Nano-scale morphology of cardiomyocyte t-tubule/sarcoplasmic reticulum junctions revealed by ultra-rapid high-pressure freezing and electron tomography.

Detailed knowledge of the ultrastructure of intracellular compartments is a prerequisite for our understanding of how cells function. In cardiac muscle cells, close apposition of transverse (t)-tubule (TT) and sarcoplasmic reticulum (SR) membranes supports stable high-gain excitation-contraction coupling. Here, the fine structure of this key intracellular element is examined in rabbit and mouse ventricular cardiomyocytes, using ultra-rapid high-pressure freezing (HPF, omitting aldehyde fixation) and electron microscopy. 3D electron tomograms were used to quantify the dimensions of TT, terminal cisternae of the SR, and the space between SR and TT membranes (dyadic cleft). In comparison to conventional aldehyde-based chemical sample fixation, HPF-preserved samples of both species show considerably more voluminous SR terminal cisternae, both in absolute dimensions and in terms of junctional SR to TT volume ratio. In rabbit cardiomyocytes, the average dyadic cleft surface area of HPF and chemically fixed myocytes did not differ, but cleft volume was significantly smaller in HPF samples than in conventionally fixed tissue; in murine cardiomyocytes, the dyadic cleft surface area was higher in HPF samples with no difference in cleft volume. In both species, the apposition of the TT and SR membranes in the dyad was more likely to be closer than 10 nm in HPF samples compared to CFD, presumably resulting from avoidance of sample shrinkage associated with conventional fixation techniques. Overall, we provide a note of caution regarding quantitative interpretation of chemically-fixed ultrastructures, and offer novel insight into cardiac TT and SR ultrastructure with relevance for our understanding of cardiac physiology.

Investigation of the safety and feasibility of AAV1/SERCA2a gene transfer in patients with chronic heart failure supported with a left ventricular assist device - the SERCA-LVAD TRIAL.

The SERCA-LVAD trial was a phase 2a trial assessing the safety and feasibility of delivering an adeno-associated vector 1 carrying the cardiac isoform of the sarcoplasmic reticulum calcium ATPase (AAV1/SERCA2a) to adult chronic heart failure patients implanted with a left ventricular assist device. The SERCA-LVAD trial was one of a program of AAV1/SERCA2a cardiac gene therapy trials including CUPID1, CUPID 2 and AGENT trials. Enroled subjects were randomised to receive a single intracoronary infusion of 1 × 1013 DNase-resistant AAV1/SERCA2a particles or a placebo solution in a double-blinded design, stratified by presence of neutralising antibodies to AAV. Elective endomyocardial biopsy was performed at 6 months unless the subject had undergone cardiac transplantation, with myocardial samples assessed for the presence of exogenous viral DNA from the treatment vector. Safety assessments including ELISPOT were serially performed. Although designed as a 24 subject trial, recruitment was stopped after five subjects had been randomised and received infusion due to the neutral result from the CUPID 2 trial. Here we describe the results from the 5 patients at 3 years follow up, which confirmed that viral DNA was delivered to the failing human heart in 2 patients receiving gene therapy with vector detectable at follow up endomyocardial biopsy or cardiac transplantation. Absolute levels of detectable transgene DNA were low, and no functional benefit was observed. There were no safety concerns in this small cohort. This trial identified some of the challenges of performing gene therapy trials in this LVAD patient cohort which may help guide future trial design.

Cardiac pacing using transmural multi-LED probes in channelrhodopsin-expressing mouse hearts.

