Pharmacophore-based, rationale design, and efficient synthesis of novel tetrahydrobenzo[b]thiophene candidates as potential dual Topo I/II inhibitors and DNA intercalators.

A series of tetrahydrobenzo[b]thiophene derivatives was designed and synthesized as dual topoisomerase (Topo) I/II inhibitors implicating potential DNA intercalation. Ethyl-2-amino-3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophene-4-carboxylate (1) was prepared by modification of the Gewald reaction procedure using a Fe2O3 nanocatalyst and then it was used as a building block for the synthesis of tetrahydrobenzo[b]thiophene candidates (2-14). Interestingly, compound 14 showed the best cytotoxic potential against hepatocellular, colorectal, and breast cancer cell lines (IC50 = 7.79, 8.10, and 3.53 µM), respectively, surpassing doxorubicin at breast cancer (IC50 = 4.17 µM). Meanwhile, the Topo I and II inhibition assay displayed that compound 3 could exhibit the best inhibitory potential among the investigated candidates (IC50 = 25.26 and 10.01 nM), respectively, in comparison to camptothecin (IC50 = 28.34 nM) and doxorubicin (IC50 = 11.01 nM), as reference standards. In addition, the DNA intercalation assay showed that compound 14 could display the best binding affinity with an IC50 value of 77.82 µM in comparison to doxorubicin (IC50 = 58.03 µM). Furthermore, cell cycle and apoptosis analyses described that compound 3 prompts the G1 phase arrest in michigan cancer foundation-7 cancer cells and increases the apoptosis ratio by 29.31% with respect to untreated cells (2.25%). Additionally, the conducted molecular docking assured the promising binding of the investigated members toward Topo I and II with potential DNA intercalation. Accordingly, the synthesized compounds could be treated as promising anticancer candidates for future optimization.

Textural properties and adsorption behavior of Zn-Mg-Al layered double hydroxide upon crystal violet dye removal as a low cost, effective, and recyclable adsorbent.

The preparation of adsorbents plays a vital role in the adsorption method. In particular, many adsorbents with high specific surface areas and unique shapes are essential for the adsorption strategy. A Zn-Mg-Al/layer double hydroxide (LDH) was designed in this study using a simple co-precipitation process. Adsorbent based on Zn-Mg-Al/LDH was used to remove crystal violet (CV) from the wastewater. The impacts of the initial dye concentration, pH, and temperature on CV adsorption performance were systematically examined. The adsorbents were analyzed both before and after adsorption using FTIR, XRD, and SEM. The roughness parameters and surface morphologies of the produced LDH were estimated using 3D SEM images. Under the best conditions (dose of adsorbent = 0.07 g and pH = 9), the maximum adsorption capacity has been achieved. Adsorption kinetics studies revealed that the reaction that led to the adsorption of CV dye onto Zn-Mg-Al/LDH was a pseudo-second-order model. Additionally, intraparticle diffusion suggests that Zn-Mg-Al/LDH has a fast diffusion constant for CV molecules (0.251 mg/(g min1/2)). Furthermore, as predicted by the Langmuir model, the maximal Zn-Mg-Al/LDH adsorption capacity of CV was 64.80 mg/g. The CV dimensionless separation factor (RL) onto Zn-Mg-Al/LDH was 0.769, indicating that adsorption was favorable. The effect of temperature was performed at 25, 35, and 45 °C in order to establish the thermodynamic parameters ∆Ho, ∆So, and ∆Go. The computed values indicated exothermic and spontaneous adsorption processes. The study presented here might be used to develop new adsorbents with enhanced adsorption capabilities for the purpose of protecting the water environment.

Correlation between Selective Motor Control of the Lower Extremities and Balance in Spastic Hemiplegic Cerebral Palsy: a randomized controlled trial.

