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Muhammad Sikandar,1 Muhammad Harris Shoaib,1 Rabia Ismail Yousuf,1 Farrukh Rafiq Ahmed,1 Fatima Ramzan Ali,1,2 Muhammad Talha Saleem,1 Kamran Ahmed,1 Sana Sarfaraz,3 Sabahat Jabeen,1 Fahad Siddiqui,1 Tazeen Husain,1 Faaiza Qazi,1 Muhammad Suleman Imtiaz1 1Department of Pharmaceutics, Faculty of Pharmacy and Pharmaceutical Sciences, University of Karachi, Karachi, 75270, Pakistan; 2Jinnah College of Pharmacy, Sohail University, Karachi, 74000, Pakistan; 3Department of Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, University of Karachi, Karachi, 75270, PakistanCorrespondence: Muhammad Harris Shoaib, Department of Pharmaceutics, Faculty of Pharmacy and Pharmaceutical Sciences, University of Karachi, Karachi, 75270, Pakistan, Email harrisshoaib2000@yahoo.com; mhshoaib@uok.edu.pkPurpose: Halloysite nanotubes (HNTs) are a versatile and highly investigated clay mineral due to their natural availability, low cost, strong mechanical strength, biocompatibility, and binding properties. The present work explores its role for retarding and controlling the drug release from the composite polymer matrix material.Methods: For this purpose, nanocomposite films comprising propranolol HCl and different concentrations of HNTs were formulated using the “solution casting method”. The menthol in a concentration of 1% w/v was used as a permeation enhancer, and its effect on release and permeation was also determined. Quality characteristics of the nanocomposite were determined, and in vitro release and permeation studies were performed using the Franz diffusion system. The data was analyzed using various mathematical models and permeation parameters. Optimized formulation was also subjected to skin irritation test, FTIR, DSC, and SEM study. Systemic absorption and disposition of propranolol HCl from the nanocomposites were predicted using the GastroPlus TCAT® model.Results: The control in drug release rate was associated with the higher concentration of HNTs. F8 released 50% of propranolol within 8 hours (drug, HNTs ratio, 1:2). The optimized formulation (F6) with drug: HNTs (2:1), exhibited drug release 80% in 4 hours, with maximum flux of 145.812 μg/cm2hr. The optimized formulation was found to be a non-irritant for skin with a shelf life of 35.46 months (28– 30 ℃). The in silico model predicted Cmax, Tmax, AUCt, and AUCinf as 32.113 ng/mL, 16.58 h, 942.34 ng/mL×h, and 1102.9 ng/mL×h, respectively.Conclusion: The study demonstrated that HNTs could be effectively used as rate controlling agent in matrix type transdermal formulations.Keywords: halloysite nanotube, transdermal, nanocomposite, controlled release, GastroPlus

Muhammad Arif Asghar,1 Rabia Ismail Yousuf,1 Muhammad Harris Shoaib,1 Muhammad Asif Asghar,2 Mehrukh Zehravi,3 Ahad Abdul Rehman,4 Muhammad Suleman Imtiaz,5 Kamran Khan5 1Department of Pharmaceutics, Faculty of Pharmacy and Pharmaceutical Sciences, University of Karachi, Karachi, Pakistan; 2Food and Feed Safety Laboratory, Food and Marine Resources Research Centre, PCSIR Laboratories Complex, Karachi, Sindh, Pakistan; 3Department of Clinical Pharmacy, College of Pharmacy for Girls, Prince Sattam Bin Abdul Aziz University, Al-Kharj, Kingdom of Saudi Arabia; 4Department of Pharmacology, Institute of Pharmaceutical Sciences, Jinnah Sindh Medical University, Karachi, Pakistan; 5Department of Pharmaceutics, Institute of Pharmaceutical Sciences, Jinnah Sindh Medical University, Karachi, PakistanCorrespondence: Rabia Ismail Yousuf; Muhammad Harris ShoaibDepartment of Pharmaceutics, Faculty of Pharmacy and Pharmaceutical Sciences, University of Karachi, Karachi, PakistanEmail rabia_pharmaceutics@yahoo.com; harrisshoaib2000@yahoo.com; mhshoaib@uok.edu.pkPurpose: The current study proposed the simple, eco-friendly and cost-effective synthesis of carboxymethyl cellulose (CMC) structured silver-based nanocomposite (CMC-AgNPs) using Syzygium aromaticum buds extract.Methods: The CMC-AgNPs were characterized by ultraviolet (UV) spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier transmission infra-red (FTIR), energy-dispersive X-ray (EDX), and dynamic light scattering (DLS) techniques. The synthesized nanocomposites were evaluated for their bactericidal kinetics, in-vivo anti-inflammatory, anti-leishmaniasis, antioxidant and cytotoxic activities using different in-vitro and in-vivo models.Results: The spherical shape nanocomposite of CMC-AgNPs was synthesized with the mean size range of 20– 30 nm, and the average pore diameter is 18.2 nm while the mean zeta potential of − 31.6 ± 3.64 mV. The highly significant (P < 0.005) antibacterial activity was found against six bacterial strains with the ZIs of 24.6 to 27.9 mm. More drop counts were observed in Gram-negative strains after 10 min exposure with CMC-AgNPs. Significant damage in bacterial cell membrane was also observed in atomic force microscopy (AFM) after treated with CMC-AgNPs. Nanocomposite showed highly significant anti-inflammatory activity in cotton pellet induced granuloma model (Phase I) in rats with the mean inhibitions of 43.13% and 48.68% at the doses of 0.025 and 0.05 mg/kg, respectively, when compared to control. Reduction in rat paw edema (Phase II) was also highly significant (0.025 mg/kg; 42.39%; 0.05 mg/kg, 47.82%). At dose of 0.05 mg/kg, CMC-AgNPs caused highly significant decrease in leukocyte counts (922 ± 83), levels of CRP (8.4 ± 0.73 mg/mL), IL-1 (177.4 ± 21.3 pg/mL), IL-2 (83.7 ± 11.5 pg/mL), IL-6 (83.7 ± 11.5 pg/mL) and TNF-α (18.3 ± 5.3 pg/mL) as compared to control group. CMC-AgNPs produced highly effective anti-leishmaniasis activity with the viable Leishmania major counts decreased up to 36.7% within 24 h, and the IC50 was found to be 28.41 μg/mL. The potent DPPH radical scavenging potential was also observed for CMC-AgNPs with the IC50 value of 112 μg/mL. Furthermore, the cytotoxicity was assessed using HeLa cell lines with the LC50 of 108.2 μg/mL.Conclusion: The current findings demonstrate positive attributes of CMC fabricated AgNPs as a promising antibacterial, anti-inflammatory, anti-leishmaniasis, and antioxidant agent with low cytotoxic potential.Keywords: silver nanoparticles, Syzygium aromaticum, carboxymethyl cellulose, green synthesis, biological applications

Tazeen Husain, Muhammad Harris Shoaib, Farrukh Rafiq Ahmed, Rabia Ismail Yousuf, Sadaf Farooqi, Fahad Siddiqui, Muhammad Suleman Imtiaz, Madiha Maboos, Sabahat Jabeen Department of Pharmaceutics, Faculty of Pharmacy and Pharmaceutical Sciences, University of Karachi, Karachi, 75270, PakistanCorrespondence: Muhammad Harris Shoaib; Farrukh Rafiq AhmedDepartment of Pharmaceutics, Faculty of Pharmacy and Pharmaceutical Sciences, University of Karachi, Karachi, 75270, PakistanEmail harrisshoaib2000@yahoo.com; farrukh.ahmed@uok.edu.pkPurpose: This study systematically investigated the potential of four model drugs (verapamil HCl, flurbiprofen, atenolol, and furosemide), each belonging to a different class of Biopharmaceutics Classification Systems (BCS) to be developed into oral modified release dosage forms after loading with halloysite nanotubes (HNTs).Methods: The drugs were studied for their loading (mass gain %) by varying solvent system, method, pH, and ratios of loading into the nanotubes using D-optimal split-plot design with the help of Design Expert software. Drug-loaded halloysites were characterized by XRD, DTA, FTIR, SEM, and HPLC-UV-based assay procedures. Dissolution studies were also performed in dissolution media with pH 1.2, 4.5, and 6.8. Moreover, the optimized samples were evaluated under stress stability conditions for determining prospects for the development of oral dosage forms.Results: As confirmed with the results of XRD and DTA, the drugs were found to be converted into amorphous form after loading with halloysite (HNTs). The drugs were loaded in the range of ∼ 7– 9% for the four drugs, with agitation providing satisfactory and equivalent loading as compared to vacuum plus agitation based reported methods. FTIR results revealed either only weak electrostatic (verapamil HCl and flurbiprofen) or no interaction with the surface structure of the HNTs. The dissolution profiling depicted significantly retarded release of drugs with Fickian diffusion from a polydisperse system as a model that suits well for the development of oral dosage forms. HPLC-UV-based assay indicated that except furosemide (BCS class IV), the other three drugs are quite suitable for development for oral dosage forms.Conclusion: The four drugs investigated undergo phase transformation with HNTs. While agitation is an optimum method for loading drugs with various physicochemical attributes into HNTs; solvent system, loading ratios and pH play an important role in the loading efficiency respective to the drug properties. The study supports the capability of developing HNT-based modified release oral dosage forms for drugs with high solubility.Keywords: halloysite nanotubes, HNT, biopharmaceutics classification system, drug release kinetics, verapamil HCl, flurbiprofen, atenolol, furosemide, XRD, DTA, FTIR, SEM

