an unnecessary update? Should modern adaptations stick more faithfully to the novels they are based on?
- In theaters: July 30, 2021
- On DVD or streaming: July 30, 2021
- Cast: Rafa Law, Michael Caine, Lena Headey
- Director: Martin Owen
- Studios: Saban Films, Sky Cinema
- Genre: Drama
- Topics: Book Characters, Friendship
- Character strengths: Empathy, Integrity, Teamwork
- Run time: 88 minutes
- MPAA rating: R
- MPAA explanation: some violence and language
- Last updated: September 28, 2021
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domain: artistic


text:
My Spoil Dog
So we mix the free dog food with Jake expensive dog food (Eukanuba). At first he ate it. But after a few meal we found that he pick put all the free dog food and put it outside his bowl and ate all the Eukanuba. I don't how he managed to pick it out because the Pal are in quite small pieces.
Then the other day I went to the pet shop they were giving away free dog biscuit so I took home and gave it to Jake. He smell it and didn't want to eat. Have to force him to eat. You can see the biscuit still on the floor. He was playing with it rather than eating it.
But if it is the Eukanuba biscuit you can see that he will be hunting for it.

domain: pet


text:
Monday, 16 June, 2014 | 0:44
TOP 25 DAYTIME and PRIMETIME TV PROGRAMS
NUTAM (National Urban TV Rating | HOUSEHOLD)
JUNE 11 2014 –WEDNESDAY
24 Oras beats rival news program
Eat Bulaga still the most viewed noontime-varierty show
Despite it's late timeslot Angel Locsin's The Legal Wife hits 31.1% vs. Ang Dalawang Mrs. Real 13.1%
More advertisers trust to AGB Nielsen such as Unilever, P and G. Statistically AGB Nielsen had 2000 panel homes in Urban Philippines while Kantar Media had only 1370.
2 major networks subscribed to AGB plus 26 other Cable/Regional Channels.
The National Urban Television Audience Measurement (NUTAM) Panel covers 95% of all urban areas of the country, with a population of more than 34 million individuals.
>Urban Luzon and Mega Manila (73%)
>Urban Visayas and Urban Mindanao (22%)
NATIONAL URBAN 95%
Source: AGB Nielsen Media Research Philippines