BACKGROUND: Children with cerebral palsy (CP) have motor deficits caused by spasticity, weakness, contractures, diminished selective motor control (SMC), and poor balance. The purpose of the current study was to evaluate the influence of mirror feedback on lower extremity selective motor control and balance in children with hemiplegic cerebral palsy. Understanding the relationship between SMC and balance will help children with hemiplegic CP receive more appropriate therapies. METHODS: Forty-seven children of both sexes diagnosed with hemiplegic CP participated in the study. Group1 (Gr1 - control group) received conventional physical therapy training while group 2 (Gr2 - intervention group) received conventional physical therapy training in addition to bilateral lower extremity mirror therapy (MT). The primary outcome measure used was Selective Control Assessment of Lower Extremity scale (SCALE), while the secondary outcome measure was the Pediatric Balance Scale (PBS). RESULTS: There were significant differences in Selective Control Assessment of Lower Extremity Scale (SCALE) and Pediatric Balance Scale (PBS) between both groups in favor of Gr2. After treatment, both groups improved significantly, yet Gr2 outperformed Gr1 by a large margin. CONCLUSION: Mirror therapy may be a useful addition to home-based motor interventions for children with hemiplegic CP due to its relative simplicity, low cost, and high patient adherence. Additionally, it may help children improve their selective motor skills and balance. TRIAL REGISTRATION: Current Controlled Trials using African Clinical Trials Registry website with ID number PACTR202105604636415 retrospectively registered on 21/01/202.

Effect of compressive therapy on sensorimotor function of the more affected upper extremity in chronic stroke patients: A randomized clinical trial.

BACKGROUND: Common upper extremity (UE) physical impairments after stroke include paresis, abnormal muscle tone, and somatosensory affection. This study evaluated the effect of passive somatosensory stimulation using compressive therapy on sensorimotor function of the more affected UE in chronic stroke patients. METHODS: Forty chronic stroke patients were enrolled in this study. They were randomized into 2 groups: Gr1 and Gr2. Three patients dropped out leaving us with a total of 37 patients completing the study. Gr1 received UE motor program for the more affected UE along with sham electrical stimulation while Gr2 had the same UE motor program along with passive somatosensory stimulation. The session duration in both groups was 85 min. Gr1 and Gr2 received a total of 36 sessions for 6 successive weeks. UE function in Gr1 and Gr2 was examined, before and after treatment using Box and Block test (BBT) and Perdue Pegboard test (PPBT) as measures of motor of both the more affected and less affected UE while the Nottingham sensory assessment (NSA) scale was used as a measure of sensory function of the more affected UE. RESULTS: There were significant improvements in motor and sensory function of the more affected UE compared to the less affected UE in both groups, measured by the BBT, PPBT, and NSA scales post-treatment (P < .05). However, the comparison between both groups regarding improvement revealed no significant change (P > .05). CONCLUSION: Upper extremity motor and passive somatosensory stimulation techniques are effective in improving sensorimotor function of the more affected UE, but none of them had the advantage over the other, in terms of improving motor and sensory function in chronic stroke patients.

Effectiveness of virtual reality-based rehabilitation versus conventional therapy on upper limb motor function of chronic stroke patients: a systematic review and meta-analysis of randomized controlled trials.

OBJECTIVE: To systematically review the available randomized controlled trials in the literature concerning the application of virtual reality (VR) rehabilitation interventions compared to conventional physical therapy, in regaining the upper limb motor function among patients with chronic stroke. METHODS: A systematic electronic database search was conducted for related studies published from inauguration and until June 25, 2020 in nine databases. Another new search was done on February 1, 2021 and no new studies were identified. RESULTS: Six studies were included in the analysis. Significant improvement was seen following the VR therapy in patients with chronic stroke, compared to their scores prior to it (SMD = 0.28; 95% CI = 0.03-0.53; p = .03). There was neither heterogeneity (I2 = 0% and P = .5) nor a risk of bias (P = .8) among the included studies. VR interventions produced a comparable effectiveness to that of the conventional rehabilitation, with no statistically significant difference (SMD = 0.15; 95% CI = -0.14-0.44; P = .3). There was neither heterogeneity (I2 = 40% and P = .1) nor a risk of bias (P = .5) among the included studies. CONCLUSIONS: The upper limb motor function of patients with chronic stroke who underwent VR-based rehabilitative intervention showed significant improvement as compared to the pre-treatment state. Our analysis also revealed no superiority of VR interventions over conservative therapies; however, the difference observed did not accomplish statistical significance.

Ternary Photodegradable Nanocomposite (BiOBr/ZnO/WO3) for the Degradation of Phenol Pollutants: Optimization and Experimental Design.