Asghar MA, Yousuf RI, Shoaib MH, et al. Int J Nanomedicine. 2021;16:5371–5393 At the request of the authors, the Editor and Publisher of International Journal of Nanomedicine wish to retract the published article. Concerns were raised regarding the alleged duplication of images in Figure 2 and Figure 9 with similar images in Figure 4 and Figure 9 from an article published by the same authors in the International Journal of Biological Macromolecules, Asghar et al, 2020 (https://doi.org/10.1016/j.ijbiomac.2020.05.197). Specifically, Figure 2C, panel AgNPs appears to have been duplicated with Figure 4A, panel AgNPs from Asghar et al, 2020. Figure 2D, panel CMC-AgNPs appears to have been duplicated with Figure 4B, panel CS-AgNPs, which has been flipped, from Asghar et al, 2020. Figure 9B, panel Streptococcus mutans appears to have been duplicated with Figure 7B, methicillin resistant Staphylococcus aureus from Asghar et al, 2020. Figure 9C, panel Staphylococcus epidermidis appears to have been duplicated with Figure 7A, vancomycin resistant Staphylococcus aureus from Asghar et al, 2020. The authors explained the TEM images in question were provided by the first author, Muhammad Arif Asghar, and on subsequent review and investigation from this author, have confirmed these images were not satisfactory. However, all the authors accept joint responsibility for the integrity of the reported research and agree with the decision to retract the article. Our decision-making was informed by our policy on publishing ethics and integrity and the COPE guidelines on retraction. The retracted article will remain online to maintain the scholarly record, but it will be digitally watermarked on each page as “Retracted”. This retraction relates to this paper

The study is based on using SeDeM expert system in developing controlled-release tramadol HCl osmotic tablets and its in-silico physiologically based pharmacokinetic (PBPK) modeling for in-vivo pharmacokinetic evaluation. A Quality by Design (QbD) based approach in developing SeDEM-driven full factorial osmotic drug delivery was applied. A 2 4 Full-factorial design was used to make the trial formulations of tramadol HCl osmotic tablets using NaCl as osmogen, Methocel K4M as rate controlling polymer, and avicel pH 101 as diluent. The preformulation characteristics of formulations (F1-F16) were determined by applying SeDeM Expert Tool. The formulation was optimized followed by in-vivo predictive pharmacokinetic assessment using PBPK "ACAT" model of GastroPlus™. The FTIR results showed no interaction among the ingredients. The index of good compressibility (ICG) values of all trial formulation blends were ≥5, suggesting direct compression is the best-suited method. Formulation F3 and F4 were optimized based on drug release at 2, 10, and 16 h with a zero-order kinetic release ( r 2 = 0.992 and 0.994). The SEM images confirmed micropores formation on the surface of the osmotic tablet after complete drug release. F3 and F4 were also stable (shelf life 29.41 and 23.46 months). The in vivo simulation of the pharmacokinetics of the PBPK in-silico model revealed excellent relative bioavailability of F3 and F4 with reference to tramadol HCl 50 mg IR formulations. The SeDeM expert tool was best utilized to evaluate the compression characteristics of selected formulation excipients and their blends for direct compression method in designing once-daily osmotically controlled-release tramadol HCl tablets. The in-silico GastroPlus™ PBPK modeling provided a thorough pharmacokinetic assessment of the optimized formulation as an alternative to tramadol HCl in vivo studies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Saleem, Shoaib, Yousuf, Ahmed, Ahmed, Siddiqui, Mahmood, Sikandar and Imtiaz.)

Background: Optical sensing solutions are being developed and adopted to classify a wide range of biological objects, including crop seeds. Performance assessment of optical classification models remains both a priority and a challenge., Methods: As training data, we acquired hyperspectral imaging data from 3646 individual tomato seeds (germination yes/no) from two tomato varieties. We performed three experimental data manipulations: (1) Object assignment error: effect of individual object in the training data being assigned to the wrong class. (2) Spectral repeatability: effect of introducing known ranges (0-10%) of stochastic noise to individual reflectance values. (3) Size of training data set: effect of reducing numbers of observations in training data. Effects of each of these experimental data manipulations were characterized and quantified based on classifications with two functions [linear discriminant analysis (LDA) and support vector machine (SVM)]., Results: For both classification functions, accuracy decreased linearly in response to introduction of object assignment error and to experimental reduction of spectral repeatability. We also demonstrated that experimental reduction of training data by 20% had negligible effect on classification accuracy. LDA and SVM classification algorithms were applied to independent validation seed samples. LDA-based classifications predicted seed germination with RMSE = 10.56 (variety 1) and 26.15 (variety 2), and SVM-based classifications predicted seed germination with RMSE = 10.44 (variety 1) and 12.58 (variety 2)., Conclusion: We believe this study represents the first, in which optical seed classification included both a thorough performance evaluation of two separate classification functions based on experimental data manipulations, and application of classification models to validation seed samples not included in training data. Proposed experimental data manipulations are discussed in broader contexts and general relevance, and they are suggested as methods for in-depth performance assessments of optical classification models., (© 2022. The Author(s).)

Opioid use disorder has been designated a worsening epidemic with over 100,000 deaths due to opioid overdoses recorded in 2021 alone. Unintentional deaths due to opioid overdoses have continued to rise inexorably. While opioid overdose antidotes such as naloxone, and nalmefene are available, these must be administered within a critical time window to be effective. Unfortunately, opioid-overdoses may occur in the absence of antidote, or may be unwitnessed, and the rapid onset of cognitive impairment and unconsciousness, which frequently accompany an overdose may render self-administration of an antidote impossible. Thus, many lives are lost because: (1) an opioid overdose is not anticipated (i.e., monitored/detected), and (2) antidote is either not present, and/or not administered within the critical frame of effectiveness. Currently lacking is a non-invasive means of automatically detecting, reporting, and treating such overdoses. To address this problem, we have designed a wearable, on-demand system that comprises a safe, compact, non-invasive device which can monitor, and effectively deliver an antidote without human intervention, and report the opioid overdose event. A novel feature of our device is a needle-stow chamber that stores needles in a sterile state and inserts needles into tissue only when drug delivery is needed. The system uses a microcontroller which continuously monitors respiratory status as assessed by reflex pulse oximetry. When the oximeter detects the wearer's percentage of hemoglobin saturated with oxygen to be less than or equal to 90%, which is an indication of impending respiratory failure in otherwise healthy individuals, the microcontroller initiates a sequence of events that simultaneously results in the subcutaneous administration of opioid antidote, nalmefene, and transmission of a GPS-trackable 911 alert. The device is compact (4 × 3 × 3 cm), adhesively attaches to the skin, and can be conveniently worn on the arm. Furthermore, this device permits a centralized remotely accessible system for effective institutional, large-scale intervention. Most importantly, this device has the potential for saving lives that are currently being lost to an alarmingly increasing epidemic., (© 2021. The Author(s).)

