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The research topic presented in this paper belongs to small training data problem in machine learning (especially in deep learning), it intends to help the work of those working in medicine by analyzing pathological X-ray recordings, using only very few images. This scenario is a particularly hot issue nowadays: How could a new disease for which only limited data are available be diagnosed using features of previous diseases? In this problem, so-called few-shot learning, the difficulty of the classification task is to learn the unique feature characteristics associated with the classes. Although there are solutions, but if the images come from different views, they will not handle these views well. We proposed an improved method, so-called Double-View Matching Network (DVMN based on the deep neural network), which solves the few-shot learning problem as well as the different views of the pathological recordings in the images. The main contribution of this is the convolutional neural network for feature extraction and handling the multi-view in image representation. Our method was tested in the classification of images showing unknown COVID-19 symptoms in an environment designed for learning a few samples, with prior meta-learning on images of other diseases only. The results show that DVMN reaches better accuracy on multi-view dataset than simple Matching Network without multi-view handling. © 2021 Scientific Association for Infocommunications. All rights reserved.

BackgroundSkin fibrosis is a cardinal feature of systemic sclerosis (SSc) and associated with significant disability. The early trajectory of skin fibrosis provides insights into the course of the disease including mortality; however, little is known about late skin fibrosis in SSc.ObjectivesThe aims of our study were to ascertain the prevalence and characteristics of late skin fibrosis in SSc.MethodsWe developed and tested three conceptual scenarios of late (>5 years after 1st non-RP sign or symptom) skin fibrosis (Figure 1):Figure 1.Conceptual models/scenarios of late skin fibrosis in SSc. A: worsening and then improvement (>3 mRSS) during the first 5 years, and then worsened again after 5 years. B: worsening for the first time after 5 years. C: worsening in the first 5 years and stayed high after 5 years (i.e., failure to improve).A. Worsening and then improvement (>3 mRSS) during the first 5 years, and then worsened again after 5 years.B. Worsening for the first time after 5 years.C. Worsening in the first 5 years and stayed high after 5 years (i.e., failure to improve).We defined skin worsening as modified Rodnan skin score (mRSS) ≥ 5 units or ≥ 25%. Using strict inclusion criteria including complete mRSS, we identified 1,043 (out of 19,115) patients within the EUSTAR database for our analysis. We further restricted analysis within 887 (out of 1,043) patients who had limited (lcSSc) or diffuse cutaneous SSc (dcSSc) at baseline.ResultsOne-fifth of patients among the whole cohort (n=208/1043, 19.9%) including in patients with lcSSc or dcSSc at baseline (n=193/887, 21.8%) developed late skin fibrosis. This was largely due to new skin worsening or failure to improve. Patients with lower baseline mRSS and lcSSc were more likely to develop late skin fibrosis. Anti-Scl-70 antibodies (Table 1) were associated with progression from baseline lcSSc to dcSSc, and anticentromere antibodies were protective.Table 1.Impact of autoantibody status on progression from baseline limited to diffuse cutaneous SSc (dcSSc).Skin worsening after 5 years (Scenario B) (n=70)Skin worsening within 5 years and failed to improve after 5-year window (Scenario C) (n=61)Progressed to dcSSc (n=23)Not progressed to dcSSc(n=47)P-valueProgressed to dcSSc (n=37)Not progressed to dcSSc(n=24)P-valueAnticentromere+ve2/22 (9.1%)19/42 (45.2%)0.00346/34 (17.6%)14/21 (66.7%)0.0002-ve20/22 (90.9%)23/42 (54.8%)28/34 (82.4%)7/21 (33.3%)Anti-Scl-70+ve15/23 (65.2%)14/44 (31.8%)0.008822/36 (61.1%)8/23 (34.8%)0.0485-ve8/23 (34.8%)30/44 (68.2%)14/36 (38.9%)15/23 (65.2%)Anti-RNA-Polymerase-III+ve0/12 (0.0%)1/22 (4.5%)1.00000/6 (0.0%)0/14 (0.0%)---ve12/12 (100%)21/22 (95.5%)6/6 (100%)14/14 (100%)ConclusionLate skin fibrosis affects approximately 20% of SSc patients >5 years after onset of disease. We have identified different patterns relevant to clinical practice and trial design. Late skin fibrosis is usually due to new worsening or failure of skin to improve. Progression from baseline limited to diffuse cutaneous SSc was associated with anti-Scl-70 antibodies, and anticentromere antibodies were protective. Late skin fibrosis is a neglected manifestation of SSc and warrants further investigation including to determine clinical outcomes and optimal therapeutic strategy.AcknowledgementsOn behalf of EUSTAR collaborators.Disclosure of InterestsMichael Hughes Speakers bureau: Speaking fees from Actelion pharmaceuticals, Eli Lilly, and Pfizer, outside of the submitted work, Suiyuan Huang: None declared, Juan Jose Alegre Sancho Speakers bureau: Speaking and/or investigational fees from Actelion pharmaceuticals, Eli Lilly, Pfizer, Boehringer Ingelheim, Roche, and GSK, outside of the submitted work, Grant/research support from: Speaking and/or investigational fees from Actelion pharmaceuticals, Eli Lilly, Pfizer, Boehringer Ingelheim, Roche, and GSK, outside of the submitted work, Patricia Carreira: None declared, Merete Engelhart: None declared, Eric Hachulla Speakers bureau: Received consulting fees/meeting fees from Johnson & Johnson, Boehringer Ingelheim, Bayer, GSK, Roche-Chugai, Sanofi-Genzyme; speaking fees from Johnson & Johnson, GSK, Roche-Chugai; and research funding from CSL Behring, GSK, Roche-Chugai and Johnson & Johnson., Consultant of: Received consulting fees/meeting fees from Johnson & Johnson, Boehringer Ingelheim, Bayer, GSK, Roche-Chugai, Sanofi-Genzyme; speaking fees from Johnson & Johnson, GSK, Roche-Chugai; and research funding from CSL Behring, GSK, Roche-Chugai and Johnson & Johnson., Jörg Henes Speakers bureau: Lectures for CHUGAI, Boehringer-Ingelheim, Eduardo Kerzberg: None declared, Maria Rosa Pozzi: None declared, Gabriela Riemekasten: None declared, Vanessa Smith: None declared, Gabriella Szucs: None declared, Marie Vanthuyne: None declared, Elisabetta Zanatta: None declared, Oliver Distler: None declared, Armando Gabrielli: None declared, Anna-Maria Hoffmann-Vold: None declared, Viginia Steen: None declared, Dinesh Khanna Shareholder of: DK has stock options in Eicos Sciences, Inc., Consultant of: Consultant for Acceleron, Amgen, Boehringer Ingelheim, CSL Behring, Chemomab, Genentech/Roche, Horizon, Mitsubishi Tanabe Pharma, Prometheus, Talaris., Grant/research support from: Has received grants from Bayer, BMS, Horizon and Pfizer (to University of Michigan).

