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.

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.

Public views about editing genes in wildlife for conservation.

Developments in CRISPR-based gene-editing technologies have generated a growing number of proposals to edit genes in wildlife to meet conservation goals. As these proposals have attracted greater attention, controversies have emerged among scientists and stakeholder groups over potential consequences and ethical implications of gene editing. Responsible governance cannot occur without consulting broader publics, yet little effort has been made to systematically assess public understandings and beliefs in relation to this new area of applied genetic engineering. We analyzed data from a survey of U.S. adults (n = 1600), collected by YouGov, and that examined respondents' concerns about gene editing in animal and plant wildlife and how those concerns are shaped by cultural dispositions toward science and beliefs about the appropriateness of intervening in nature at the genetic level. On average, respondents perceived more risk than benefit in using these tools. Over 70% agreed that gene editing in wildlife could be "easily used for the wrong purposes." When evaluating the moral acceptability of gene editing in wildlife, respondents evaluated applications to improve survival in endangered wildlife as more morally acceptable than applications to decrease abundance in a population or eliminate a population. Belief in the authority of scientific knowledge was positively related to favorable views of the benefits, risks, and moral acceptability of editing genes in wildlife. The belief that editing genes in wildlife inappropriately intervenes in nature predicted relatively more concern about risks and moral acceptability and skepticism about benefits. Given high levels of concern and skepticism about gene editing in wildlife for conservation among the U.S. public, a take-it-slow approach to making decisions about when or whether to use these tools is advisable. Early opinions, including those uncovered in this study, are likely to be provisional. Thus, consulting the public should be an ongoing process.

Opinión Pública sobre la Edición de Genes en la Conservación de Fauna Resumen El desarrollo de tecnologías de edición de genes basada en CRISPR ha generado un número creciente de propuestas para editar los genes de especies silvestres y así lograr los objetivos de conservación. Conforme estas propuestas han atraído la atención, han surgido controversias entre los grupos de científicos y accionistas sobre las consecuencias potenciales y las implicaciones éticas de la edición de genes. La gobernanza responsable no puede ocurrir sin consultar a una audiencia más amplia, y aun así se han realizado muy pocos esfuerzos por evaluar sistemáticamente las creencias y el entendimiento público relacionados con esta nueva etapa de ingeniería genética aplicada. Analizamos los resultados de una encuesta en línea realizado por YouGov entre adultos de los Estados Unidos (n = 1,600), la cual examinaba las preocupaciones de los respondientes sobre la edición genética en animales y plantas y cómo estas preocupaciones están moldeadas por la disposición cultural hacia la ciencia y las creencias sobre lo correcto que es intervenir en la naturaleza a nivel genético. En promedio, los respondientes percibieron más un riesgo que un beneficio al usar estas herramientas. Más del 70% estuvo de acuerdo en que la edición genética de especies silvestres podría "usarse fácilmente para los propósitos equivocados". Cuando evaluamos la aceptación moral de la edición genética de la fauna, los respondientes calificaron a las aplicaciones para incrementar la supervivencia de las especies amenazadas como más aceptables moralmente que las aplicaciones para disminuir la abundancia de una población o eliminarla totalmente. La creencia en la autoridad del conocimiento científico estuvo relacionada positivamente con la opinión favorable sobre los beneficios, riesgos y la aceptación moral de la edición genética de especies silvestres. La creencia de que la edición genética de especies silvestres interviene inapropiadamente con la naturaleza predijo relativamente una mayor preocupación por los riesgos y la aceptación moral y mayor escepticismo sobre los beneficios de la edición genética. Debido a los altos niveles de preocupación y escepticismo sobre la edición genética de especies silvestres para la conservación mostrados por el público estadunidense, se recomienda utilizar una estrategia de hazlo-con-calma para decidir cuándo o si usar estas herramientas. Es probable que las primeras opiniones, incluyendo las descubiertas con este estudio, sean provisionales. Por lo tanto, consultar al público debe ser un proceso continuo.

[Laboratory practices: diagnostics and antibiotics resistance testing of Neisseria gonorrhoeae in Germany]. / Die deutsche Laborlandschaft zu Diagnostik und Antibiotikaresistenztestung bei Neisseria gonorrhoeae.