domain: geography


text:
Treatment and broadening access to mental health support. Https Github.Com Betterhelp Multisite Pull 1667 Issuecomment-300320316 .
Users with a convenient method to get in touch with certified therapists by means of text, audio, or video messages. Talkspace’s platform offers a safe and secure and confidential environment for individuals seeking treatment.
II. Secret Functions and Benefits
Available and Hassle-free:.
Talkspace eliminates geographical barriers, allowing users to gain access to treatment from anywhere, anytime, utilizing their mobile phone or computer system.
It caters to individuals who may face challenges participating in in-person sessions due to place, mobility issues, or hectic schedules.
Wide Range of Therapists and Specialties:.
Talkspace provides access to a diverse network of certified therapists with knowledge in numerous locations, such as stress and anxiety, anxiety, relationships, and more.
Users can select a therapist who lines up with their specific needs and preferences, guaranteeing individualized care.
Talkspace’s asynchronous messaging function enables users to communicate with their therapist throughout the day, at their benefit.
This function offers people with the versatility to reveal their thoughts and emotions in real-time or as they occur, promoting continuous assistance.
Audio and Video Sessions:.
In addition to messaging, Talkspace provides audio and video sessions for a more immersive therapeutic experience.
These sessions can be scheduled based upon the user’s preferences and offer an opportunity for more interactive and immediate discussions.
Boosted Personal Privacy and Confidentiality:.
Talkspace complies with stringent personal privacy requirements, making sure safe communication and securing user information.
The platform utilizes file encryption and other security steps to keep the privacy of therapy sessions.
III. Factors To Consider and Limitations
This term paper aims to supply a thorough analysis of the co-occurrence of depression and anxiety, 2 prevalent psychological health disorders that often manifest together. The paper examines existing literature to check out the correlation in between anxiety and stress and anxiety, their shared threat aspects, neurobiological foundations, and treatment implications. Understanding the complex relationship between these disorders is important for effective diagnosis, treatment, and improved mental health outcomes.
Briefly present the significance of depression and anxiety as different disorders and their frequent co-occurrence.
Highlight the impact of comorbidity on individuals’ wellness, functioning, and treatment results.
Occurrence and Diagnostic Criteria
Present statistics on the prevalence of depression and anxiety conditions individually and their high rates of comorbidity.
Discuss the diagnostic requirements for major depressive condition (MDD), generalized stress and anxiety disorder (GAD), and other relevant anxiety disorders.
Shared Risk Elements
Check out common threat elements that contribute to the development and upkeep of anxiety and anxiety, such as genetic predisposition, childhood misfortune, and characteristic.
Go over how shared danger elements may account for the high comorbidity rates observed in scientific populations.
BetterUp is an online coaching and counseling platform that focuses on individual and professional development. While BetterUp uses training services, it does not use therapy services like BetterHelp. BetterUp is ideal for people who wish to concentrate on personal development and profession development rather than mental health concerns.
In conclusion, BetterHelp therapy uses numerous advantages over conventional in-person treatment, including therapist, convenience, and cost choice. While there are other online treatment platforms available, BetterHelp sticks out for its large network of therapists and affordable prices strategies. Ultimately, the choice between online therapy and traditional in-person treatment boils down to individual preference and specific needs.
Depression is a common psychological health condition that affects countless individuals worldwide. Treatment can assist by supplying a safe area to talk about your feelings and emotions. A therapist can help you recognize unfavorable thought patterns and behaviors and deal with you to develop coping methods and favorable practices.
Stress and anxiety is another typical mental health condition that can be debilitating. Treatment can help by teaching you relaxation techniques, such as deep breathing and mindfulness, and working with you to establish coping strategies to handle stress and anxiety triggers.
PTSD, or post-traumatic stress disorder, is a mental health condition that can develop after experiencing or experiencing a traumatic occasion. Treatment can help by offering a safe area to process the trauma and establish coping strategies to handle the symptoms of PTSD.
OCD, or obsessive-compulsive disorder, is a mental health condition defined by intrusive thoughts and compulsive behaviors. Treatment can help by teaching you how to recognize and manage these thoughts and behaviors, in addition to develop coping strategies to manage the symptoms of OCD.
Bipolar affective disorder is a mental health condition characterized by severe mood swings, varying from depressive episodes to manic episodes. Therapy can help by offering support and assistance in managing these mood swings, establishing coping techniques, and improving communication skills.
Eating disorders, such as anorexia and bulimia, are psychological health conditions that can have severe physical effects. Treatment can assist by dealing with the underlying mental and psychological issues that contribute to the eating disorder, in addition to establishing techniques to manage the physical signs.
Drug abuse can be a difficult routine to break, but treatment can be an effective tool in managing addiction. Therapy can help by resolving the underlying psychological and mental issues that contribute to drug abuse, along with establishing methods to manage cravings and activates.
Relationship concerns, such as communication issues and dispute, can have a considerable impact on mental health. Therapy can assist by supplying a safe area to go over these problems and establish techniques to enhance interaction and resolve conflict.
Sorrow and loss Https Github.Com Betterhelp Multisite Pull 1667 Issuecomment-300320316
Grief and loss can be a difficult experience to browse, however therapy can assist by supplying assistance and guidance through the grieving procedure. A therapist can help you identify and manage the emotions connected with sorrow and loss, as well as develop coping strategies to move on.
Tension is a typical experience for many people, but it can have unfavorable impacts on psychological health. Treatment can help by teaching relaxation methods and establishing coping strategies to handle stress, along with identifying and resolving the underlying psychological and mental issues that add to stress.
In conclusion, treatment can be a reliable tool in handling a vast array of psychological health conditions, from anxiety and anxiety to substance abuse and relationship issues. If you are having problem with your mental health, consider seeking the assistance and guidance of a licensed therapist.
Seeing a therapist can have various benefits for a person’s psychological health and wellbeing. Here are a few of the benefits of seeing a therapist from a psychological viewpoint:
Talkspace provides resources for users in crisis, however it might not provide immediate support in emergency situation scenarios.
It is necessary for people experiencing a severe crisis to get in touch with emergency services or a regional mental health crisis line.
IV. The Future of Online Therapy
Talkspace has played a substantial function in shaping the future of treatment by harnessing the power of technology to expand access to mental health assistance. As digital platforms continue to progress, online treatment is likely to become significantly incorporated into mental healthcare systems worldwide. Nevertheless, it is essential to strike a balance in between the benefit of online treatment and the customized care and subtleties of traditional in-person sessions.
Talkspace has changed the field of mental health therapy by offering an available, practical, and protected platform for.
Understanding Insurance Coverage:.
Numerous insurance coverage strategies provide coverage for mental health services, consisting of treatment sessions.
It is essential to examine insurance coverage, comprehend coverage limits, co-pays, and any pre-authorization requirements related to therapy.
In-Network vs. Out-of-Network Providers:.
Insurance coverage strategies usually have a network of favored providers with whom they have actually negotiated rates.
In-network therapists frequently have lower out-of-pocket costs for insured individuals, while out-of-network suppliers may involve higher costs.
Utilizing Worker Help Programs (EAPs):.
Many business use EAPs, which offer a limited number of therapy sessions at no cost to employees.
EAPs can be a valuable resource for staff members seeking short-term counseling and support.