Introduction The prevalence of chronic renal disease (CKD) is continuously increasing in developed countries. Uremic cardiomyopathy characterized by left ventricular hypertrophy (LVH) and diastolic dysfunction (DD) is a common cardiovascular complication of CKD. Cardiac microvascular low-grade inflammation and altered expression of endothelium derived Neuregulin-1 (NRG-1) are contributed to left ventricular DD. Our aim was to charachterize the effects of CKD on the expression of NRG-1 and 2) NRG-1 treatment on myocardial hypertrophy, diastolic dysfunction and renal function in the rat model of CKD. Methods Male Wistar rats were used and randomized into 3 groups: 1) Sham-operated,2) CKD induced by 5/6 nephrectomy (CKD) and 3) NRG-1-treated CKD group (CKD+NRG-1). In this group, 2 weeks after the CKD induction, the rats were treated with recombinant human NRG-1 (rhNRG-1) at the dose of 10 μg/kg/d for consecutive 10 days with tail vein injection of NRG-1. Serum and urea creatinine levels were measured to verify the development of CKD and transthoracic echocardiography was performed to monitor cardiac morphology and function. Furthermore, total RNA was isolated and RT-qPCR was performed to evaluate the expression levels of inflammatory chemokine and cytokines (TNF-α, TGF-β). In addition, NRG-1 protein levels were assessed in both kidney and heart tissue by ELISA. To clarify the underling anti-fibrotic mechanism, human ventricular cardiac fibroblasts (HCF) were cultured and treated with the TGF-β (20 ng/ml), and TGF-β + hrNRG-1 for 24 h, respectively. Confocal microscopy was used to detect α-smooth muscle actin (α-SMA) expression, marker for fibroblast to myofibroblast transtion. Results 10 weeks after the 5/6 nephrectomy, serum carbamide and creatinine levels were significantly increased and creatinine clearence was significantly decreased as compared to sham-operated animals proving the development of chronic kidney disease (CKD). This was accompanied by a significant decrease in NRG-1 protein expression levels in both cardiac and kidney tissue. Of note, NRG-1 treatment markedly reduced these changes, suggesting its renoprotective effects in CKD. In addition, In CKD animals, the significantly increased anterior, posterior and septal wall thicknesses with decreased end-diastolic and end-systolic diameters proved the development of concentric left ventricular hypertrophy. In CKD, the septal e' was significantly decreased and E/e' increased indicating the developemnt of diastolic dysfunction. These parameters were significantly improved by NRG-1 treatment. Mechanistically, NRG-1 treatment reduced the expression of inflammatory cytokines in compared to untreat group. Furthermore, TGF-β induced α-SMA and Col I upregulation was markedly reduced by hrNRG-1 treatment. Conclusions NRG-1 treatment improved both renal and cardiac funtion in CKD, via a mechansim including the anti-inflammatory and anti-fibrotic properties of NRG-1. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): Österreichischer Austauschdienst