Recent years have seen a world-wide increase in antimicrobial resistance (AMR) in cases of infection with Neisseria gonorrhoeae (NG). NG infection is not notifiable in Germany and there is a lack of information available about the spread and AMR of NG infections. The objective of the study was to provide information on diagnostic methods and AMR testing in cases of NG infections in German laboratories. A cross-sectional survey was undertaken in Germany between June and August 2013 using an online questionnaire. Laboratories performing NG diagnostics were identified and described with regard to the diagnostic methods used, the number of tests performed, the antibiotics tested and the AMR observed, in addition to general laboratory information. In total, 188 of the 521 participating laboratories performed NG diagnostics; these were included in the further statistical analysis. 92.6 % of the 188 laboratories performed culture. A median of 60 (IQR 15-270) samples per quarter (SPQ) were tested, with an overall positivity rate of 4.1 and 6.9 % among men. Most (82.1 %) of the 151 laboratories performing NG culture tested for AMR as well. The most frequently tested antibiotics were ciprofloxacin (94.8 %), penicillin (93.1 %), doxycycline (70.0 %) and ceftriaxone (67.2 %). The most frequently observed AMR ever were those against ciprofloxacin (87.1 %), penicillin (78.3 %), doxycycline (56.6 %) and azithromycin (35.1 %; all percentages refer to laboratories). The laboratories used different standards regarding susceptibility criteria. The emergence and spread of AMR shows that it is crucial to assess and monitor the scope and trends of multidrug-resistant gonorrhea. The data collected on diagnostic methods and AMR testing in cases of NG infections in German laboratories constitute an important basis for future monitoring.

[Phlebology in German departments of dermatology. An analysis on behalf of the German Society of Phlebology]. / Phlebologie an deutschen Hautkliniken. Eine Bestandsaufnahme im Auftrag der Deutschen Gesellschaft für Phlebologie.

BACKGROUND: Phlebologic diseases have become extremely common and have major socio-economic impact. However, the percentage of dermatologists working in phlebology appears to be decreasing according to the data of the German Society of Phlebology (DGP). METHODS: To investigate the reasons for this development, we--on behalf of the DGP--sent a questionnaire to 120 German Departments of Dermatology in autumn 2012. RESULTS: In 76 returned questionnaires, the number of physicians with additional fellowship training in phlebology averaged 1.5; the average number of those who fulfill the criteria for training fellows in phlebology was 0.9. In 71.1 % of the departments there was a phlebologist. A special phlebologic outpatient clinic existed in 73.7 % of the departments. Sonography with Doppler (89.5 %) and duplex (86.8 %) was used as the most frequent diagnostic tool. For therapy, compression (94.7 %), sclerotherapy (liquid 78.9 %, foam 63.2 %, catheter 18.4 %), endoluminal thermic procedures (radio wave 28.9 %, laser 17.1 %) and surgery (especially crossectomy and stripping 67.1 %, phlebectomy of tributaries 75 %) were used. The average number of treatments was very heterogenous in the different departments. CONCLUSIONS: Phlebology definitely plays an important role in dermatology. Most departments fulfill the formal criteria for the license to conduct advanced training in phlebology. A wide spectrum of phlebological diagnostic and therapeutic procedures is available.

Anaerobic co-digestion of sewage sludge and primary clarifier skimmings for increased biogas production.

The objective of the study was to identify the impact of co-digesting clarifier skimmings on the overall methane generation from the treatment plant and additional energy value of the increased methane production. Biogas production from co-digesting clarifier skimmings and sewage sludge in pilot-scale fed-batch mesophilic anaerobic digesters has been evaluated. The digester was fed with increasing quantities of clarifier skimmings loads: 1.5, 2.6, 3.5 and 7.0 g COD equivalent/(L·d) (COD: chemical oxygen demand). Average volatile solids reduction of 65% was achieved in the scum-fed digester, compared with 51% in the control digester. Average 69% COD removal was achieved at highest scum loading (7 g COD eq/(L·d)) with approximate methane yield of 250 L CH(4)/kg COD fed (4 ft(3)/lb COD fed). The results show that scum as co-substrate in anaerobic digestion systems improves biogas yields while a 29% increase in specific CH(4) yield could be achieved when scum load is 7 g COD eq/(L·d). Based on the pilot-scale study results and full-scale data from South East Water Pollution Control Plant and Northeast Water Pollution Control Plant the expected annual energy recovery would be approximately 1.7 billion BTUs or nearly 0.5 million kWh.

Leukosialin (CD43)-major histocompatibility class I molecule interactions involved in spontaneous T cell conjugate formation.