domain: medical


text:
Gastronaut Studios is an independent game development/publishing company committed to producing fun, sociable games that are accessible to all gamers.
Location: Capitol Hill, Seattle, Washington
Gel: Set & Match
Platform: Xbox 360
Genre: Xbox Live Arcade
Release Date: 5/20/2009
Gel: Set & Match offers brain-busting fun for everyone with its unique take on puzzle gameplay. Match colored blocks together and they melt, but throw a melting block into another one and the chain continues! In Puzzle Mode, blocks are arranged to test your wits. Action mode throws in a side-scrolling twist and challenges you to clear a path. Battl ... more

domain: entertainment


News Flash Home
The original item was published from 5/19/2017 12:44:28 PM to 5/21/2017 8:05:02 AM.
Warrenville Community News
Posted on: May 19, 2017
[ARCHIVED] Police Department Special Hours
The Police Department front lobby and Records Division will be open for special hours on Saturday, May 20, 2017, from 7:00 a.m. to 3:00 p.m.
Persons coming to the Police Department in need of assistance may use the red telephone located between in the front lobby entrance doors to connect with the 9-1-1 dispatch center, or may dial 9-1-1 from their cellular phone. Police emergency response will not be affected.


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By Joe Miller
* each one of those North Carolina hikes is the cream-of-the-crop
* Full-color topographical maps, elevation profiles, and over a hundred lovely path photographs
* Trails-at-a-Glance chart to assist locate the hikes you will want speedy and easily
* comprises information on North Carolina public lands acquisition and regulations
North Carolina is the appropriate mountaineering nation: dramatic mountain trails within the Southern Appalachians, plenty of rolling trails close to excessive inhabitants facilities within the Piedmont, diversified hikes that few humans find out about at the Coastal simple. the numerous climates of the kingdom make for a wide range of climbing possibilities, certain to supply a few favorites for everybody. no matter if you thrive off of an severe heart-pounding climb with a sweeping vista as a gift or want a smoother terrain jam-packed with fascinating historic tidbits, there's a vintage hike for you.
Read or Download 100 Classic Hikes in North Carolina PDF
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Throughout the first seven versions of this enduring textual content, A. Viola Mitchell shared her wisdom and talents with legions of educators, camp administrators, and counselors who participated within the prepared camp move. This vintage, very popular quantity has now been completely up-to-date to supply a 21st-century view of the tendencies, philosophies, and practices of prepared camping out.
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Additional resources for 100 Classic Hikes in North Carolina