Optogenetics enables cell-type specific monitoring and actuation via light-activated proteins. In cardiac research, expressing light-activated depolarising ion channels in cardiomyocytes allows optical pacing and defibrillation. Previous studies largely relied on epicardial illumination. Light penetration through the myocardium is however problematic when moving to larger animals and humans. To overcome this limitation, we assessed the utility of an implantable multi light-emitting diode (LED) optical probe (IMLOP) for intramural pacing of mouse hearts expressing cardiac-specific channelrhodopsin-2 (ChR2). Here we demonstrated that IMLOP insertion needs approximately 20 mN of force, limiting possible damage from excessive loads applied during implantation. Histological sections confirmed the confined nature of tissue damage during acute use. The temperature change of the surrounding tissue was below 1 K during LED operation, rendering the probe safe for use in situ. This was confirmed in control experiments where no effect on cardiac action potential conduction was observed even when using stimulation parameters twenty-fold greater than required for pacing. In situ experiments on ChR2-expressing mouse hearts demonstrated that optical stimulation is possible with light intensities as low as 700 µW/mm2; although stable pacing requires higher intensities. When pacing with a single LED, rheobase and chronaxie values were 13.3 mW/mm2 ± 0.9 mW/mm2 and 3 ms ± 0.6 ms, respectively. When doubling the stimulated volume the rheobase decreased significantly (6.5 mW/mm2 ± 0.9 mW/mm2). We have demonstrated IMLOP-based intramural optical pacing of the heart. Probes cause locally constrained tissue damage in the acute setting and require low light intensities for pacing. Further development is necessary to assess effects of chronic implantation.

Mitochondrial Deformation During the Cardiac Mechanical Cycle.

Cardiomyocytes both cause and experience continual cyclic deformation. The exact effects of this deformation on the properties of intracellular organelles are not well characterized, although they are likely to be relevant for cardiomyocyte responses to active and passive changes in their mechanical environment. In the present study we provide three-dimensional ultrastructural evidence for mechanically induced mitochondrial deformation in rabbit ventricular cardiomyocytes over a range of sarcomere lengths representing myocardial tissue stretch, an unloaded "slack" state, and contracture. We also show structural indications for interaction of mitochondria with one another, as well as with other intracellular elements such as microtubules, sarcoplasmic reticulum and T-tubules. The data presented here help to contextualize recent reports on the mechanosensitivity and cell-wide connectivity of the mitochondrial network and provide a structural framework that may aide interpretation of mechanically-regulated molecular signaling in cardiac cells. Anat Rec, 302:146-152, 2019. © 2018 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

A new look at the heart-novel imaging techniques.

The development and successful implementation of cutting-edge imaging technologies to visualise cardiac anatomy and function is a key component of effective diagnostic efforts in cardiology. Here, we describe a number of recent exciting advances in the field of cardiology spanning from macro- to micro- to nano-scales of observation, including magnetic resonance imaging, computed tomography, optical mapping, photoacoustic imaging, and electron tomography. The methodologies discussed are currently making the transition from scientific research to routine clinical use, albeit at different paces. We discuss the most likely trajectory of this transition into clinical research and standard diagnostics, and highlight the key challenges and opportunities associated with each of the methodologies.

Quantitative assessment of passive electrical properties of the cardiac T-tubular system by FRAP microscopy.

Well-coordinated activation of all cardiomyocytes must occur on every heartbeat. At the cell level, a complex network of sarcolemmal invaginations, called the transverse-axial tubular system (TATS), propagates membrane potential changes to the cell core, ensuring synchronous and uniform excitation-contraction coupling. Although myocardial conduction of excitation has been widely described, the electrical properties of the TATS remain mostly unknown. Here, we exploit the formal analogy between diffusion and electrical conductivity to link the latter with the diffusional properties of TATS. Fluorescence recovery after photobleaching (FRAP) microscopy is used to probe the diffusion properties of TATS in isolated rat cardiomyocytes: A fluorescent dextran inside TATS lumen is photobleached, and signal recovery by diffusion of unbleached dextran from the extracellular space is monitored. We designed a mathematical model to correlate the time constant of fluorescence recovery with the apparent diffusion coefficient of the fluorescent molecules. Then, apparent diffusion is linked to electrical conductivity and used to evaluate the efficiency of the passive spread of membrane depolarization along TATS. The method is first validated in cells where most TATS elements are acutely detached by osmotic shock and then applied to probe TATS electrical conductivity in failing heart cells. We find that acute and pathological tubular remodeling significantly affect TATS electrical conductivity. This may explain the occurrence of defects in action potential propagation at the level of single T-tubules, recently observed in diseased cardiomyocytes.

Research on anisotropy of fusion-produced protons and neutrons emission from high-current plasma-focus discharges.