Resting T cells spontaneously adhere in a selective manner to potent accessory cells, such as dendritic cells (DC) and lymphoblastoid B blasts (LCL). Here we demonstrate that leukosialin (CD43) and major histocompatibility complex class I molecules (MHC-I) might play a critical role in this process. T cell conjugate formation with monocyte-derived DC (md-DC) and LCL could be strongly inhibited by either preincubating T cells with Fab fragments of CD43 monoclonal antibody (mAb) 6F5 or by preincubating md-DC or LCL with MHC-I mAb W6/32. Intact CD43 mAb 6F5, in contrast to monovalent Fab fragments, enhanced T cell adhesiveness by transactivating CD2 binding to CD58 molecules. Interestingly, induction of this proadhesive signal via CD43 with intact 6F5 mAb was found to revert mAb W6/32-mediated inhibition of T cell conjugate formation. These observations indicated that CD43 cross-linkage mimics and monovalent mAb 6F5 inhibits interaction of T cell CD43 with a stimulatory ligand on opposing cells, presumably MHC-I. For the demonstration of direct physical interaction between CD43 on T cells and MHC-I-coated beads it was necessary, however, to ligate CD2 on T cells with a stimulatory pair of CD2 mAbs (VIT13 plus TS2/18). This suggests that CD2 ligation crosswise upregulates CD43 binding avidity for MHC-I and that both adhesion molecule pairs (CD43/MHC-I and CD2/CD58) act in concert to induce and mediate T cell conjugate formation with certain cell types.

Measurement and analysis of sarcomere length in rat cardiomyocytes in situ and in vitro.

Sarcomere length (SL) is an important determinant and indicator of cardiac mechanical function; however, techniques for measuring SL in living, intact tissue are limited. Here, we present a technique that uses two-photon microscopy to directly image striations of living cells in cardioplegic conditions, both in situ (Langendorff-perfused rat hearts and ventricular tissue slices, stained with the fluorescent marker di-4-ANEPPS) and in vitro (acutely isolated rat ventricular myocytes). Software was developed to extract SL from two-photon fluorescence image sets while accounting for measurement errors associated with motion artifact in raster-scanned images and uncertainty of the cell angle relative to the imaging plane. Monte-Carlo simulations were used to guide analysis of SL measurements by determining error bounds as a function of measurement path length. The mode of the distribution of SL measurements in resting Langendorff-perfused heart is 1.95 mum (n = 167 measurements from N = 11 hearts) after correction for tissue orientation, which was significantly greater than that in isolated cells (1.71 mum, n = 346, N = 9 isolations) or ventricular slice preparations (1.79 mum, n = 79, N = 3 hearts) under our experimental conditions. Furthermore, we find that edema in arrested Langendorff-perfused heart is associated with a mean SL increase; this occurs as a function of time ex vivo and correlates with tissue volume changes determined by magnetic resonance imaging. Our results highlight that the proposed method can be used to monitor SL in living cells and that different experimental models from the same species may display significantly different SL values under otherwise comparable conditions, which has implications for experiment design, as well as comparison and interpretation of data.

Acetoclastic methanogens in an anaerobic digester could be susceptible to trace metal supplementation.

The objective of this study was to investigate the effects of nutrient supplementation on anaerobic biomass. While many studies emphasized the importance of supplementing trace metals such as iron, cobalt, and nickel for maximum methanogenic activity, there is no evidence whether such supplements, even at relatively low concentration, could perturb anaerobic biomass. Effects of supplementing nutrients, including yeast extract, on anaerobic biomass from two full-scale mesophilic digesters, operating under different conditions, at the North East Water Pollution Control Plant in Philadelphia, Pennsylvania, USA, were assessed using biochemical methane potential tests. The results show that acetoclastic methanogens from a recently cleaned digester was not stimulated by nutrient supplementation at relatively low concentrations and a slight perturbation was observed when supplementation was at a relatively high concentration. Furthermore, greater degree of susceptibility to the trace metal supplementation was observed for biomass from another digester that had not been cleaned for over 10 years, thus it had reduced active volume due to grit accumulation. For instance, supplementation of 200 mg/L of iron as FeCl(2)·4H(2)O to the biomass from the reduced-active-volume digester caused 17% reduction in CH(4) production, as compared to a control which did not receive any supplements, while the same concentration had no effect on the biomass from full-active-volume digester. Results strongly suggest that acetoclastic methanogens stressed due to reduced hydraulic/solids retention time may be susceptible to trace metal addition. Therefore, trace metal supplementation for anaerobic digesters should be considered on a case by case basis.