Objectives: The persistence pattern of anti-migraine drugs' use among migraineurs is very low in the United States and different European countries. However, the cost and persistence of antimigraine drugs in Asian countries have not been well-studied. Hence, the present study aimed to evaluate the treatment cost and persistence among migraineurs in Pakistan., Methods: Data from prescriptions collected from migraineurs who visited the Outpatient Department (OPD) of different public and private sector tertiary-care hospitals of Karachi, Pakistan were used to conduct this retrospective cohort study from 2017 to 2019. The minimum follow up period for each migraineur was about 12 months for persistence analysis while dropped-out patients data were also included in survival analysis as right censored data. Pairwise comparisons from Cox regression/hazards ratio were used to assess the predictors of persistence with the reference category of non-binary variables i.e. hazard ratio = 1 for low frequency migraineurs and NSAIDs users. Persistence with anti-migraine drugs was estimated using the Kaplan-Meier curve along with the Log Rank test., Results: A total of 1597 patients were included in this study, 729 (45.6%) were male and 868 (54.3%) were female. Non-steroidal anti-inflammatory drugs (NSAIDs) were the most prescribed class of drug initially for all classes of migraineurs (26.1%). Of them, 57.3% of migraineurs discontinued their treatment, 28.5% continued while 14.8% were switched to other treatment approaches. Persistence with initial treatment was more profound in female (58.8%) patients compared to males while the median age of continuers was 31 years. The total cost of migraine treatment in the entire study cohort was 297532.5 Pakistani Rupees ($1901.1). By estimating the hazard ratios (HR) using the Cox regression analysis, it can be observed that patients with high frequency (HR, 1.628; 95%CI, 1.221-2.179; p<0.0001) migraine, depression (HR, 1.268; 95%CI, 1.084-1.458; p<0.0001), increasing age (HR, 1.293; 95%CI, 1.092-1.458; p<0.0001), combination analgesics (HR, 1.817; 95%CI, 0.841-2.725; p = 0.0004) and prophylaxis drugs (HR, 1.314; 95%CI, 0.958-1.424; p<0.0001) users were at a higher risk of treatment discontinuation. However, patients with chronic migraine (HR, 0.881; 95%CI, 0.762-0.912; p = 0.0002), epileptic seizure (HR, 0.922; 95%CI, 0.654-1.206; p = 0.0002), other comorbidities (HR, 0.671; 95%CI, 0.352-1.011; p = 0.0003) and users of triptan(s) (HR, 0.701; 95%CI, 0.182-1.414; p = 0.0005) and triptan(s) with NSAIDs (HR, 0.758; 95%CI, 0.501-1.289; p<0.0001) had more chances to continue their initial therapy., Conclusion: Similar to western countries, the majority of migraineurs exhibited poor persistence to migraine treatments. Various factors of improved persistence were identified in this study., Competing Interests: The authors have read the journal’s policy and the authors of this manuscript have the following competing interests: MHR is a paid employee of Reckett Benckiser. There are no patents, products in development or marketing products to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Biochar is an effective material for the removal of heavy metals from wastewater. Operational conditions, such as metal initial concentration, temperature, contact time as well as the presence of competing ions can impact the effectiveness of the treatment process. While several models have been proposed for modelling the adsorption process, no model currently exists that accounts for the mutual interactions of key process parameters on the adsorption capacity in multi-solute systems. The aim of this study is to address this gap in knowledge by formulating a multi-input multi-output (MIMO) model, which takes into account the effect of mutual interactions of key factors while predicting heavy metals adsorption capacity of the biochar in single and multi-solute systems. In this study, we use machine learning models, specifically several ANN models, radial basis and gradient boosting algorithms to model the MIMO process. The results of our models provide highly accurate predictions (R 2  > 0.99). The generalized regression network provided the best match to the experimental data. This approach can allow operators to predict how the adsorption system will respond to changes in the operations and hence provide them with a tool for process optimization., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Veins exhibit spontaneous contractile activity, a phenomenon generally termed vasomotion. This is mediated by spontaneous rhythmical contractions of mural cells (i.e. smooth muscle cells (SMCs) or pericytes) in the wall of the vessel. Vasomotion occurs through interconnected oscillators within and between mural cells, entraining their cycles. Pharmacological studies indicate that a key oscillator underlying vasomotion is the rhythmical calcium ion (Ca 2+ ) release-refill cycle of Ca 2+ stores. This occurs through opening of inositol 1,4,5-trisphosphate receptor (IP 3 R)- and/or ryanodine receptor (RyR)-operated Ca 2+ release channels in the sarcoplasmic/endoplasmic (SR/ER) reticulum and refilling by the SR/ER reticulum Ca 2+ ATPase (SERCA). Released Ca 2+ from stores near the plasma membrane diffuse through the cytosol to open Ca 2+ -activated chloride (Cl - ) channels, this generating inward current through an efflux of Cl - . The resultant depolarisation leads to the opening of voltage-dependent Ca 2+ channels and possibly increased production of IP 3 , which through Ca 2+ -induced Ca 2+ release (CICR) of IP 3 Rs and/or RyRs and IP 3 R-mediated Ca 2+ release provide a means by which store oscillators entrain their activity. Intercellular entrainment normally involves current flow through gap junctions that interconnect mural cells and in many cases this is aided by additional connectivity through the endothelium. Once entrainment has occurred the substantial Ca 2+ entry that results from the near-synchronous depolarisations leads to rhythmical contractions of the mural cells, this often leading to vessel constriction. The basis for venous/venular vasomotion has yet to be fully delineated but could improve both venous drainage and capillary/venular absorption of blood plasma-associated fluids.

Modern endoscopes play a significant role in diagnosing various gastrointestinal (GI) tract related diseases where the visual quality of endoscopic images helps improving the diagnosis. This article presents an image enhancement method for color endoscopic images that consists of three stages, and hence termed as "Tri-scan" enhancement: (1) tissue and surface enhancement: a modified linear unsharp masking is used to sharpen the surface and edges of tissue and vascular characteristics; (2) mucosa layer enhancement: an adaptive sigmoid function is employed on the R plane of the image to highlight micro-vessels of the superficial layers of the mucosa and submucosa; and (3) color tone enhancement: the pixels are uniformly distributed to create an enhanced color effect to highlight the subtle micro-vessels, mucosa and tissue characteristics. The proposed method is used on a large data set of low contrast color white light images (WLI). The results are compared with three existing enhancement techniques: Narrow Band Imaging (NBI), Fuji Intelligent Color Enhancement (FICE) and i-scan Technology. The focus value and color enhancement factor show that the enhancement level achieved in the processed images is higher compared to NBI, FICE and i-scan images.

Rationale: Heart failure is a multimodal disorder, of which disrupted Ca 2+ homeostasis is a hallmark. Central to Ca 2+ homeostasis is the major cardiac Ca 2+ release channel - the ryanodine receptor (RyR2) - whose activity is influenced by associated proteins, covalent modification and by Ca 2+ and Mg 2+ . That RyR2 is remodelled and its function disturbed in heart failure is well recognized, but poorly understood., Objective: To assess Ca 2+ and Mg 2+ regulation of RyR2 from left ventricles of healthy, cystic fibrosis and failing hearts, and to correlate these functional changes with RyR2 modifications and remodelling., Methods and Results: The function of RyR2 from left ventricular samples was assessed using lipid bilayer single-channel measurements, whilst RyR2 modification and protein:protein interactions were determined using Western Blots and co-immunoprecipitation. In all failing hearts there was an increase in RyR2 activity at end-diastolic cytoplasmic Ca 2+ (100nM), a decreased cytoplasmic [Ca 2+ ] required for half maximal activation (K a ) and a decrease in inhibition by cytoplasmic Mg 2+ . This was accompanied by significant hyperphosphorylation of RyR2 S 2808 and S 2814 , reduced free thiol content and a reduced interaction with FKBP12.0 and FKBP12.6. Either dephosphorylation of RyR2 using PP1 or thiol reduction using DTT eliminated any significant difference in the activity of RyR2 from healthy and failing hearts. We also report a subgroup of RyR2 in failing hearts that were not responsive to regulation by intracellular Ca 2+ or Mg 2+ ., Conclusion: Despite different aetiologies, disrupted RyR2 Ca 2+ sensitivity and biochemical modification of the channel are common constituents of failing heart RyR2 and may underlie the pathological disturbances in intracellular Ca 2+ signalling., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The aim of the study was to develop a reservoir-type transdermal patch for a controlled delivery of dexibuprofen and to evaluate its in vivo anti-inflammatory activity in Albino Wistar rats. In order to develop these patches, six formulations of dexibuprofen microemulsion comprising ethyl oleate, Tween 80: PG (2 : 1), and water were prepared by simplex lattice design and characterized. The reservoir compartment was filled with these microemulsions and in vitro release and skin permeation were assessed. The optimized patch was obtained on the basis of the responses: Q 24 and flux. The impact of drug loading, surface area, membrane thickness, adhesive, and agitation speed on drug release and permeation was also studied. The skin sensitivity reaction and in vivo anti-inflammatory activity of optimized patch were evaluated. Stability study at three different temperatures for three months was carried out. The result suggests that a membrane based patch with zero-order release rate, Q 24 of 79.13 ± 3.08%, and maximum flux of 331.17  µ g/cm 2 h can be obtained exhibiting suitable anti-inflammatory activity with no visible skin sensitivity reaction. The outcomes of stability study recommend storage of patches at 4°C having shelf-life of 6.14 months. The study demonstrates that the reservoir-type transdermal patch of dexibuprofen microemulsion has a potential of delivering drug across skin in controlled manner with required anti-inflammatory activity.