The paper concerns fast protons and neutrons from D-D fusion reactions in a Plasma-Focus-1000U facility. Measurements were performed with nuclear-track detectors arranged in "sandwiches" of an Al-foil and two PM-355 detectors separated by a polyethylene-plate. The Al-foil eliminated all primary deuterons, but was penetrable for fast fusion protons. The foil and first PM-355 detector were penetrable for fast neutrons, which were converted into recoil-protons in the polyethylene and recorded in the second PM-355 detector. The "sandwiches" were irradiated by discharges of comparable neutron-yields. Analyses of etched tracks and computer simulations of the fusion-products behavior in the detectors were performed.

Glucocorticoids promote structural and functional maturation of foetal cardiomyocytes: a role for PGC-1α.

Glucocorticoid levels rise dramatically in late gestation to mature foetal organs in readiness for postnatal life. Immature heart function may compromise survival. Cardiomyocyte glucocorticoid receptor (GR) is required for the structural and functional maturation of the foetal heart in vivo, yet the molecular mechanisms are largely unknown. Here we asked if GR activation in foetal cardiomyocytes in vitro elicits similar maturational changes. We show that physiologically relevant glucocorticoid levels improve contractility of primary-mouse-foetal cardiomyocytes, promote Z-disc assembly and the appearance of mature myofibrils, and increase mitochondrial activity. Genes induced in vitro mimic those induced in vivo and include PGC-1α, a critical regulator of cardiac mitochondrial capacity. SiRNA-mediated abrogation of the glucocorticoid induction of PGC-1α in vitro abolished the effect of glucocorticoid on myofibril structure and mitochondrial oxygen consumption. Using RNA sequencing we identified a number of transcriptional regulators, including PGC-1α, induced as primary targets of GR in foetal cardiomyocytes. These data demonstrate that PGC-1α is a key mediator of glucocorticoid-induced maturation of foetal cardiomyocyte structure and identify other candidate transcriptional regulators that may play critical roles in the transition of the foetal to neonatal heart.

Valdensinia heterodoxa Peyronel as a New Pathogen of Blueberry in Poland.

Valdensia leaf blight on blueberry in Poland was reported in one commercial nursery plantation near Prazmów, Mazovia voivodship, where heavy defoliation was observed on cv. Bluecrop, grown in nursery pots, in August 2011. Older fruiting bushes were only slightly affected by the disease. Initial symptoms of the disease were small, oval to circular zonated necrosis surrounded with dark brown borders that enlarged on the leaves throughout the canopy. Multicellular, hyaline or light brown, star-shaped conidiospores were observed on the necrotic areas. The mean length of 50 conidiospores from the end of head to the end of arm apex was 307 to 348 µm (4). Eight single-spore isolates of the fungus were obtained. Single conidiospores were picked up from necrotic spots on leaves and transferred with sterile needle on potato dextrose agar (PDA) and incubated at 20°C under ambient light. After 10 days of incubation, total DNA was extracted. Amplification of the internal transcribed spacer (ITS) region of rDNA was done using primers ITS1F and ITS4A (1). PCRs were carried out as follows: initial denaturation at 94°C for 2 min, denaturation at 94°C for 1 min, annealing at 57°C for 1 min, extension at 72°C for 1 min, and final extension at 72°C for 5 min for 28 cycles (Applied Biosystems Veriti 96 Wel Thermal Cycler). Amplicons, which were approximately 520 bp, were sequenced and nucleotide sequences were analyzed by Clustal W2EBI. The sequences of all eight isolates showed 100% similarity to each other and were compared with sequences stored in GenBank using BLAST. Sequences were 525 bp long and showed 100% homology to Valdensinia heterodoxa Peyronel, Sclerotiniaceae (anamorph: Valdensia heterodoxa Peyronel) from Japan and Norway (Accession Nos. AB663682 and Z81447, respectively) (3). The sequence from one isolate was submitted to GenBank (Accession No. KF212190). To fulfill Koch's postulates, each of the eight isolates was used to inoculate 20 healthy young leaves of Vaccinium corymbosum L. cv. Bluecrop and bilberry (V. myrtillus L.) (10 leaves per plant). Mycelial plugs 5 mm in diameter were taken from PDA cultures, approximately 20 days old, and used as inoculum and placed in the center of each leaf and moistened with sterile distilled water. Mycelium-free plugs were used as control. Inoculated leaves were placed in plastic box and incubated at 20°C in laboratory for 5 days, at which time small necrotic lesions consistent with initial symptoms of the disease were observed. Isolates obtained from these symptoms were morphologically identical to those used for inoculation. Control leaves did not show any disease symptoms. Valdensia leaf blight occurrence may be attributed to rainy July and August 2011 and long presence of water on soil surface. In Poland, Valdensinia heterodoxa causes heavy defoliation of Vaccinium myrtillus in pine stands and is a common pathogen of some herbaceous plants (2). To our knowledge, this is the first report of Valdensia leaf blight on highbush blueberry in Poland. References: (1) I. Larena et al. 75:187, 1999. (2) W. Mulenko and S. Woodward. Mycologist 10:69, 1996. (3) S. Nekoduka et al. J. Gen. Plant Pathol. 78:151, 2012. (4) S. Zhao and S. F. Shamoun. Mycology 1:113, 2010.