A few years ago, waterfowlers were in an age of steel the only material allowed for shot while hunting ducks and geese.
This year, however, technological advances approved by Washington and Idaho will give hunters two new shot choices that come closer to approximating the punch of lead.
The choices this year include steel shot, as well as bismuth-tin and tungsten-iron.
Lead pellets were outlawed nationwide for hunting waterfowl nearly a decade ago because heavy concentrations of spent shot could poison birds that picked them up while feeding.
Soft iron pellets, known as steel shot, replaced lead for several years and continue to be the most common form of non-toxic shot on the market.
However, many hunters object to steel shot because it is not compatible with some older-model guns. Some hunters blame steel shot for a higher percentage of crippling losses because the shot is lighter and loses its bird-killing energy at a shorter range than lead.
Pellets made of bismuth and tin were approved by the U.S. Fish and Wildlife Service a few years ago, and most states added their approval.
Now, the federal agency has approved tungsten-iron shot on a trial basis for this season. As a result, the Idaho and Washington wildlife commissions are allowing tungsteniron non-toxic shot, as long as the size does not exceed 0.20 (size T).
Bismuth-tin and tungsten-iron shells presently cost more than steel shot, but early experiences indicate that both shot types are effective.
Bismuth is soft enough to shoot in guns previously used only with lead pellets. Tungsten-iron pellets are comparable in hardness to steel shot and maintain their spherical integrity as well as steel, but they have nearly the mass as lead pellets and they retain similar down-range energy.


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UNM’s Ethnic Centers on the Budget Chopping Block
By Marisa Demarco
What’s a Little Historical Amnesia Between Neighbors?
Somos Primos campaign opens dialogue between Hispanic New Mexicans and Mexican immigrants
By Simon McCormack
The Radford Files
The Fuzzy, Superficial World of Balance
By Benjamin Radford
Art Start at Albuquerque Museum of Art and History
One-hour tours of the museum’s galleries and sculpture garden for preschoolers and their caregivers.
Stories in the Night Sky & ABQ Concert Band at Anderson-Abruzzo Balloon Museum
Community Movement Series: Ecstatic Dance at Studio SwayMore Recommented Events ››