Introduction: The resting ECG is the most commonly used tool to assess cardiac electrophysiology. Previous studies have estimated heritability of ECG parameters based on these snapshots of the cardiac electrical activity. In this study we set out to determine whether analysis of heart rate specific data from Holter ECGs allows more complete assessment of the heritability of ECG parameters., Methods and Results: Holter ECGs were recorded from 221 twin pairs and analyzed using a multi-parameter beat binning approach. Heart rate dependent estimates of heritability for QRS duration, QT interval, Tpeak-Tend and Theight were calculated using structural equation modeling. QRS duration is largely determined by environmental factors whereas repolarization is primarily genetically determined. Heritability estimates of both QT interval and Theight were significantly higher when measured from Holter compared to resting ECGs and the heritability estimate of each was heart rate dependent. Analysis of the genetic contribution to correlation between repolarization parameters demonstrated that covariance of individual ECG parameters at different heart rates overlap but at each specific heart rate there was relatively little overlap in the genetic determinants of the different repolarization parameters., Conclusions: Here we present the first study of heritability of repolarization parameters measured from Holter ECGs. Our data demonstrate that higher heritability can be estimated from the Holter than the resting ECG and reveals rate dependence in the genetic-environmental determinants of the ECG that has not previously been tractable. Future applications include deeper dissection of the ECG of participants with inherited cardiac electrical disease.

In this paper, we present a real-time preprocessing algorithm for image enhancement for endoscopic images. A novel dictionary based color mapping algorithm is used for reproducing the color information from a theme image. The theme image is selected from a nearby anatomical location. A database of color endoscopy image for different location is prepared for this purpose. The color map is dynamic as its contents change with the change of the theme image. This method is used on low contrast grayscale white light images and raw narrow band images to highlight the vascular and mucosa structures and to colorize the images. It can also be applied to enhance the tone of color images. The statistic visual representation and universal image quality measures show that the proposed method can highlight the mucosa structure compared to other methods. The color similarity has been verified using Delta E color difference, structure similarity index, mean structure similarity index and structure and hue similarity. The color enhancement was measured using color enhancement factor that shows considerable improvements. The proposed algorithm has low and linear time complexity, which results in higher execution speed than other related works.

The Kv11.1 or hERG potassium channel is responsible for one of the major repolarising currents (IKr) in cardiac myocytes. Drug binding to hERG can result in reduction in IKr, action potential prolongation, acquired long QT syndrome and fatal cardiac arrhythmias. The current guidelines for pre-clinical assessment of drugs in development is based on the measurement of the drug concentration that causes 50% current block, i.e., IC50. However, drugs with the same apparent IC50 may have very different kinetics of binding and unbinding, as well as different affinities for the open and inactivated states of Kv11.1. Therefore, IC50 measurements may not reflect the true risk of drug induced arrhythmias. Here we have used an in silico approach to test the hypothesis that drug binding kinetics and differences in state-dependent affinity will influence the extent of cardiac action potential prolongation independent of apparent IC50 values. We found, in general that drugs with faster overall kinetics and drugs with higher affinity for the open state relative to the inactivated state cause more action potential prolongation. These characteristics of drug-hERG interaction are likely to be more arrhythmogenic but cannot be predicted by IC50 measurement alone. Our results suggest that the pre-clinical assessment of Kv11.1-drug interactions should include descriptions of the kinetics and state dependence of drug binding. Further, incorporation of this information into sophisticated in silico models should be able to better predict arrhythmia risk and therefore more accurately assess safety of new drugs in development., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Dantrolene is the first line therapy of malignant hyperthermia. Animal studies suggest that dantrolene also protects against heart failure and arrhythmias caused by spontaneous Ca(2+) release. Although dantrolene inhibits Ca(2+) release from the sarcoplasmic reticulum of skeletal and cardiac muscle preparations, its mechanism of action has remained controversial, because dantrolene does not inhibit single ryanodine receptor (RyR) Ca(2+) release channels in lipid bilayers. Here we test the hypothesis that calmodulin (CaM), a physiologic RyR binding partner that is lost during incorporation into lipid bilayers, is required for dantrolene inhibition of RyR channels. In single channel recordings (100 nM cytoplasmic [Ca(2+)] + 2 mM ATP), dantrolene caused inhibition of RyR1 (rabbit skeletal muscle) and RyR2 (sheep) with a maximal inhibition of Po (Emax) to 52 ± 4% of control only after adding physiologic [CaM] = 100 nM. Dantrolene inhibited RyR2 with an IC50 of 0.16 ± 0.03 µM. Mutant N98S-CaM facilitated dantrolene inhibition with an IC50 = 5.9 ± 0.3 nM. In mouse cardiomyocytes, dantrolene had no effect on cardiac Ca(2+) release in the absence of CaM, but reduced Ca(2+) wave frequency (IC50 = 0.42 ± 0.18 µM, Emax = 47 ± 4%) and amplitude (IC50 = 0.19 ± 0.04 µM, Emax = 66 ± 4%) in the presence of 100 nM CaM. We conclude that CaM is essential for dantrolene inhibition of RyR1 and RyR2. Its absence explains why dantrolene inhibition of single RyR channels has not been previously observed., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

Recent human imaging studies indicate that reduced blood flow through pial collateral vessels ('collateral failure') is associated with late infarct expansion despite stable arterial occlusion. The cause for 'collateral failure' is unknown. We recently showed that intracranial pressure (ICP) rises dramatically but transiently 24 hours after even minor experimental stroke. We hypothesized that ICP elevation would reduce collateral blood flow. First, we investigated the regulation of flow through collateral vessels and the penetrating arterioles arising from them during stroke reperfusion. Wistar rats were subjected to intraluminal middle cerebral artery (MCA) occlusion (MCAo). Individual pial collateral and associated penetrating arteriole blood flow was quantified using fluorescent microspheres. Baseline bidirectional flow changed to MCA-directed flow and increased by >450% immediately after MCAo. Collateral diameter changed minimally. Second, we determined the effect of ICP elevation on collateral and watershed penetrating arteriole flow. Intracranial pressure was artificially raised in stepwise increments during MCAo. The ICP increase was strongly correlated with collateral and penetrating arteriole flow reductions. Changes in collateral flow post-stroke appear to be primarily driven by the pressure drop across the collateral vessel, not vessel diameter. The ICP elevation reduces cerebral perfusion pressure and collateral flow, and is the possible explanation for 'collateral failure' in stroke-in-progression.

Transfer cell morphology is characterized by a polarized ingrowth wall comprising a uniform wall upon which wall ingrowth papillae develop at right angles into the cytoplasm. The hypothesis that positional information directing construction of wall ingrowth papillae is mediated by Ca(2+) signals generated by spatiotemporal alterations in cytosolic Ca(2+) ([Ca(2+)]cyt) of cells trans-differentiating to a transfer cell morphology was tested. This hypothesis was examined using Vicia faba cotyledons. On transferring cotyledons to culture, their adaxial epidermal cells synchronously trans-differentiate to epidermal transfer cells. A polarized and persistent Ca(2+) signal, generated during epidermal cell trans-differentiation, was found to co-localize with the site of ingrowth wall formation. Dampening Ca(2+) signal intensity, by withdrawing extracellular Ca(2+) or blocking Ca(2+) channel activity, inhibited formation of wall ingrowth papillae. Maintenance of Ca(2+) signal polarity and persistence depended upon a rapid turnover (minutes) of cytosolic Ca(2+) by co-operative functioning of plasma membrane Ca(2+)-permeable channels and Ca(2+)-ATPases. Viewed paradermally, and proximal to the cytosol-plasma membrane interface, the Ca(2+) signal was organized into discrete patches that aligned spatially with clusters of Ca(2+)-permeable channels. Mathematical modelling demonstrated that these patches of cytosolic Ca(2+) were consistent with inward-directed plumes of elevated [Ca(2+)]cyt. Plume formation depended upon an alternating distribution of Ca(2+)-permeable channels and Ca(2+)-ATPase clusters. On further inward diffusion, the Ca(2+) plumes coalesced into a uniform Ca(2+) signal. Blocking or dispersing the Ca(2+) plumes inhibited deposition of wall ingrowth papillae, while uniform wall formation remained unaltered. A working model envisages that cytosolic Ca(2+) plumes define the loci at which wall ingrowth papillae are deposited., (© The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Presented is a new power-efficient colour generation algorithm for wireless capsule endoscopy (WCE) application. In WCE, transmitting colour image data from the human intestine through radio frequency (RF) consumes a huge amount of power. The conventional way is to transmit all R, G and B components of all frames. Using the proposed dictionary-based colour generation scheme, instead of sending all R, G and B frames, first one colour frame is sent followed by a series of grey-scale frames. At the receiver end, the colour information is extracted from the colour frame and then added to colourise the grey-scale frames. After a certain number of grey-scale frames, another colour frame is sent followed by the same number of grey-scale frames. This process is repeated until the end of the video sequence to maintain the colour similarity. As a result, over 50% of RF transmission power can be saved using the proposed scheme, which will eventually lead to a battery life extension of the capsule by 4-7 h. The reproduced colour images have been evaluated both statistically and subjectively by professional gastroenterologists. The algorithm is finally implemented using a WCE prototype and the performance is validated using an ex-vivo trial.