Background: Heart failure (HF) is a burden to patients and health care systems. The objectives of HF treatment are to improve health related quality of life (HRQoL) and reduce mortality and morbidity. We aimed to evaluate determinants of health-related quality of life (HRQoL) in patients with iron deficiency and HF treated with intravenous (i.v.) iron substitution or placebo. Methods: A randomised, double-blind, placebo-controlled trial (n = 459) in iron-deficient chronic heart failure (CHF) patients with or without anaemia studied clinical and HRQoL benefits of i.v. iron substitution using ferric carboxymaltose (FCM) over a 24-week trial period. Multivariate analysis was carried out with various clinical variables as independent variables and HRQoL measures as dependent variables. Results: Mean change from baseline of European Quality of Life - 5 Dimensions (EQ-5D) (value set-based) utilities (on a 0 to 100 scale) at week 24 was 8.91 (i.v. iron) and 0.68 (placebo; p < 0.01). In a multivariate analysis excluding baseline HRQoL, a higher exercise tolerance and i.v. iron substitution positively influenced HRQoL, whereas impaired renal function and a history of stroke had a negative effect. The level of HRQoL was also influenced by country of residence. When baseline HRQoL was factored in, the multivariate model remained stable. Conclusion: In this study, i.v. iron substitution, exercise tolerance, stroke, country of residence and renal function influenced measures of HRQoL in patients with heart failure and iron deficiency. (C) 2013 The Authors. Published by Elsevier Ireland Ltd. All rights reserved.

Background and Aims The effect of red palm oil (RPO) supplementation on infarct size after ischaemia/reperfusion in a cholesterol enriched diet-induced hyperlipidemic animal model has not been reported. Previous studies reported results on the effect of RPO in a normal diet, whilst evidence of protection has been linked to improved functional recovery, prosurvival kinase, anti-apoptosis and NO-cGMP. Therefore, we aimed to investigate the effects of dietary RPO supplementation in a cholesterol-enriched diet-induced hyperlipidemic rat model and to investigate the involvement of matrix metalloproteinase 2 (MMP2) inhibition as a possible mechanism of protection. Materials and Methods Male Wistar rats were fed either a standard rat chow diet (Norm) or a 2% cholesterol-enriched diet (Chol) for nine weeks. Additionally, two more groups received the same treatment, however, at the week 4, diet was supplemented with RPO for the last five weeks (Norm+RPO and Chol+RPO), respectively. After the feeding period hearts were isolated, perfused according to Langendorff and subjected to 30 minutes of normothermic global ischaemia followed by two hours of reperfusion. Infarct size was measured by 2,3,5-triphenyltetrazolium chloride staining at the end of reperfusion. Results Cholesterol-enriched diet increased myocardial infarct size from 23.5 ± 3.0% to 37.2 ± 3.6% (p < 0.05) when compared to normal diet. RPO supplementation significantly reduced infarct size either in Norm+RPO or in Chol+RPO (to 9.2 ± 1.0% and 26.9 ± 3.0%), respectively. Infarct size in Chol+RPO was comparable to the Norm group. MMP2 activity before ischaemia was significantly reduced in the Chol+RPO group when compared to the Chol group. However, the MMP2 activity of the hearts of the RPO fed rats was significantly increased when compared to the normal diet group after ischaemia. Conclusions For the first time it was shown that dietary RPO supplementation attenuated the increased susceptibility of the hearts in cholesterol fed rats to ischaemia/reperfusion injury. This was shown by reduced infarct size. For the first time we also show that red palm oil supplementation altered pre-ischaemic levels of MMP-2, which may indicate that myocardial MMP2 may be implicated as a possible role player in RPO mediated protection against ischaemia/reperfusion injury in hearts of cholesterol supplemented rats.