Spatially resolved high-resolution x-ray spectroscopy of high-current plasma-focus discharges.

Soft x-ray emission from a Mather-type plasma-focus device (PF-1000) operated at ∼400 kJ was measured. The high density and temperature plasma were generated by the discharge in the deuterium-argon gas mixture in the modified (high-current) plasma-focus configuration. A spherically bent mica crystal spectrograph viewing the axial output of the pinch region was used to measure the x-ray spectra. Spatially resolved spectra including the characteristic x-ray lines of highly ionized Ar and continua were recorded by means of an x-ray film. The x-ray emission of PF-1000 device was studied at different areas of the pinch.

Mass immunisation campaign in a Roma settled community created an opportunity to estimate its size and measles vaccination uptake, Poland, 2009.

During a mass immunisation campaign following an outbreak of measles in a Roma community settled in the town of Pulawy, Poland, we performed an estimation of the size of this Roma population and an assessment of its vaccination uptake. We obtained a list of Roma residing in Pulawy from the local municipality and estimated using a simple capture-recapture formula that Pulawy had 377 Roma residents (43% under 20 years old), which was 27% more than the 295 registered at the municipality. During the vaccination campaign, demographic information was recorded that could be linked to information from the municipality list as well as to prior immunisation status. Among the people whose data were recorded during the vaccination campaign, 14% were not registered at the primary healthcare centres, and were therefore deprived of access to healthcare. Among 102 screened subjects under the age of 20 years, 51% were vaccinated according to schedule. Vaccine uptake for the first dose of measles-containing vaccine was 56% (54/96) and for the second dose 37% (18/49). The present study indicates the need to get a better demographic overview of Roma communities living in Poland and to understand the barriers limiting their access to healthcare and social services. Organisation of catch-up immunisations of this vulnerable population is necessary.

Spotlight on measles 2010: A measles outbreak in a Roma population in Pulawy, eastern Poland, June to August 2009.

We describe a local indigenous outbreak of measles in a susceptible Roma community, which occurred in Pulawy, a town of 50,000 citizens in the Lubelskie province (eastern Poland) during summer 2009. From 22 June to 30 August 2009, 32 measles cases were reported, and additionally nine possible cases were actively identified. A mass immunisation campaign was organised to stop measles transmission in the Roma community. Active surveillance of rash-febrile illnesses allowed documentation of the impact of mass immunisation in preventing further measles spread in the Roma community, and the surrounding population of Pulawy.

Preparation of a respiratory syncytial virus human reference serum for use in the quantitation of neutralization antibody.

Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infection in infants, young children and the elderly. Yet, the development of a vaccine to protect against RSV infection still remains an unmet need. At present, immune responses to experimental vaccines under investigation are usually evaluated by ELISA and/or by neutralization assays against RSV. However, both types of assays are generally performed somewhat differently at different laboratories. An important step towards standardization of serology is the use of a standard human reference serum enabling normalization of results generated within and between laboratories. To fill this need, we prepared and characterized a human reference serum against the A2 strain of respiratory syncytial virus. The serum represents a pool of more than 400 individual human sera obtained from commercial sources. The sera were screened and selected on the basis of individual RSV neutralization titers. A final neutralization titer of 973 (95% C.I., 884-1072) was assigned to the final reference serum pool after it was tested three times in the presence of 10% guinea pig complement and a titer of 286 (95% C.I., 243-337) was assigned to the serum when it was tested in the absence of an exogenous complement source. Sterilely reconstituted lyophilized aliquots of the serum exhibited a stable neutralization titer for at least 1 month at room temperature and at 4 degrees C, as well as after 5 weekly freeze-and-thaw cycles at -20 degrees C. In the lyophilized state, the neutralization titer of the lyophilized reagent was stable for at least 6 months, the last time point tested. Two additional smaller pools of serum with high and medium neutralization titers of 2692 and 575, respectively, were also produced in parallel for use as positive controls and were designated as control sera. The reference serum can be used to normalize neutralization and/or other RSV-specific assay results from different laboratories and the control sera can be used for quality control purposes or as part of a panel to test operator proficiency. Individual lyophilized aliquots of the reference and control sera may be obtained from the US National Institute of Allergy and Infectious Diseases (NIAID) Reference Reagent Repository.