Modern endoscopes play an important role in diagnosing various gastrointestinal (GI) tract related diseases. The improved visual quality of endoscopic images can provide better diagnosis. This paper presents an efficient color image enhancement method for endoscopic images. It is achieved in two stages: image enhancement at gray level followed by space variant chrominance mapping color reproduction. Image enhancement is achieved by performing adaptive sigmoid function and uniform distribution of sigmoid pixels. Secondly, a space variant chrominance mapping color reproduction is used to generate new chrominance components. The proposed method is used on low contrast color white light images (WLI) to enhance and highlight the vascular and mucosa structures of the GI tract. The method is also used to colorize grayscale narrow band images (NBI) and video frames. The focus value and color enhancement factor show that the enhancement level in the processed image is greatly increased compared to the original endoscopic image. The overall contrast level of the processed image is higher than the original image. The color similarity test has proved that the proposed method does not add any additional color which is not present in the original image. The algorithm has low complexity with an execution speed faster than other related methods.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) causes sudden cardiac death due to mutations in cardiac ryanodine receptors (RyR2), calsequestrin, or calmodulin. Flecainide, a class I antiarrhythmic drug, inhibits Na(+) and RyR2 channels and prevents CPVT. The purpose of this study is to identify inhibitory mechanisms of flecainide on RyR2. RyR2 were isolated from sheep heart, incorporated into lipid bilayers, and investigated by single-channel recording under various activating conditions, including the presence of cytoplasmic ATP (2 mM) and a range of cytoplasmic [Ca(2+)], [Mg(2+)], pH, and [caffeine]. Flecainide applied to either the cytoplasmic or luminal sides of the membrane inhibited RyR2 by two distinct modes: 1) a fast block consisting of brief substate and closed events with a mean duration of ∼1 ms, and 2) a slow block consisting of closed events with a mean duration of ∼1 second. Both inhibition modes were alleviated by increasing cytoplasmic pH from 7.4 to 9.5 but were unaffected by luminal pH. The slow block was potentiated in RyR2 channels that had relatively low open probability, whereas the fast block was unaffected by RyR2 activation. These results show that these two modes are independent mechanisms for RyR2 inhibition, both having a cytoplasmic site of action. The slow mode is a closed-channel block, whereas the fast mode blocks RyR2 in the open state. At diastolic cytoplasmic [Ca(2+)] (100 nM), flecainide possesses an additional inhibitory mechanism that reduces RyR2 burst duration. Hence, multiple modes of action underlie RyR2 inhibition by flecainide., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.)

Regulation of the cardiac ryanodine receptor (RyR2) by intracellular Ca(2+) and Mg(2+) plays a key role in determining cardiac contraction and rhythmicity, but their role in regulating the human RyR2 remains poorly defined. The Ca(2+)- and Mg(2+)-dependent regulation of human RyR2 was recorded in artificial lipid bilayers in the presence of 2 mM ATP and compared with that in two commonly used animal models for RyR2 function (rat and sheep). Human RyR2 displayed cytoplasmic Ca(2+) activation (K(a) = 4 µM) and inhibition by cytoplasmic Mg(2+) (K(i) = 10 µM at 100 nM Ca(2+)) that was similar to RyR2 from rat and sheep obtained under the same experimental conditions. However, in the presence of 0.1 mM Ca(2+), RyR2s from human were 3.5-fold less sensitive to cytoplasmic Mg(2+) inhibition than those from sheep and rat. The K(a) values for luminal Ca(2+) activation were similar in the three species (35 µM for human, 12 µM for sheep, and 10 µM for rat). From the relationship between open probability and luminal [Ca(2+)], the peak open probability for the human RyR2 was approximately the same as that for sheep, and both were ~10-fold greater than that for rat RyR2. Human RyR2 also showed the same sensitivity to luminal Mg(2+) as that from sheep, whereas rat RyR2 was 10-fold more sensitive. In all species, modulation of RyR2 gating by luminal Ca(2+) and Mg(2+) only occurred when cytoplasmic [Ca(2+)] was <3 µM. The activation response of RyR2 to luminal and cytoplasmic Ca(2+) was strongly dependent on the Mg(2+) concentration. Addition of physiological levels (1 mM) of Mg(2+) raised the K(a) for cytoplasmic Ca(2+) to 30 µM (human and sheep) or 90 µM (rat) and raised the K(a) for luminal Ca(2+) to ~1 mM in all species. This is the first report of the regulation by Ca(2+) and Mg(2+) of native RyR2 receptor activity from healthy human hearts., (© 2014 Walweel et al.)

Background: In mammals, lymph is propelled centrally primarily via the phasic contractions of collecting lymphatic vessels, known as lymphatic pumping. Electrophysiological studies conducted in guinea pig and sheep mesenteric lymphatic vessels indicate that contractions are initiated in the lymphatic muscle by nifedipine-sensitive action potentials (APs). Lymphatic pumping is highly sensitive to luminal fluid loading and the mechanical properties of this stretch-induced pumping have been consistently studied, in particular in rat mesenteric lymphatic vessels. However, membrane potential (Vm) and the electrophysiological events underlying stretch-induced lymphatic pumping have not been investigated in the rat. The aim of this study was thus to examine the properties of rat mesenteric lymphatic muscle Vm under resting conditions and to assess changes in Vm caused by distension., Methods and Results: Lymphatic muscle Vm was measured with sharp intracellular microelectrodes either in unstretched conditions or under isometric tension provided by a wire-myograph. In unstretched vessels, Vm was -48 ± 2 mV (n=30). APs (amplitude ∼25 mV) were observed at a frequency of ∼8/min and were abolished by nifedipine. Under isometric tension, Vm was less polarized (-36 ± 1 mV, n=23), even at minimum tension. Increase in tension led to increase in contraction strength and contraction/AP frequency, while Vm was slightly hyperpolarized and AP amplitude not markedly altered., Conclusions: In our experimental conditions, rat lymphatic muscle has electrophysiological characteristics similar to that in other species. It responds to an increase in isometric tension with an increase in AP frequency, but resting Vm is not significantly affected.

Background: This study examines the use of topical pharmacological agents as a snakebite first aid where slowing venom reaching the circulation prevents systemic toxicity. It is based on the fact that toxin molecules in most snake venoms are large molecules and generally first enter and traverse the lymphatic system before accessing the circulation. It follows on from a previous study where it was shown that topical application of a nitric oxide donor slowed lymph flow to a similar extent in humans and rats as well as increased the time to respiratory arrest for subcutaneous injection of an elapid venom (Pseudonaja textilis, Ptx; Eastern brown snake) into the hind feet of anaesthetized rats., Methodology/principal Findings: The effects of topical application of the L-type Ca(2+) channel antagonist nifedipine and the local anesthetic lignocaine in inhibiting lymph flow and protecting against envenomation was examined in an anaesthetized rat model. The agents significantly increased dye-measured lymph transit times by 500% and 390% compared to controls and increased the time to respiratory arrest to foot injection of a lethal dose of Ptx venom by 60% and 40% respectively. The study also examined the effect of Ptx venom dose over the lethal range of 0.4 to 1.5 mg/kg finding a negative linear relationship between increase in venom dose and time to respiratory arrest., Conclusions/significance: The findings suggest that a range of agents that inhibit lymphatic flow could potentially be used as an adjunct treatment to pressure bandaging with immobilization (PBI) in snakebite first aid. This is important given that PBI (a snakebite first aid recommended by the Australian National Health and Medical research Council) is often incorrectly applied. The use of a local anesthetic would have the added advantage of reducing pain.

This paper presents an image enhancement and space-variant color reproduction method based on adaptive sigmoid function for endoscopic image. At first, using YCBCR conversion matrix, the color image is separated into luminance and chrominance components. The adaptive sigmoid function with two controlling parameters is applied on the uniformly distributed luminance pixels. The space-variant color reproduction generates new chrominance components by transferring and modifying old chrominance based on texture information. Finally, new luminance and chrominance components are converted into RGB color image. The proposed method highlights some of the tissue and vascular characteristics as well as pit patterns in lesion and polyp. The performance of the proposed scheme is compared with other related methods in terms of image quality, focus value, efficiency of color reproduction and statistic of visual representation.