Introduction : Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases capable of degrading extracellular matrix, including the basement membrane. MMPs are associated with various physiological processes such as morphogenesis, angiogenesis, and tissue repair. Moreover, due to the novel non-matrix related intra- and extracellular targets of MMPs, dysregulation of MMP activity has been implicated in a number of acute and chronic pathological processes, such as arthritis, acute myocardial infarction, chronic heart failure, chronic obstructive pulmonary disease, inflammation, and cancer metastasis. MMPs are considered as viable drug targets in the therapy of the above diseases. Methods : For the development of selective MMP inhibitor molecules, reliable methods are necessary for target validation and lead development. Here, we discuss the major methods used for MMP assays, focusing on substrate zymography. We highlight some problems frequently encountered during sample preparations, electrophoresis, and data analysis of zymograms. Results and Discussion : Z ymography is a widely used technique to study extracellular matrix-degrading enzymes, such as MMPs, from tissue extracts, cell cultures, serum or urine. This simple and sensitive technique identifies MMPs by the degradation of their substrate and by their molecular weight and therefore helps to understand the widespread role of MMPs in different pathologies and cellular pathways.

From January to November 2006, 223 norovirus outbreaks were reported in Hungary. For 128 out of 223 outbreaks (57%) the norovirus agent was laboratory confirmed

In anticipation of a future vaccination program against rotavirus disease, a longitudinal survey has been set up to evaluate the epidemiologic features of rotavirus infections. In this report hospitalisation data and serotyping results are compiled from an epidemiologic survey conducted in Baranya County, Hungary. It was found that rotavirus-associated hospitalisation constituted a major part of infectious gastroenteritis cases (range, 14.9% to 28.5%). A higher proportion of rotavirus-positive cases was recorded when the serotype of predominant strains changed from G1 (1996-1999) to G4 (1999-2000), however, due to the short time period it was not possible to demonstrate a firm association between serotype prevalence and rotavirus-associated hospitalisation rate. In the future, such studies might help to understand if serotype-specific immunity against rotavirus infection plays an important role at the population level and if (re-)emerging rotavirus strains make an impact on the annual disease burden.

Extracellular application of glutamate elicited cytoplasmic Ca2+ transients in freshly dissociated rat neurones of the dorsal cochlear nucleus (DCN) (identified as pyramidal cells) with half-maximal concentration of 513 micromol/l while saturating doses (5 mmol/l) of this neurotransmitter caused transients of 46.1 +/- 3.0 nmol/l on an average. The genesis of these glutamate-evoked Ca2+ transients required extracellular Ca2+. When [Mg2+]o was 1 mmol/l, the NMDA receptor antagonist AP5 (100 micromol/l) had no effects while 100 micromol/l CNQX and 10 micromol/l NBQX, inhibitors of the AMPA receptors, greatly decreased the glutamate-induced Ca2+ transients (a decrease of 92 and 57%, respectively). When facilitating the activation of the NMDA receptors (50 micromol/l glycine, 20 micromol/l [Mg2+]o) in the presence of 100 micromol/l CNQX, Ca2+ transients of 55.4 +/- 13.1 nmol/l could be produced. Block of the voltage-gated Ca2+ channels (200 micromol/l Cd2+) decreased the Ca2+ transients to approx. 50%. The data indicate that under our control experimental circumstances the glutamate-induced Ca2+ transients of the isolated DCN neurones are produced mainly by Ca2+ entry through voltage-gated Ca2+ channels and AMPA receptors. However, when the activation of the NMDA receptors may take place, these receptors also contribute significantly to the genesis of the glutamate-evoked cytoplasmic [Ca2+] elevations.