The degradation of environmental contaminants with photocatalysts has bright prospects for application in the control of pollution. In this study, BiOBr/ZnO/WO3 heterojunctions have been documented to be reliable visible-light photocatalysts for phenol deterioration. X-ray diffraction, X-ray photoelectron spectroscopy, Fourier-transform infrared spectroscopy, scanning electron microscopy, transmission electron microscopy, photoluminescence spectral analysis, electrochemical impedance spectroscopy (EIS), EIS Bode plots, linear sweep voltammetry, and UV-visible diffuse reflectance spectroscopy were employed to describe the heterojunction's structure in addition to its optical features. The results revealed that the BiOBr/ZnO/WO3 ternary photocatalyst displayed more degradation activity in comparison to single-phase ZnO, WO3, or BiOBr, which is also higher than that of binary mixture photocatalysts with a phenol degradation efficiency of 90%. The influence of degradation variables, for instance, the potential of hydrogen (pH) and the initial organic contaminant content besides the heterojunction dose, on the deterioration efficiency was optimized using the response surface methodology. The degradation efficiency reached 95% under the optimal conditions of 0.08 g/0.03 L catalyst dose, a pH of 9, and an initial organic contaminant content of 10 mg L-1. However, the optimal phenol degradation efficiency of 39.37 mg g-1 was achieved under the conditions of 0.08 g/0.03 L catalyst dose, pH of 9, and 200 mg L-1 initial phenol concentration.

A Comparative Study on the Effect of Task Specific Training on Right Versus Left Chronic Stroke Patients.

Functional impairment of the upper limb (UL) after stroke is a great problem. Finding methods that can improve UL function after stroke is a major concern to all medical service providers. This study was intended to compare the effect of upper limb task specific training (TST) on brain excitability of the affected hemisphere and motor function improvements in patients with left and right stroke. Forty male patients with mild impairment of UL functions were divided into two equal groups; G1 consisted of patients with left hemisphere affection (right side stroke) while G2 consisted of patients with right hemisphere affection (left side stroke). All patients received TST for the affected UL for one hour, three sessions per week for six consecutive weeks. Evaluation was performed twice, pre-, and post-treatment. Outcome measures used were Wolf Motor Function Test (WMFT) and Box and Block Test (BBT) as measures of UL motor function and Quantitative Electroencephalogram (QEEG) of motor and sensory areas of the affected hemisphere as a measure of brain reorganization post-stroke. Both groups showed improvement in motor function of the affected UL measured by WMFT and BBT with reported significant difference between them. G1 showed greater improvement in motor function of the affected UL post-treatment compared to G2. Additionally, there was a significant increase in peak frequency of motor and sensory areas with higher and significant excitability in G1 only. These findings imply that brain reorganization in the left hemisphere responded more to TST compared to the right hemisphere. Based on findings of the current study, we can recommend adding TST to the physical therapy program in stroke patients with left hemisphere lesions.

Poly(I:C) source, molecular weight and endotoxin contamination affect dam and prenatal outcomes, implications for models of maternal immune activation.

The viral mimetic polyinosinic:polycytidylic acid (poly(I:C)) is increasingly used to induce maternal immune activation (mIA) to model neurodevelopmental disorders (NDDs). Robust and reproducible phenotypes across studies are essential for the generation of models that will enhance our understanding of NDDs and enable the development of improved therapeutic strategies. However, differences in mIA-induced phenotypes using poly(I:C) have been widely observed, and this has prompted the reporting of useful and much needed methodological guidelines. Here, we perform a detailed investigation of molecular weight and endotoxin variations in poly(I:C) procured from two of the most commonly used suppliers, Sigma and InvivoGen. We demonstrate that endotoxin contamination and molecular weight differences in poly(I:C) composition lead to considerable variability in maternal IL-6 response in rats treated on gestational day (GD)15 and impact on fetal outcomes. Specifically, both endotoxin contamination and molecular weight predicted reductions in litter size on GD21. Further, molecular weight predicted a reduction in placental weight at GD21. While fetal body weight at GD21 was not affected by poly(I:C) treatment, male fetal brain weight was significantly reduced by poly(I:C), dependent on supplier. Our data are in agreement with recent reports of the importance of poly(I:C) molecular weight, and extend this work to demonstrate a key role of endotoxin on relevant phenotypic outcomes. We recommend that the source and batch numbers of poly(I:C) used should always be stated and that molecular weight variability and endotoxin contamination should be minimised for more robust mIA modelling.