by Mike Breen
Local bassist first artist announced for 2014 London Bass Guitar Show clinics
Veteran Cincinnati bass wizard Freekbass (of the band Freekbass, as well as Freekbot, the Kelly Richey Band and Headtronics) is the first Master Class leader to be announced for The London Bass Guitar Show 2014. The event (March 1-2 at the Olympia Conference Centre London) will draw some of the world's greatest bassists to London to "promote the art of bass playing to the general public." The show will showcase new bass products and gear, plus feature clinics and master classes throughout the event. Past London Bass Guitar Show participants include Mark King, Gary Willis, TM Stevens, Paul Turner, Marco Mendoza, Yolanda Charles, Doug Wimbish, Neil Murray and Dave Marks, Peter Hook, Igor Saavedra, Wojtek Pilichowski, Guy Pratt, Andy Irvine, Jonas Hellborg, Grog & Die So Fluid, Malcolm Joseph, Andrew Levy, David Ellefson, Jah Wobble, Lee Rocker, Nate Watts and Phil Mulford. Here's some footage from last year's event: The bassist is gearing up for the next album (click here to download Freekbass' album Concentrate for free) from his eponymous band. Freekbass is working on the new album at Lexington's Shangri-La Studios with Duane Lundy, who has done work with My Morning Jacket, Ben Sollee and others. The album will reportedly feature special guests DJ Spooky, Adam Deitch (a producer/drummer who has worked with everyone from John Scofield to 50 Cent) and Steve Molitz (Particle, Headtronics). This Wednesday, Freekbot (Freebass + DJ/beatmaster/electronicist Tobotius) will perform a recently announced show at Longworth's in Mt. Adams, 9:30 p.m.-midnight.
by Mike Breen
Chris Sherman of funky Cincy bands Freekbass and Freekbot joins Jyn Yates as Kelly Richey Band's new rhythm section
While not necessarily “weird,” when fans discovered that bad-ass, longtime local Blues artist Kelly Richey had started a side-business as a “life coach,” undoubtedly a few thought they misheard or misread. It’s not really a logical step from stunning Blues guitarist and vocalist to personal life coach for hire. Likewise, when I first got an email about the new lineup of Richey’s band, I did a double take. Again, not that it was particularly unusual news — I just didn’t see it coming. (The music world could use a lot more moments like that.)Joining Richey in the new version of KRB is experienced drummer Jyn Yates from Kentucky and Chris Sherman, the guiding force behind popular local Funk group Freekbass (and his new duo project, Freekbot). While Sherman (who goes by his stage name, Freekbass) is best known for his huge Funk grooves, dabbling in modern EDM and rather flamboyant stage outfits at times, Richey’s sound skates the line between hard Blues and Rock; the most flash from her live shows come from out of her fingers as she plays and mouth as she sings.On the surface, the pairing seems on par with Buckethead joining Bonnie Raitt’s band or Flea sitting in with Stevie Ray Vaughan. In other words, it should be quite interesting to hear what the trio comes up with. The first single, “Fast Drivin’ Mama,” offers a hint — the song is a rocking slice of riffing Blues Funk. It’s not drastically different from Richey’s usual sound, but the groove is appropriately huge. Here's the debut single/video for "Mama":The new KRB trio was assembled to record the next Kelly Richey Band album, which the musicians have been working on at Shangri-La Studios in Lexington, Ky. The album — Sweet Spirit — is scheduled for release March 1, followed by tour dates that will take them from Florida to Canada. The trio’s debut show is Saturday in Northern Illinois (Mishawaka, to be exact), but local fans won’t have to wait long to check out the new KRB. The group performs on Jan. 11 at Legends Nightclub in Cheviot. Showtime is 8 p.m. and there is a $10 cover at the door.
0 Comments · Wednesday, May 9, 2012
Tickets for the first Afghan Whigs
concert in 13 years go on sale this Friday through ticketmaster.com. The
band is kicking off its (so far) primarily European reunion tour dates
with a May 23 show at New York’s Bowery Ballroom.
by Mike Breen
Indie darling Annie Clark and her St. Vincent are at Bogart's tonight promoting their new album, Strange Mercy. Check out Jason Gargano's recent feature story on Clark from last week's CityBeat here. Opening the show is Austin-based Shearwater, Jonathan Meiburg's orchestral Indie project that is on tour in support of the new Animal Joy album on Sub Pop Records. (Read more here.) Showtime is 9 p.m. and tickets are $20. Here's a recent live clip of Clark and Co. performing "Surgeon."• Local Boogie Woogie piano master Ricky Nye has been taking his pals in the Paris Blues Band around the region for several performance dates. If you missed his show last week at the small room at Baker Hunt Arts & Cultural Center, you can catch the whole crew at Covington's Chez Nora tonight at 7:30 p.m. for free. Read Brian Baker's 2010 feature on the band ("The French Connection") here. And here's a clip for Ricky and his Paris bros from last year's visit.