The cochlear nucleus is the site in the auditory pathway where the primary sensory information carried by the fibres of the acoustic nerve is transmitted to the second-order neurones. According to the generally accepted view this transmission is not a simple relay process but is considered as the first stage where the decoding of the auditory information begins. This notion is based on the diverse neurone composition and highly ordered structure of the nucleus, on the complex electrophysiological properties and activity patterns of the neurones, on the activity of local and descending modulatory mechanisms and on the presence of a highly sophisticated intracellular Ca2+ homeostasis. This review puts emphasis on introducing the experimental findings supporting the above statements and on the questions which should be answered in order to gain a better understanding of the function of the cochlear nucleus.

Although multivitamin products are widely used as dietary supplements, the effects of these products were not investigated in hypercholesterolemia which is a major risk factor of cardiovascular diseases. Therefore, here we investigated if a preparation of different minerals, vitamins, phytosterols and trace elements (MVT) developed for human use affects the severity of experimental hyperlipidemia as well as myocardial ischemia/reperfusion injury. Male Wistar rats were fed a normal or a 2% cholesterol plus 0.2% cholic acid-enriched diet for 12 weeks to induce hyperlipidemia. From week 8, rats in both groups were fed with an MVT preparation or placebo for 4 weeks. Serum triglyceride and cholesterol levels were measured at week 0, 8 and 12. At week 12, hearts were isolated, perfused according to Langendorff and subjected to a 30-min coronary occlusion followed by 120 min reperfusion. At week 8, cholesterol plus cholic acid-fed rats showed significantly higher serum cholesterol level as compared to normal rats, however, serum triglyceride level did not change. At week 12, MVT treatment significanty decreased serum cholesterol level in the hyperlipidemic group, however, MVT treatment failed to decrease infarct size in the hyperlipidemic hearts. In the normolipidemic group, MVT treatment did not influence the serum cholesterol level or the infarct size. We conclude that MVT treatment reduces serum cholesterol level in hyperlipidemia, however, it fails to decrease infarct size.

We have previously reported that chronic oral treatment with the HMG-CoA reductase inhibitor lovastatin shows cardioprotection but interacts with the cardioprotective effect of postconditioning and that farnesol, a key intermediate of the mevalonate pathway, is cardioprotective possibly via enhancement of protein geranylgeranylation. Here we studied the interaction of the HMG-CoA reductase inhibitor atorvastatin and farnesol with postconditioning. Male Wistar rats were orally treated with vehicle (2.5% methylcellulose), 10 mg/kg/day atorvastatin, 1 mg/kg/day farnesol, or their combination, respectively, for 12 days. At the end of the treatments, hearts were isolated and perfused according to Langendorff. Hearts were subjected to 30 minutes of coronary occlusion and 120 minutes of reperfusion with or without a postconditioning protocol induced by six intermittent periods of ischemia/reperfusion of 10-s duration each. At the end of the perfusion protocol, infarct size was determined by standard triphenyltetrazolium chloride staining and expressed as % of the area at risk. We found that postconditioning significantly decreased infarct size (19.5±4.1% vs. 40.0±2.9%, p

Introduction: We have previously shown that autophagy is necessary for the ischemic preconditioning (IPC) of the heart and that IPC is compromised in the setting of hypercholesterolemia (HC). The mTOR pathway, an upstream modulator of autophagy, has been shown to be activated in HC. The purpose of this study was to test the hypothesis that HC results in down regulation of autophagy due in part to activation of mTOR signalling and to investigate whether HC modulates programmed cell death mechanism such as apoptosis and necroptosis in the heart. Methods: Male Wistar rats were fed either normal chow or with 2% cholesterol and 1% cholic acid-enriched diet for 10 weeks. HC rats exhibited a 41% increase in plasma total cholesterol level over that of rats fed standard chow (2.89mmol/L vs. 4.09mmol/L) at the end of diet period. Animals were sacrificed, hearts were excised and briefly washed out, then left ventricles were snap-frozen for determination of autophagy, mTOR pathway, apoptosis, and necroptosis-related proteins by Western blot. Results: HC was associated with a significant reduction in LC3-II and in Beclin-1 expression compared to control values. Consistent with mTOR activation, S6 phosphorylation, an mTOR target, was increased in HC animals (p=0.021). Bax/Bcl2 ratio and cleaved caspase-3 signal increases in HC animlas, while no difference in the expression of RIP3 or MLKL was detected between treatments. Conclusion: These data indicate that hypercholesterolemia suppresses basal cardiac autophagy and that the decrease in autophagy may be a result of an upregulated mTOR pathway; however the exact signaling mechanism is yet to be elucidated. Reduced autophagy was accompanied by signs of increased apoptosis, while cardiac necroptosis was not modulated by HC. Decreased basal autophagy and elevated apoptosis may be responsible for the loss of cardioprotection reported in hypercholesterolemic states.