Maximal shortening velocity during plantar flexion: Effects of pre-activity and initial stretching state.

We investigated the effects of the initial length of the muscle-tendon unit (MTU) and muscle pre-activation on muscle-tendon interactions during plantarflexion performed at maximal velocity. Ultrasound images of gastrocnemius medialis were obtained on 11 participants in three conditions: (a) active plantarflexion performed at maximal velocity from three increasingly stretched positions (10°, 20°, and 30° dorsiflexion), (b) passive plantarflexion induced by a quick release of the ankle joint from the same three positions, and (c) pre-activation, which consisted of a maximal isometric contraction of the plantarflexors at 10° of dorsiflexion followed by a quick release of ankle joint. During the active condition at maximal velocity, initial MTU stretch positively influenced ankle joint velocity (+15.3%) and tendinous tissues shortening velocity (+37.6%) but not the shortening velocity peak value reached by muscle fascicle. The muscle fascicle was shortened during the passive condition; however, its shortening velocity never exceeded peak velocity measured in the active condition. Muscle pre-activation resulted in a considerable increase in ankle joint (+114.7%) and tendinous tissues velocities (+239.1%), although we observed a decrease in muscle fascicle shortening velocity. During active plantarflexion at maximal velocity, initial MTU length positively influences ankle joint velocity by increasing the contribution of tendinous tissues. Although greater initial stretch of the plantarflexors (ie, 30° dorsiflexion) increased the passive velocity of the fascicle during initial movement, its peak velocity was not affected. As muscle pre-activation prevented reaching the maximal muscle fascicle shortening velocity, this condition should be used to characterize tendinous tissues rather than muscle contractile properties.

Cytokine profile in gingival crevicular fluid and plasma of patients with aggressive periodontitis.

OBJECTIVE: This study aimed to determine the content of cytokines in gingival crevicular fluid (GCF) as well as in plasma of Sudanese patients with aggressive periodontitis (AgP) and healthy controls (HC). MATERIALS AND METHODS: Nineteen AgP patients and 19 HC were included. The mean probing pocket depth and clinical attachment level of the GCF sampled sites in patients were both ≥5 mm. The GCF and plasma levels of 27 cytokines were determined using 27-multiplex fluorescent bead-based immunoassays. Ratios were calculated among cytokines of the T-helper cell subsets Th1 and Th2. Descriptive statistics, the Mann-Whitney U-test and Spearman's rho rank correlation coefficient analysis were used. RESULTS: Interferon-γ was the only cytokine found in significantly lower levels in GCF of patients compared with HC. Levels of interleukin (IL)-10, IL-13, IL-1Ra, monocyte chemoattractant protein-1 (MCP-1), regulated on activation normal T-cell expressed and secreted (RANTES), granulocyte-colony-stimulating factor (G-CSF), and granulocyte-macrophage-CSF (GM-CSF) were significantly lower in plasma of AgP compared with HC. The ratios of Th1:Th2 in GCF and Treg:Th17 in plasma were significantly lower in AgP. CONCLUSIONS: The lower levels of cytokines detected systemically in plasma of AgP patients may have an impact on the immune response. The lower ratio of Th1:Th2 cytokines in GCF samples of AgP patients suggests a role for Th2 at the local site of disease.

Fast Low-to-High Confinement Mode Bifurcation Dynamics in a Tokamak Edge Plasma Gyrokinetic Simulation.

Transport barrier formation and its relation to sheared flows in fluids and plasmas are of fundamental interest in various natural and laboratory observations and of critical importance in achieving an economical energy production in a magnetic fusion device. Here we report the first observation of an edge transport barrier formation event in an electrostatic gyrokinetic simulation carried out in a realistic diverted tokamak edge geometry under strong forcing by a high rate of heat deposition. The results show that turbulent Reynolds-stress-driven sheared E×B flows act in concert with neoclassical orbit loss to quench turbulent transport and form a transport barrier just inside the last closed magnetic flux surface.

Novel polyacrylamide-based solid scale inhibitor.