The factors responsible for the regulation of regenerative calcium-induced calcium release (CICR) during Ca(2+) spark evolution remain unclear. Cardiac ryanodine receptor (RyR) gating in rats and sheep was recorded at physiological Ca(2+), Mg(2+), and ATP levels and incorporated into a 3D model of the cardiac dyad, which reproduced the time course of Ca(2+) sparks, Ca(2+) blinks, and Ca(2+) spark restitution. The termination of CICR by induction decay in the model principally arose from the steep Ca(2+) dependence of RyR closed time, with the measured sarcoplasmic reticulum (SR) lumen Ca(2+) dependence of RyR gating making almost no contribution. The start of CICR termination was strongly dependent on the extent of local depletion of junctional SR Ca(2+), as well as the time course of local Ca(2+) gradients within the junctional space. Reducing the dimensions of the dyad junction reduced Ca(2+) spark amplitude by reducing the strength of regenerative feedback within CICR. A refractory period for Ca(2+) spark initiation and subsequent Ca(2+) spark amplitude restitution arose from 1), the extent to which the regenerative phase of CICR can be supported by the partially depleted junctional SR, and 2), the availability of releasable Ca(2+) in the junctional SR. The physical organization of RyRs within the junctional space had minimal effects on Ca(2+) spark amplitude when more than nine RyRs were present. Spark amplitude had a nonlinear dependence on RyR single-channel Ca(2+) flux, and was approximately halved by reducing the flux from 0.6 to 0.2 pA. Although rat and sheep RyRs had quite different Ca(2+) sensitivities, Ca(2+) spark amplitude was hardly affected. This suggests that moderate changes in RyR gating by second-messenger systems will principally alter the spatiotemporal properties of SR release, with smaller effects on the amount released., (Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Calcium-induced calcium release (CICR) is an inherently regenerative process due to the Ca(2+)-dependent gating of ryanodine receptors (RyRs) in the sarco/endoplasmic reticulum (SR) and is critical for cardiac excitation-contraction coupling. This process is seen as Ca(2+) sparks, which reflect the concerted gating of groups of RyRs in the dyad, a specialised junctional signalling domain between the SR and surface membrane. However, the mechanism(s) responsible for the termination of regenerative CICR during the evolution of Ca(2+) sparks remain uncertain. Rat cardiac RyR gating was recorded at physiological Ca(2+), Mg(2+) and ATP levels and incorporated into a 3D model of the cardiac dyad which reproduced the time-course of Ca(2+) sparks, Ca(2+) blinks and Ca(2+) spark restitution. Model CICR termination was robust, relatively insensitive to the number of dyadic RyRs and automatic. This emergent behaviour arose from the rapid development and dissolution of nanoscopic Ca(2+) gradients within the dyad. These simulations show that CICR does not require intrinsic inactivation or SR calcium sensing mechanisms for stability and cessation of regeneration that arises from local control at the molecular scale via a process we call 'induction decay'., (Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.)

Here we investigate how ß-adrenergic stimulation of the heart alters regulation of ryanodine receptors (RyRs) by intracellular Ca(2+) and Mg(2+) and the role of these changes in SR Ca(2+) release. RyRs were isolated from rat hearts, perfused in a Langendorff apparatus for 5 min and subject to 1 min perfusion with 1 µM isoproterenol or without (control) and snap frozen in liquid N2 to capture their phosphorylation state. Western Blots show that RyR2 phosphorylation was increased by isoproterenol, confirming that RyR2 were subject to normal ß-adrenergic signaling. Under basal conditions, S2808 and S2814 had phosphorylation levels of 69% and 15%, respectively. These levels were increased to 83% and 60%, respectively, after 60 s of ß-adrenergic stimulation consistent with other reports that ß-adrenergic stimulation of the heart can phosphorylate RyRs at specific residues including S2808 and S2814 causing an increase in RyR activity. At cytoplasmic [Ca(2+)] <1 µM, ß-adrenergic stimulation increased luminal Ca(2+) activation of single RyR channels, decreased luminal Mg(2+) inhibition and decreased inhibition of RyRs by mM cytoplasmic Mg(2+). At cytoplasmic [Ca(2+)] >1 µM, ß-adrenergic stimulation only decreased cytoplasmic Mg(2+) and Ca(2+) inhibition of RyRs. The Ka and maximum levels of cytoplasmic Ca(2+) activation site were not affected by ß-adrenergic stimulation. Our RyR2 gating model was fitted to the single channel data. It predicted that in diastole, ß-adrenergic stimulation is mediated by 1) increasing the activating potency of Ca(2+) binding to the luminal Ca(2+) site and decreasing its affinity for luminal Mg(2+) and 2) decreasing affinity of the low-affinity Ca(2+)/Mg(2+) cytoplasmic inhibition site. However in systole, ß-adrenergic stimulation is mediated mainly by the latter.

Lymphatic vessels serve as a route by which interstitial fluid, protein and other macromolecules are returned to the blood circulation and immune cells and antigens gain access to lymph nodes. Lymph flow is an active process promoted by rhythmical contraction-relaxation events occurring in the collecting lymphatic vessels. This lymphatic pumping is an intrinsic property of the lymphatic muscles in the vessel wall and consequent to action potentials. Compromised lymphatic pumping may affect lymph and immune cell transport, an action which could be particularly detrimental during inflammation. Importantly, many inflammatory mediators alter lymphatic pumping. Vasoactive intestinal peptide (VIP) is a neuro- and immuno-modulator thought to be released by nerve terminals and immune cells in close proximity to lymphatic vessels. We demonstrated the presence of the peptide in lymphatic vessels and in the lymph and examined the effects of VIP on mesenteric collecting lymphatic vessels of the guinea pig using pharmacological bioassays, intracellular microelectrode electrophysiology, immunofluorescence and quantitative real-time PCR. We showed that VIP alters lymphatic pumping by decreasing the frequency of lymphatic contractions and hyperpolarizing the lymphatic muscle membrane potential in a concentration-dependent manner. Our data further suggest that these effects are mainly mediated by stimulation of the VIP receptor VPAC2 located on the lymphatic muscle and the downstream involvement of protein kinase A (PKA) and ATP-sensitive K⁺ (KATP) channels. Inhibition of lymphatic pumping by VIP may compromise lymph drainage, oedema resolution and immune cell trafficking to the draining lymph nodes.

Calcium plays important role in biological systems where it is involved in diverse mechanisms such as signaling, muscle contraction and neuromodulation. Action potentials are generated by dynamic interaction of ionic channels located on the plasma-membrane and these drive the rhythmic activity of biological systems such as the smooth muscle and the heart. However, ionic channels are not the only pacemakers; an intimate interaction between intracellular Ca(2+) stores and ionic channels underlie rhythmic activity. In this review we will focus on the role of Ca(2+) stores in regulation of rhythmical behavior.

We have characterized the currents that flow during the interspike interval in mouse locus coeruleus (LC) neurons, by application of depolarizing ramps and pulses, and compared our results with information available for rats. A tetrodotoxin (TTX)-sensitive current was the only inward conductance active during the interspike interval; no TTX-insensitive Na(+) or oscillatory currents were detected. Ca(2+)-free and Ba(2+)-containing solutions failed to demonstrate a Ca(2+) current during the interspike interval, although a Ca(2+) current was activated at membrane potentials positive to -40 mV. A high- tetraethylammonium chloride (TEA) (15 mM) sensitive current accounted for almost all the K(+) conductance during the interspike interval. Ca(2+)-activated K(+), inward rectifier and low-TEA (10 muM) sensitive currents were not detected within the interspike interval. Comparison of these findings to those reported for neonatal rat LC neurons indicates that the pacemaker currents are similar, but not identical, in the two species with mice lacking a persistent Ca(2+) current during the interspike interval. The net pacemaking current determined by differentiating the interspike interval from averaged action potential recordings closely matched the net ramp-induced currents obtained either under voltage clamp or after reconstructing this current from pharmacologically isolated currents. In summary, our results suggest the interspike interval pacemaker mechanism in mouse LC neurons involves a combination of a TTX-sensitive Na(+) current and a high TEA-sensitive K(+) current. In contrast with rats, a persistent Ca(2+) current is not involved., (Copyright 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

