Experimental data manipulations to assess performance of hyperspectral classification models of crop seeds and other objects.

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.

Hypoxia driven opioid targeted automated device for overdose rescue.

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.

Venous Vasomotion.

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 (Ca2+) release-refill cycle of Ca2+ stores. This occurs through opening of inositol 1,4,5-trisphosphate receptor (IP3R)- and/or ryanodine receptor (RyR)-operated Ca2+ release channels in the sarcoplasmic/endoplasmic (SR/ER) reticulum and refilling by the SR/ER reticulum Ca2+ATPase (SERCA). Released Ca2+ from stores near the plasma membrane diffuse through the cytosol to open Ca2+-activated chloride (Cl-) channels, this generating inward current through an efflux of Cl-. The resultant depolarisation leads to the opening of voltage-dependent Ca2+ channels and possibly increased production of IP3, which through Ca2+-induced Ca2+ release (CICR) of IP3Rs and/or RyRs and IP3R-mediated Ca2+ 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 Ca2+ 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.

Tri-Scan: A Three Stage Color Enhancement Tool for Endoscopic Images.

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.

Heritability of ECG Biomarkers in the Netherlands Twin Registry Measured from Holter ECGs.

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.

Color reproduction and processing algorithm based on real-time mapping for endoscopic images.

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.

In silico assessment of kinetics and state dependent binding properties of drugs causing acquired LQTS.

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.

Color Enhancement in Endoscopic Images Using Adaptive Sigmoid Function and Space Variant Color Reproduction.

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.

Essential Role of Calmodulin in RyR Inhibition by Dantrolene.

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.

Intracranial pressure elevation reduces flow through collateral vessels and the penetrating arterioles they supply. A possible explanation for 'collateral failure' and infarct expansion after ischemic stroke.

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.

Polarized and persistent Ca²âº plumes define loci for formation of wall ingrowth papillae in transfer cells.

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.

Differences in the regulation of RyR2 from human, sheep, and rat by Ca²âº and Mg²âº in the cytoplasm and in the lumen of the sarcoplasmic reticulum.

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.

Electrophysiological properties of rat mesenteric lymphatic vessels and their regulation by stretch.

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.

Pharmacological approaches that slow lymphatic flow as a snakebite first aid.

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.

ß-Adrenergic stimulation increases RyR2 activity via intracellular Ca2+ and Mg2+ regulation.

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.

Mechanisms of VIP-induced inhibition of the lymphatic vessel pump.

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 oscillations and pacemaking.

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.

Ketamine anesthesia helps preserve neuronal viability.

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.

SR Ca2+ store refill--a key factor in cardiac pacemaking.

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).

Synchronization of Ca2+ oscillations: a coupled oscillator-based mechanism in smooth muscle.

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.