To investigate the relationship between antiprothrombin (aPT) and antiannexin V (aANX) autoantibodies of IgG isotype and thrombosis in patients with systemic autoimmune diseases. To compare the clinical relevance of these antibodies to that of anticardiolipin (aCL), anti-beta2-glycoprotein I antibodies (anti-beta2-GPI), and lupus anticoagulant (LAC).Serum IgG aPT, aANX, aCL, and anti-beta2-GPI levels were measured by solid phase enzyme immunoassay in the sera of 70 patients with systemic autoimmune diseases, 35 with antiphospholipid syndrome (APS) and 35 without APS. Medical records were analyzed, and associations of the antibodies with clinical features of APS were assessed.Patients with APS had higher frequency of aPT (p = 0.001) and aANX (p = 0.002) compared to patients without APS. Thrombotic events occurred more frequently in those with aPT or aANX than those without (p = 0.005, p = 0.006, respectively). The presence of aPT and aANX was found to be highly specific for APS.Measurement of aPT and aANX antibodies may be of value in confirming the diagnosis of APS, and in evaluating risk of venous and arterial thrombosis in patients with systemic autoimmune diseases.

To monitor cytosolic [Ca2+] over a wide range of concentrations in functioning skeletal muscle cells, we have used simultaneously the rapid but relatively low affinity calcium indicator antipyrylazo III (AP III) and the slower but higher affinity indicator fura-2 in single frog twitch fibers cut at both ends and voltage clamped with a double vaseline gap system. When both dyes were added to the end pool solution the cytosolic fura-2 concentration reached a steady level equal to the end pool concentration within approximately 2.5 h, a time when the AP III concentration was still increasing. For depolarizing pulses of increasing amplitude, the fura-2 fluorescence signal approached saturation when the simultaneously recorded AP III absorbance change was far from saturation. Comparison of simultaneously recorded fura-2 and AP III signals indicated that the mean values of the on and off rate constants for calcium binding to fura-2 in 18 muscle fibers were 1.49 x 10(8) M-1 s-1 and 11.9 s-1, respectively (mean KD = 89 nM), if all AP III in the fiber is assumed to behave as in calibrating solution and to be in instantaneous equilibrium with [Ca2+]. [Ca2+] transients calculated from the fura-2 signals using these rate constants were consistent with the [Ca2+] transients calculated from the AP III signals. Resting [Ca2+] or small changes in [Ca2+] which could not be reliably monitored with AP III could be monitored with fura-2 with little or no interference from changes in [Mg2+] or from intrinsic signals. The fura-2 signal was also less sensitive to movement artifacts than the AP III signal. After a [Ca2+] transient the fura-2 signal demonstrated a relatively small elevation of [Ca2+] that was maintained for many seconds.