• Former local singer with Uncle Six (and in other local projects) Noah Hunt returns to Greater Cincy tonight with the man who took him away from us, Blues/Rock guitar whiz Kenny Wayne Shepherd. Hunt has been singing with Shepherd's band since 1997. On Noah's website it says, "Noah and Kenny are like brothers, and continue to record and tour the world together." Local crew Grooveshire opens up tonight's show at Covington's Madison Theater at 8 p.m. Tickets are $25. Here's a clip from the KWS Band's "Never Lookin' Back" from the most recent album, How I Go. • Indie trio The Spring Standards consists of three longtime friends who knock out harmonies as if they were related and have been singing together since birth. James Cleare, Heather Robb and James Smith formed the band in the Delaware Valley during their teens, but moved to Brooklyn to launch their career. The Standards’ 2008 debut, the EP No One Will Know, was produced by Rhett Miller of The Old 97’s; their debut full-length, Would Things be Different, was self-released in 2010. The band recently issued a superb “double EP,” Yellow/Gold, which features 12 engrossing tracks’ worth of the trio’s crafty Pop, which occasionally conjures the warm, fuzzy feelings of “AM Gold” Soft Rock from the ’70s, but also pumps things up to a more rocking level, such as on the Carsian tune "Here We Go" (check the clip below). The threesome performs tonight at MOTR Pub. Local Folk Pop act Shiny and the Spoon opens the free, 10 p.m. show.• Freekbot, the local Electronic/Funk/Dance duo featuring local funkateer Chris Sherman (aka Freekbass) and award-winning turntablist/producer Tobe Donohue (Tobotius), has not played a ton of local shows, spending a lot of time on the road as a touring unit instead. But that changes starting tonight when the duo begins a Wednesday residency at MVP Bar & Grill in Silverton (formerly Play By Play). Each week, the musicians will be joined by a different artist to “give things a different flavor,” says Sherman. For tonight's inaugural event — dubbed “Full Spectrum Wednesdays” — Yusef Quotah from You, You’re Awesome and Brian Olive’s band sits in on keys. On May 23, longtime Jazz saxophonist Randy Villars (currently touring with Bootsy Collins) is the special guest. In June, Sherman says they’ll have a different DJ or band open the shows, as well. Here's the twosome at work.
by Mike Breen
There are oodles of live musical options tonight all over our area tonight. Here's a quick rundown.• Dayton/Columbus band The Werks — self-described as a "Psychedelic Dance Funk Rock Improv" group — celebrate the release of their new self-titled album, the band's third, a self-issued full-length. Opening up the 10:30 p.m. show at Covington's Madison Theater is Freekbot, the dance-inducting duo project of Chris "Freekbass" Sherman and Tobe "Tobotius" Donohue. Below, check out the Freekbot track "Get Up" from a recent Rock Against Cancer benefit album (which also includes a track from locals Skeetones). Tickets for tonight's show are $10.Get Up by FREEKBOT• Inventive banjo genius Bela Fleck has reconvened the original lineup of his Bela Fleck and the Flecktones band. The progressive/Jazz/Roots/World/experimental troupe got back together to record Rocket Science (released around this time last spring), the first recording by the original four Flecktones in nearly two decades. They're touring behind the album and coming to Millett Hall on the campus of Miami University in Oxford tonight for a 7:30 p.m. concert. Tickets range from $20-$50. (Read more here.)Here's a behind the scenes look at the recording of Rocket Science:• Much buzzed about singer/songwriter and Garage/Rock/Blues explorer Hanni El Khatib performs a free show tonight at around 10 p.m. at MOTR Pub. Sundelles open up. Check out our interview with El Khatib in this week's CityBeat here and then check out the video for his song, "Loved One," below.• Elsewhere tonight: Eclectic local label Grasshopper Juice celebrates its third anniversary with a two-floor party/show at downtown's Mainstay featuring the diverse lineup of Revenge Pinata, Sometimes, Playfully Yours, The B.E.A.T., Blastronauts, Dub Lock and DWB. (9 p.m.; $5); a great display of fresh local Hip Hop can be found at Mayday in Northside. Valley High, Trademark Aaron, Zoo Crew, DJ Sinceer and DJ Benigma perform. (10 p.m.; free); acclaimed local Celtic Rock crew Roger Drawdy and the Firestarters perform at a benefit for the Keegan's Spirit Foundation, which assist families affected by heart disease. The show is this evening at Molly Malone's in Covington. (7 p.m.; $10); radio show Salina Underground hosts a free "Record Store Day Eve" bash at Northside Tavern tonight at 10 p.m. featuring the great lineup of The Sweep, newcomers The Chance Brothers and Brian Olive. (10 p.m.; free); great local Americana ensemble Mangolia Mountain presents its album release party at the Ballroom at the Taft Theatre tonight (read more here). (8 p.m.; $13); get your 420 on at Stanley's Pub tonight as the club hosts a "420 Party" with Reggae band The Cliftones and the Grateful Dead-lovin' Jerry's Little Band. (9:30 p.m.; $7); and — where I know MOST of you will be — schmaltzy singer/songwriter Barry Manilow turns the Bank of Kentucky Center in Highland Heights into the Copacabana when he performs at 7:30 p.m. tonight ($34.99-$124.99). We kid the Barster, of course. I mean, have you heard the dude rap?Click here for even more local live music events tonight.
0 Comments · Tuesday, February 7, 2012
If a duo can qualify as a “supergroup,” then Freekbot is a legit local music supergroup. Freekbot features Freekbass (a.k.a.
bassist/singer Chris Sherman of Funk crew Freekbass) and Tobotius
(a.k.a. Tobe Donohue, producer and founder of world-renowned turntable
crew Animal Crackers) in an Electronic duo configuration.