Growth retardation and osteomalacia as a result of ifosfamide nephrotoxicity in a 3-year-old boy whose genotype reveals the genes encoding glutathione S-transferases GSTM1 and GSTT1.

A case of a child with growth retardation and prolonged osteomalacia, as a result of chronic renal tubulopathy, following successful therapy for a sacral-coccygeal germinal tumour, is described. The male patient was enrolled into the research programme for the evaluation of the association between deletion of the genes encoding a number of classes of glutathione S-transferases (GST) and adverse reactions to alkylating agents. His genotype revealed the genes encoding glutathione transferase classes GSTM1 and GSTT1, but these enzymes did not provide adequate protection for the tubular cells, from the toxic effects of ifosfamide metabolites. Intense chemotherapy resulted in an increased risk of chronic side effects. Further studies are necessary for increased understanding of the inter-individual variability in the extent and nature of ifosfamide nephrotoxicity.

[Selected indices of nutritional status and food habits among young ballet dancers]. / Wybrane wskazniki stanu odzywienia oraz upodobania zywieniowe mlodziezy szkoly baletowej.

The work has been aimed at assessment of nutritional status on the base of some selected anthropometric factors and at evaluation of food habits among schoolchildren of the ballet dance school at Gdansk. The 58 boys and girls were examined. The height, body mass and body fat were measured, and the Body Mass Index values calculated. The results were compared to those obtained among children at Poznan and Kielce. The survey of food habits was made with the especially prepared questionnaire. The substantial deficiency of body fat and relatively low body mass were observed. The BMI value below 18 was found for 35% of subjects, and between 20 and 25 only for 23% of subjects. The wrong food habits were found, especially low number of meals and their irregular consumption.

Kynurenic acid does not protect against nicotine-induced seizures in mice.

Kynurenic acid, an antagonist of glutamatergic ionotropic receptors and alpha7 nicotinic cholinergic receptors failed to affect nicotine-induced convulsions in mice which may indicate that alpha7 nicotinic receptor-mediated events play no role in seizure activity produced by nicotine.

Comparison of acetylation phenotype with genotype coding for N-acetyltransferase (NAT2) in children.

The present study focused on evaluation of the extent to which genotype coding for N-acetyltransferase agrees with acetylation phenotype in children at various ages. In 82 Caucasian children aged from 1 mo to 17 y (57 boys and 25 girls) and including 37 infants, the acetylation phenotype was evaluated from the urinary metabolic ratio of 5-acetylamino-6-formylamino-3-methyluracil (AFMU) to 1-methylxanthine (1X) after oral administration of caffeine. At the same time, by use of PCR and restriction analysis of amplified fragments of the N-acetyltransferase gene, four nucleotide transitions were identified: 481C-->T (KpnI), 590 G-->A (TaqI), 803 A-->G (DdeI), and 857 G-->A (BamHI). The wild-type allele was detected in 27 (33%) children, and the slow acetylation genotype was found in 55 (67%) children. The results of the study show that the metabolic ratio AFMU/1X could be calculated only in 72 children, because in 10 (14%) infants <20 wk of age, AFMU was not detected. Determination of the relation between the acetylation phenotype and genotype revealed that 18 children (23%) containing at least one wild-type allele had AFMU/1X <0.4 (slow acetylation activity) and 7 (8%) of genotypically slow acetylators presented high metabolic ratio (high acetylation activity). We concluded that the disagreement between the acetylation phenotype and genotype is more often found in the group of children characterized by low AFMU/1X and that in small children only N-acetyltransferase genotype studies enable the detection of genetic acetylation defect.