This study presents a theoretical analysis of the role of store Ca(2+) uptake on sinoatrial node (SAN) cell pacemaking. Two mechanisms have been shown to be involved in SAN pacemaking, these being: 1) the membrane oscillator model where rhythm generation is based on the interaction of voltage-dependent membrane ion channels and, 2) the store oscillator model where cyclical release of Ca(2+) from intracellular Ca(2+) stores depolarizes the membrane through activation of the sodium-calcium exchanger (NCX). The relative roles of these oscillators in generation and modulation of pacemaker rate have been vigorously debated and have many consequences. The main new outcomes of our study are: 1) uptake of Ca(2+) by intracellular Ca(2+) stores increases the maximum diastolic potential (MDP) by reducing the cytosolic Ca(2+) concentration [Ca(2+)](c) and hence decreasing the NCX current; 2) this hyperpolarization enhances recruitment of key pacemaker currents (e.g. the hyperpolarization-activated HCN current (I(f)) and T-type Ca(2+) current (I(T-Ca))); 3) the resultant enhanced Ca(2+) entry during the pacemaker depolarization increases [Ca(2+)](c) causing advancement of the store Ca(2+) release cycle and increased NCX current. In overview, the novel feature of our study is an investigation of the role of store Ca(2+) uptake on SAN pacemaking. This occurs during the early diastolic period and causes enhanced I(f), I(T-Ca) and store release (and hence I(NCX)) during the later diastolic period. There is thus a symbiotic interaction between the two pacemaker "clocks" over the entire diastolic period, this providing robust and highly malleable SAN pacemaking. Accounting for store Ca(2+) uptake also provides insight into hitherto unexplained SAN behaviour, as we exemplify for the sinus bradycardia exhibited in catecholaminergic polymorphic ventricular tachycardia (CPVT)., (Copyright 2010 Elsevier Ltd. All rights reserved.)

The dissociative anesthetic ketamine that acts as an N-methyl-D-aspartate (NMDA) antagonist has been reported to improve neurological damage after experimental ischemic challenges. Here we show that deep anesthesia with ketamine before euthanasia by decapitation improves the quality of neonatal mouse neuronal brain slice preparations. Specifically we found that neurons of the locus coeruleus (LC) and hypoglossal motor neurons had significantly higher input resistances, and LC neurons that generally are difficult to voltage control, could be more reliably voltage clamped compared to control neurons., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

1. Mechanisms underlying the generation and propagation of gastrointestinal slow wave depolarizations have long been controversial. The present review aims to collate present knowledge on this subject with specific reference to slow waves in gastric smooth muscle. 2. At present, there is strong agreement that interstitial cells of Cajal (ICC) are the pacemaker cells that generate slow waves. What has been less clear is the relative role of primary types of ICC, including the network in the myenteric plexus (ICC-MY) and the intramuscular network (ICC-IM). It is concluded that both ICC-MY and ICC-IM are likely to serve a major role in slow wave generation and propagation. 3. There has been long-standing controversy as to how slow waves 'propagate' circumferentially and down the gastrointestinal tract. Two mechanisms have been proposed, one being action potential (AP)-like conduction and the other phase wave-based 'propagation' resulting from an interaction of coupled oscillators. Studies made on single bundle gastric strips indicate that both mechanisms apply with relative dominance depending on conditions; the phase wave mechanism is dominant under circumstances of rhythmically generating slow waves and the AP-like propagation is dominant when the system is perturbed. 4. The phase wave mechanism (termed Ca(2+) phase wave) uses cyclical Ca(2+) release as the oscillator, with coupling between oscillators mediated by several factors, including: (i) store-induced depolarization; (ii) resultant electrical current flow/depolarization through the pacemaker cell network; and (iii) depolarization-induced increase in excitability of downstream Ca(2+) stores. An analogy is provided by pendulums in an array coupled together by a network of springs. These, when randomly activated, entrain to swing at the same frequency but with a relative delay along the row giving the impression of a propagating wave. 5. The AP-like mechanism (termed voltage-accelerated Ca(2+) wave) propagates sequentially like a conducting AP. However, it is different in that it depends on regenerative store Ca(2+) release and resultant depolarization rather than regenerative activation of voltage-dependent channels in the cell membrane. 6. The applicability of these mechanisms to describing propagation in large intact gastrointestinal tissues, where voltage-dependent Ca(2+) entry is also likely to be functional, is discussed.

Entrained oscillations in Ca(2+) underlie many biological pacemaking phenomena. In this article, we review a long-range signaling mechanism in smooth muscle that results in global outcomes of local interactions. Our results are derived from studies of the following: (a) slow-wave depolarizations that underlie rhythmic contractions of gastric smooth muscle; and (b) membrane depolarizations that drive rhythmic contractions of lymphatic smooth muscle. The main feature of this signaling mechanism is a coupled oscillator-based synchronization of Ca(2+) oscillations across cells that drives membrane potential changes and causes coordinated contractions. The key elements of this mechanism are as follows: (a) the Ca(2+) release-refill cycle of endoplasmic reticulum Ca(2+) stores; (b) Ca(2+)-dependent modulation of membrane currents; (c) voltage-dependent modulation of Ca(2+) store release; and (d) cell-cell coupling through gap junctions or other mechanisms. In this mechanism, Ca(2+) stores alter the frequency of adjacent stores through voltage-dependent modulation of store release. This electrochemical coupling is many orders of magnitude stronger than the coupling through diffusion of Ca(2+) or inositol 1,4,5-trisphosphate, and thus provides an effective means of long-range signaling.

Guinea pig mesenteric lymphatic vessels exhibit rhythmic constrictions induced by action potential (AP)-like spikes and initiated by entrainment of spontaneous transient depolarizations (STDs). To characterize STDs and the signaling mechanisms responsible for their occurrence, we used intracellular microelectrodes, Ca2+ imaging, and pharmacological agents. In our investigation of the role of intracellular Ca2+ released from Ca2+ stores, we observed that intracellular Ca2+ transients accompanied some STDs, although there were many exceptions where Ca2+ transients occurred without accompanying STDs. STD frequency and amplitude were markedly affected by activators/inhibitors of inositol 1,4,5-trisphosphate receptors (IP3Rs) but not by treatments known to alter Ca2+ release via ryanodine receptors. A role for Ca2+-activated Cl(-) (Cl(Ca)) channels was indicated, as STDs were dependent on the Cl(-) but not Na+ concentration of the superfusing solution and were inhibited by the Cl(Ca) channel blockers niflumic acid (NFA), anthracene 9-carboxylic acid, and 5-nitro-2-(3-phenylpropylamino)benzoic acid but not by the volume-regulated Cl(-) blocker DIDS. Increases in STD frequency and amplitude induced by agonist stimulation were also inhibited by NFA. Nifedipine, the hyperpolarization-activated inward current blocker ZD-7288, and the nonselective cation/store-operated channel blockers SKF-96365, Gd3+, and Ni2+ had no or marginal effects on STD activity. However, nifedipine, 2-aminoethoxydiphenyl borate, NFA, SKF-96365, Gd3+, and Ni2+ altered the occurrence of spontaneous APs. Our findings support a role for Ca2+ release through IP3Rs and a resultant opening of Cl(Ca) channels in STD generation and confirm the importance of these events in the initiation of lymphatic spontaneous APs and subsequent contractions. The abolition of spontaneous APs by blockers of other excitatory ion channels suggests a contribution of these conductances to lymphatic pacemaking.

This study presents an investigation of pacemaker mechanisms underlying lymphatic vasomotion. We tested the hypothesis that active inositol 1,4,5-trisphosphate receptor (IP(3)R)-operated Ca(2+) stores interact as coupled oscillators to produce near-synchronous Ca(2+) release events and associated pacemaker potentials, this driving action potentials and constrictions of lymphatic smooth muscle. Application of endothelin 1 (ET-1), an agonist known to enhance synthesis of IP(3), to quiescent lymphatic smooth muscle syncytia first enhanced spontaneous Ca(2+) transients and/or intracellular Ca(2+) waves. Larger near-synchronous Ca(2+) transients then occurred leading to global synchronous Ca(2+) transients associated with action potentials and resultant vasomotion. In contrast, blockade of L-type Ca(2+) channels with nifedipine prevented ET-1 from inducing near-synchronous Ca(2+) transients and resultant action potentials, leaving only asynchronous Ca(2+) transients and local Ca(2+) waves. These data were well simulated by a model of lymphatic smooth muscle with: 1), oscillatory Ca(2+) release from IP(3)R-operated Ca(2+) stores, which causes depolarization; 2), L-type Ca(2+) channels; and 3), gap junctions between cells. Stimulation of the stores caused global pacemaker activity through coupled oscillator-based entrainment of the stores. Membrane potential changes and positive feedback by L-type Ca(2+) channels to produce more store activity were fundamental to this process providing long-range electrochemical coupling between the Ca(2+) store oscillators. We conclude that lymphatic pacemaking is mediated by coupled oscillator-based interactions between active Ca(2+) stores. These are weakly coupled by inter- and intracellular diffusion of store activators and strongly coupled by membrane potential. Ca(2+) store-based pacemaking is predicted for cellular systems where: 1), oscillatory Ca(2+) release induces depolarization; 2), membrane depolarization provides positive feedback to induce further store Ca(2+) release; and 3), cells are interconnected. These conditions are met in a surprisingly large number of cellular systems including gastrointestinal, lymphatic, urethral, and vascular tissues, and in heart pacemaker cells.