In the early 1990’s the WWW revolutionized information. 10 years later, the Internet became more mature & programmable. We saw the rise of the so-called Web2, which brought us social media and e-commerce platforms. It revolutionized social interactions, bringing producers and consumers of information, goods and services closer together, and allowed us to enjoy P2P interactions on a global scale. But always with a middleman: a platform acting as a trusted intermediary between A and B who did not know or trust each other. While these platforms have done an amazing job at creating a P2P economy, with an ever more sophisticated content discovery layer, they also dictate all rules of the transactions, and these platforms own all of our data.
In this context, Blockchain seems to be a driving force of the next generation Internet, the Decentralized Web, or Web3. Blockchain can bring us true P2P transactions without a middleman, and Bitcoin is the first use case. While Bitcoin is P2P money without banks and bank managers, the same technology that brought us Bitcoin could now allow us to build ride sharing without Uber, apartment sharing without Airbnb, and social media without Facebook and Twitter.
Killing the Server: Redesigning Data Structures
We first had the computer, and then we started connecting computers over the internet protocol. In the early days of personal computers, we used to save data on a floppy disc, eject it, walk over to the colleague that needed the file, insert the floppy disc into that person’s computer, and copy the file onto their computer so they could use it. Data was centrally stored one physical device, in order to transfer data, copies needed to be made. The internet made the transfer of these copies faster and massively reduced transaction costs.
30 years into mass adoption of the internet, our data architectures are mostly still client-server based. Which means that our data is centrally stored on one computer, and retrieved via the Internet by another computer over the Internet. Even though we live in an ever more connected world, where every device whether toaster or fridge are also connected to the internet, data is still centrally stored: on our devices, on the USB stick or even in the cloud. Which raises issues of trust: Can I trust those people and institutions that store my data against any form of corruption: internal or external, man-made or machine failure, on purpose or by accident? Such centralized data structures have a unique point of failure points of failure. It’s as if we never invented the Internet.
From Data Monarchy to Data Democracy
P2P data architectures have existed since the 1990’s where they rose to fame with file sharing programs like BitTorrent and Tor browser. In combination with cryptography and game theoretic incentive mechanisms, Blockchain has taken P2P architectures to a new level. We can now start to move from centralized data structures where all data is stored on a central computer to more decentralized or fully distributed data architectures.
In the Web3, we are redefining data structure, given that we live in a connected world. It is important to note that Blockchain is only one of many technologies in this Decentralized Web Stack. While Blockchain is a great P2P way to record who did what and when, it is not ideal for storing large amounts of data, for two reasons: (1) scalability: blockchains are too slow, and (2) doesn’t allow privacy by design: never store private data on the Blockchain.
Web3 Technology Stack
Similar to building a normal web or mobile applications, creating a dApp commonly requires a few things: computation, file storage, external data, monetization, and payments. The community has made a lot of progress building the ecosystem in the past 4 years. While it was borderline impossible to build a dApp in 2014, in 2017 it’s feasible to build a basic dApp that requires minimal computation and file storage. The Web3 ecosystem has come a long way to develop a technology stack that devs can build upon. Here a listing of a few selected graphics:
The transition from client-server internet to the decentralized web will be gradual rather than radical. As the decentralized web stack is still maturing, the transition seems to be shifting from centralized to partially decentralized to fully decentralized. Furthermore, it is important to point out that while decentralized architectures are more fault tolerant and attack resistant, they are also slower. While it is likely that the future of the internet will be more decentralized, this does not mean that we will get rid of centralized systems altogether. Centralized systems also have advantages and will likely prevail, but only for specific use cases.
- The dApp Developer Stack: The Blockchain Industry Barometer, by Fred Ehrsam
- Blockchain will usher in the era of decentralised computing, Bruce Pon, BigchainDB
- Fat Protocols, Joel Monegro
- The Shared Data Layer of The Blockchain Application Stack, Joel Monegro
- Web 3.0 Revisited — Part One: “Across Chains and Across Protocols”, Stephane Tual
- Blockchain Infrastructure Landscape: A First Principles Framing, Trent McConaghy