Novel solid-state phosphorus-containing polymer was synthesized, characterized and investigated as anti-scaling agent for the removal of alkaline earth metals ions from water. An optimization protocol for the sorption process of the metal ions on the polymer surface was proposed and executed. The protocol involved parameters such as pH, contact time, polymer dose, and the initial concentration of the metal ion. The optimum pH was found to be around seven for all of the tested metal ions. The maximum sorption capacities of the prepared polymer were 667, 794, 769 and 709 (mg/g) for Mg, Ca, Sr and Ba ions, respectively. Evaluation of the sorption process from the isotherm, kinetic and thermodynamic points of view was also studied. The experimental evidence revealed that the sorption obeys Langmuir isotherm model and follows a pseudo-second order mechanism. Moreover, the sorption process is exothermic. Possibility of polymer reuse was also investigated.

Ten-year cardiovascular risk assessment in university students.

Cardiovascular disease (CVD) is responsible for more than half of all deaths in the European region. The aim of the study was to compare body composition, blood pressure, total cholesterol (TC) and high density lipoprotein cholesterol (HDL-C), family history, activity behaviors, and the 10-year risk of having a heart attack between 166 university students (21.62 ± 2.59 yrs) from Utah (USA) and 198 students (22.11 ± 2.51 yrs) from Hungary. Ninety-two percent of the Hungarian students and 100% of the Utah students had an estimated 10-year Framingham risk score of 1% or less. The high prevalence of low risk was primarily due to the young age of study participants, healthy body composition and non-smoking behavior. Hungarians who had higher 10-year risk of heart attack had significantly higher waist hip ratio (WHR), TC, diastolic blood pressure (DBP) and were smokers compared to those Hungarians with lower risk. The self-reported physical activity levels between the two groups of students were not different. In conclusion the young men and women who participated in this study were, for the most part healthy; however the smoking habits and the lower physical activity of the Hungarian students likely elevated their risk of CVD.

Ratio of pro-resolving and pro-inflammatory lipid mediator precursors as potential markers for aggressive periodontitis.

Aggressive periodontitis (AgP) is a rapidly progressing type of periodontal disease in otherwise healthy individuals which causes destruction of the supporting tissues of the teeth. The disease is initiated by pathogenic bacteria in the dental biofilm, and the severity of inflammation and attachment loss varies with the host response. Recently, there has been an increased interest in determining the role of lipid mediators in inflammatory events and the concept of pro-inflammatory and pro-resolution lipid mediators has been brought into focus also in periodontal disease. The present study aimed to determine the profile of omega-3 or n3- as well as omega-6 or n6- polyunsaturated fatty acids (PUFAs) and PUFA-metabolites of linoleic acid, arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in gingival crevicular fluid (GCF), saliva and serum in AgP patients and healthy controls. In total, 60 selected n3- and n6-PUFAs and various PUFA metabolites were measured using high performance liquid chromatography-tandem electrospray ionisation mass spectrometry (HPLC-ESI-MS-MS). Of these, 51 could be quantified in this study. The concentrations of the majority were low in saliva samples compared with serum and GCF, but were mainly higher in AgP patients compared with healthy controls in all three kinds of sample. Ratios of n3- to n6-PUFAs (DHA + EPA)/AA were significantly lower in the GCF of AgP patients than in the healthy controls. Furthermore, various ratios of the direct precursors of the pro-resolution lipid mediators (precursors of resolvins and protectins) were calculated against the precursors of mainly pro-inflammatory lipid mediators. These ratios were mainly lower in GCF and saliva of AgP patients, compared with healthy controls, but only reached significance in GCF (P<0.05). To conclude, the ratios of precursors of pro-resolution/pro-inflammatory lipid mediators seem to be more relevant for describing the disease status of AgP than the concentration of specific lipid mediators.

Genotype-dependent responses to levels of sibling competition over maternal resources in mice.