The effects of calcitonin gene-related peptide (CGRP) on constriction frequency, smooth muscle membrane potential (V(m)), and endothelial V(m) of guinea pig mesenteric lymphatics were examined in vitro. CGRP (1-100 nM) caused an endothelium-dependent decrease in the constriction frequency of perfused lymphatic vessels. The endothelium-dependent CGRP response was abolished by the CGRP-1 receptor antagonist CGRP-(8-37) (1 microM) and pertussis toxin (100 ng/ml). This action of CGRP was also blocked by the nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine (L-NNA; 10 microM), an action that was reversed by the addition of L-arginine (100 microM). cGMP, adenylate cyclase, cAMP-dependent protein kinase (PKA), and ATP-sensitive K+ (K+(ATP)) channels were all implicated in the endothelium-dependent CGRP response because it was abolished by methylene blue (20 microM), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (10 microM), dideoxyadenosine (10 microM), N-[2-(p-bromociannamylamino)-ethyl]-5-isoquinolinesulfonamide-dichloride (H89; 1 microM) and glibenclamide (10 microM). CGRP (100 nM), unlike acetylcholine, did not alter endothelial intracellular Ca2+ concentration or V(m). CGRP (100 nM) hyperpolarized the smooth muscle V(m), an effect inhibited by L-NNA, H89, or glibenclamide. CGRP (500 nM) also caused a decrease in constriction frequency. However, this was no longer blocked by CGRP-(8-37). CGRP (500 nM) also caused smooth muscle hyperpolarization, an action that was now not blocked by L-NNA (100 microM). It was most likely mediated by the activation of the cAMP/PKA pathway and the opening of K+(ATP) channels because it was abolished by H89 or glibenclamide. We conclude that CGRP, at low to moderate concentrations (i.e., 1-100 nM), decreases lymphatic constriction frequency primarily by the stimulation of CGRP-1 receptors coupled to pertussis toxin-sensitive G proteins and the release of NO from the endothelium or enhancement of the actions of endogenous NO. At high concentrations (i.e., 500 nM), CGRP also directly activates the smooth muscle independent of NO. Both mechanisms of activation ultimately cause the PKA-mediated opening of K+(ATP) channels and resultant hyperpolarization.

Many blood and lymphatic vessels undergo spontaneous rhythmical constrictions. Such activity is an intrinsic property of the smooth muscle in the walls of these vessels and is induced or enhanced by a wide range of activators including pressure-induced distension and sympathetic neurotransmitters in both blood vessels and lymphatic vessels. This review considers present understanding of vasomotion.

Slow waves are rhythmic depolarizations that underlie mechanical activity of many smooth muscles. Slow waves result through rhythmic Ca(2+) release from intracellular Ca(2+) stores through inositol 1,4,5-trisphosphate (IP(3)) sensitive receptors and Ca(2+)-induced Ca(2+) release. Ca(2+) oscillations are transformed into membrane depolarizations by generation of a Ca(2+)-activated inward current. Importantly, the store Ca(2+) oscillations that underlie slow waves are entrained across many cells over large distances. It has been shown that IP(3) receptor-mediated Ca(2+) release is enhanced by membrane depolarization. Previous studies have implicated diffusion of Ca(2+) or the second messenger IP(3) across gap junctions in synchronization of Ca(2+) oscillations. In this study, a novel mechanism of Ca(2+) store entrainment through depolarization-induced IP(3) receptor-mediated Ca(2+) release is investigated. This mechanism is significantly different from chemical coupling-based mechanisms, as membrane potential has a coupling effect over distances several orders of magnitude greater than either diffusion of Ca(2+) or IP(3) through gap junctions. It is shown that electrical coupling acting through voltage-dependent modulation of store Ca(2+) release is able to synchronize oscillations of cells even when cells are widely separated and have different intrinsic frequencies of oscillation.

Ca2+ imaging and multiple microelectrode recording procedures were used to investigate a slow wave-like electrical rhythmicity in single bundle strips from the circular muscle layer of the guinea-pig gastric pylorus. The 'slow waves' (SWs) consisted of a pacemaker and regenerative component, with both potentials composed of more elementary events variously termed spontaneous transient depolarizations (STDs) or unitary potentials. STDs and SW pacemaker and regenerative potentials exhibited associated local and distributed Ca2+ transients, respectively. Ca2+ transients were often larger in cellular regions that exhibited higher basal Ca2+ indicator-associated fluorescence, typical of regions likely to contain intramuscular interstitial cells of Cajal (ICCIM). The emergence of rhythmicity arose through entrainment of STDs resulting in pacemaker Ca2+ transients and potentials, events that exhibited considerable spatial synchronicity. Application of ACh to strips exhibiting weak rhythmicity caused marked enhancement of SW synchronicity. SWs and underlying Ca2+ increases exhibited very high 'apparent conduction velocities' ('CVs') orders of magnitude greater than for sequentially conducting Ca2+ waves. Central interruption of either intercellular connectivity or inositol 1,4,5-trisphosphate receptor (IP3R)-mediated store Ca2+ release in strips caused SWs at the two ends to run independently of each other, consistent with a coupled oscillator-based mechanism. Central inhibition of stores required much wider regions of blockade than inhibition of connectivity indicating that stores were voltage-coupled. Simulations, made using a conventional store array model but now including depolarization coupled to IP3R-mediated Ca2+ release, predicted the experimental findings. The linkage between membrane voltage and Ca2+ release provides a means for stores to interact as strongly coupled oscillators, resulting in the emergence of Ca2+ phase waves and associated pacemaker potentials. This distributed pacemaker triggers regenerative Ca2+ release and resultant SWs.

A qualitative mathematical model is presented that examines membrane potential feedback on synthesis of inositol 1,4,5-trisphosphate (IP(3)), and its role in generation and modulation of slow waves. Previous experimental studies indicate that slow waves show voltage dependence, and this is likely to result through membrane potential modulation of IP(3). It is proposed that the observed response of the tissue to current pulse, pulse train, and maintained current injection can be explained by changes in IP(3), modulated through a voltage-IP(3) feedback loop. Differences underlying the tissue responses to current injections of opposite polarities are shown to be due to the sequence of events following such currents. Results from this model are consistent with experimental findings and provide further understanding of these experimental observations. Specifically, we find that membrane potential can induce, abolish, and modulate slow wave frequency by altering the excitability of the tissue through the voltage-IP(3) feedback loop.

1. Intracellular recordings made in single bundle strips of a visceral smooth muscle revealed rhythmic spontaneous membrane depolarizations termed slow waves (SWs). These exhibited 'pacemaker' and 'regenerative' components composed of summations of more elementary events termed spontaneous transient depolarizations (STDs). 2. STDs and SWs persisted in the presence of tetrodotoxin, nifedipine and ryanodine, and upon brief exposure to Ca2+-free Cd2+-containing solutions; they were enhanced by ACh and blocked by BAPTA AM, cyclopiazonic acid and caffeine. 3. SWs were also inhibited in heparin-loaded strips. SWs were observed over a wide range of membrane potentials (e.g. -80 to -45 mV) with increased frequencies at more depolarized potentials. 4. Regular spontaneous SW activity in this preparation began after 1-3 h superfusion of the tissue with physiological saline following the dissection procedure. Membrane depolarization applied before the onset of this activity induced bursts of STD-like events (termed the 'initial' response) which, when larger than threshold levels initiated regenerative responses. The combined initial-regenerative waveform was termed the SW-like action potential. 5. Voltage-induced responses exhibited large variable latencies (typical range 0.3-4 s), refractory periods of approximately 11 s and a pharmacology that was indistinguishable from those of STDs and spontaneous SWs. 6. The data indicate that SWs arise through more elementary inositol 1,4,5-trisphosphate (IP3) receptor-induced Ca2+ release events which rhythmically synchronize to trigger regenerative Ca2+ release and induce inward current across the plasmalemma. The finding that action potentials, which were indistinguishable from SWs, could be evoked by depolarization suggests that membrane potential modulates IP3 production. Voltage feedback on intracellular IP3-sensitive Ca2+ release is likely to have a major influence on the generation and propagation of SWs.