Intramembrane charge movement and myoplasmic free calcium transients (delta[Ca2+]) were monitored in voltage-clamped segments of isolated frog muscle fibres cut at both ends and mounted in a double Vaseline-gap chamber. The fibres were stretched to sarcomere lengths of 3.5-4.6 micron to minimize mechanical movement and the related optical artifacts. The over-all calcium removal capability of each fibre was characterized by analysing the decay of delta[Ca2+] following pulses of several different amplitudes and durations. The rate of sarcoplasmic reticulum (s.r.) calcium release was then calculated for each delta[Ca2+] using the calcium removal properties determined for that fibre. The calculated calcium release wave form reached a relatively early peak and then declined appreciably during a 100-150 ms depolarizing pulse. The voltage dependence of the peak rate of calcium release was steeper and was centred at more positive membrane potentials than the steady-state voltage dependence of charge movement in the same fibres. A considerable fraction of the total intramembrane charge was moved at potentials at which delta[Ca2+] and calcium release were only a few per cent of maximum. This 'subthreshold' charge may correspond to charge moved in preliminary transitions that precede a final charge transition that activates release. A 'stepped on' pulse protocol was used to experimentally separate the subthreshold charge movement from the charge movement of the final transitions that may control calcium release. The stepped on pulse consisted of a set 50 ms pre-pulse to a potential just at or below the potential for detectable delta[Ca2+] followed immediately by a test pulse of varying amplitude and duration. For a wide range of test pulse amplitudes and durations in the stepped on protocol the peak rate of calcium release was linearly related to the charge movement during the test pulse. This result points to a tight control of activation of s.r. calcium release by intramembrane charge movement. The voltage dependence of both charge movement and of the rate of calcium release could be fitted simultaneously with a three-state, two-transition sequential model in which charge moves in both transitions but only the final transition activates s.r. calcium release. A model with three identical and independent charged gating particles per channel gave an equally good fit to the data. Both models closely fit the charge movement and release data except within about 10 mV of the voltage at which release became detectable, where release varied more steeply with membrane potential than predicted by either model.(ABSTRACT TRUNCATED AT 400 WORDS)

1. Free intracellular calcium transients (delta[Ca2+] were monitored in cut segments of frog skeletal muscle fibres voltage clamped in a double Vaseline-gap chamber and stretched to sarcomere lengths that eliminated fibre movement. The measured calcium transients were used to calculate the rate of calcium release from the sarcoplasmic reticulum (s.r.) as previously described (Melzer, Rios & Schneider, 1984, 1987). 2. Conditioning pulses were found to suppress the rate of calcium release in test pulses applied after the conditioning pulse. Various combinations of conditioning and test pulses were used to investigate the basis of the suppression of calcium release by the conditioning pulse. 3. Using a constant test pulse applied at varying intervals after a constant conditioning pulse, recovery from suppression of release was found to occur in two phases. During the fast phase of recovery, which was completed within about 1 s, the rate of calcium release was smaller and had a different wave form than the unconditioned control release. The early peak in release that is characteristic of the control release wave form was absent or depressed. During the slow phase of recovery, which required about 1 min for completion, the release wave form was the same as control but was simply scaled down compared to the control. 4. Conditioning pulses also slowed the rate of decay of delta[Ca2+] after a constant test pulse, probably due to an increased occupancy by calcium of slowly equilibrating myoplasmic sites that bind some of the calcium released by the conditioning pulse. Since calcium binding to these sites contributes to the decay of delta[Ca2+], their increased occupancy would slow the decay of delta[Ca2+] following the test pulse. This effect was used to estimate the calcium occupancy of the slowly equilibrating sites. 5. Comparison of the time course of the slow recovery from suppression of release following a constant conditioning pulse with the time course of the loss of calcium from the slowly equilibrating myoplasmic calcium binding sites indicated that the two processes occurred in parallel. 6. Using a set 1 s recovery period and a constant test pulse but varying the amplitude and/or duration of the conditioning pulse, the degree of slowly recovering suppression of release was found to be directly related to the amount of calcium remaining outside of the s.r. at the start of the test pulse. 7. Points 3, 5 and 6 above indicate that the slow recovery from suppression of release may be due to slow recovery from depletion of calcium from the s.r.(ABSTRACT TRUNCATED AT 400 WORDS)

An epidemic of acute haemorrhagic conjunctivitis (AHC) occurred in and around Chandigarh, north India, during June, July and August 1981. Considering the difficulty of virus isolation, the indirect immunofluorescence test was used for the demonstration of virus-specific antigen in the cytoplasm of exfoliated conjunctival cells, using reference antisera. The epidemic appeared to be primarily due to enterovirus type 70. The method was found suitable for the rapid diagnosis of AHC cases and for detecting subclinical infection among healthy subjects and convalescent carriers. During follow-up, it was observed that some of the AHC cases were shedding virus-infected conjunctival cells for a prolonged period, even after clinical recovery and during convalescence, before they became free from virus. In the absence of virus isolation, this method can be considered as suitable for the epidemiological monitoring of AHC.