Research on phenotypic plasticity has often focused on how a given genotype responds to the changing physical environments such as temperature or diet. However, for many species the social environment has an equally important role because of competition for resources. During early development, the level of competition for limited (maternally provided) resources will often depend critically on the number of siblings. Therefore, competition among siblings should drive the evolution of genes that allow flexible responses to realized levels of competition and maternal resource availability. However, it is unknown whether genetically based differences between individuals exist in their response to the social environment that affect their future development. Using a quantitative trait locus approach in an experimental population of mice we demonstrate that effects of sibling number on body weight depend on individual genotype at seven loci, over and above the general negative litter size effect. Overall, these litter size-by-genotype interactions considerably modified the degree to which increasing litter size caused reduced weight. For example at one locus this effect leads to a 7% difference in body weight at week 7 between individuals experiencing the extremes of the normal range of litter sizes in our population (five to nine litter mates). The observed interaction between genotype and the competitive environment can produce differences in body weight that are similar in magnitude to the main effect of litter size on weight. Our results show that different genotypes respond to the social environment differentially and that interaction effects of genotype with litter size can be as important as genotype-independent effects of litter size.

Selective abortion and the evolution of genomic imprinting.

Mothers can determine which genotypes of offspring they will produce through selective abortion or selective implantation. This process can, at some loci, favour matching between maternal and offspring genotype whereas at other loci mismatching may be favoured (e.g. MHC, HLA). Genomic imprinting generally renders gene expression monoallelic and could thus be adaptive at loci where matching or mismatching is beneficial. This hypothesis, however, remains unexplored despite evidence that loci known to play a role in genetic compatibility may be imprinted. We develop a simple model demonstrating that, when matching is beneficial, imprinting with maternal expression is adaptive because the incompatible paternal allele is not detected, protecting offspring from selective abortion. Conversely, when mismatching is beneficial, imprinting with paternal expression is adaptive because the maternal genotype is more able to identify the presence of a foreign allele in offspring. Thus, imprinting may act as a genomic 'cloaking device' during critical periods in development when selective abortion is possible.

Sit to stand transfer: performance in rising power, transfer time and sway by age and sex in senior athletes.

OBJECTIVE: To observe the differences in performance variables of the sit to stand transfer (as measured on the NeuroCom Balance Master) in a population of senior athletes. METHOD: A convenience sample of 173 subjects aged 50 years and older. Data were obtained from voluntary participation in a health fair offered at the annual Huntsman World Senior Games in St George, Utah, USA. All sit to stand tests were performed on the NeuroCom Balance Master. The measured parameters were weight transfer time (WTT), rising power (force exerted to rise), and centre of gravity sway (COG sway) during the rising phase. RESULTS: A significant difference was found between stratified age groups (50-64 and 65+ years) on rising power. There was also a sex difference in rising power. No significant differences were found in weight transfer time or COG sway. CONCLUSION: While rising power decreases with increasing age in senior athletes, WTT and COG sway remain similar regardless of age or sex. The maintenance of these other two variables (WTT and COG sway) may be attributable to physical activity and/or participation in sport.

Mitochondria suppress local feedback activation of inositol 1,4, 5-trisphosphate receptors by Ca2+.

The concerted action of inositol 1,4,5-trisphosphate (IP3) and Ca2+ on the IP3 receptor Ca2+ release channel (IP3R) is a fundamental step in the generation of cytosolic Ca2+ oscillations and waves, which underlie Ca2+ signaling in many cells. Mitochondria appear in close association with regions of endoplasmic reticulum (ER) enriched in IP3R and are particularly responsive to IP3-induced increases of cytosolic Ca2+ ([Ca2+]c). To determine whether feedback regulation of the IP3R by released Ca2+ is modulated by mitochondrial Ca2+ uptake, the interactions between ER and mitochondrial Ca2+ pools were examined by fluorescence imaging of compartmentalized Ca2+ indicators in permeabilized hepatocytes. IP3 decreased luminal ER Ca2+ ([Ca2+]ER), and this was paralleled by an increase in mitochondrial matrix Ca2+ ([Ca2+]m) and activation of Ca2+-sensitive mitochondrial metabolism. Remarkably, the decrease in [Ca2+]ER evoked by submaximal IP3 was enhanced when mitochondrial Ca2+ uptake was blocked with ruthenium red or uncoupler. Moreover, subcellular regions that were relatively deficient in mitochondria demonstrated greater sensitivity to IP3 than regions of the cell with a high density of mitochondria. These data demonstrate that Ca2+ uptake by the mitochondria suppresses the local positive feedback effects of Ca2+ on the IP3R, giving rise to subcellular heterogeneity in IP3 sensitivity and IP3R excitability. Thus, mitochondria can play an important role in setting the threshold for activation and establishing the subcellular pattern of IP3-dependent [Ca2+]c signaling.