Overexpression of connexin 43 using a retroviral vector improves electrical coupling of skeletal myoblasts with cardiac myocytes in vitro.

BACKGROUND: Organ transplantation is presently often the only available option to repair a damaged heart. As heart donors are scarce, engineering of cardiac grafts from autologous skeletal myoblasts is a promising novel therapeutic strategy. The functionality of skeletal muscle cells in the heart milieu is, however, limited because of their inability to integrate electrically and mechanically into the myocardium. Therefore, in pursuit of improved cardiac integration of skeletal muscle grafts we sought to modify primary skeletal myoblasts by overexpression of the main gap-junctional protein connexin 43 and to study electrical coupling of connexin 43 overexpressing myoblasts to cardiac myocytes in vitro. METHODS: To create an efficient means for overexpression of connexin 43 in skeletal myoblasts we constructed a bicistronic retroviral vector MLV-CX43-EGFP expressing the human connexin 43 cDNA and the marker EGFP gene. This vector was employed to transduce primary rat skeletal myoblasts in optimised conditions involving a concomitant use of the retrovirus immobilising protein RetroNectin and the polycation transduction enhancer Transfectam. The EGFP-positive transduced cells were then enriched by flow cytometry. RESULTS: More than four-fold overexpression of connexin 43 in the transduced skeletal myoblasts, compared with non-transduced cells, was shown by Western blotting. Functionality of the overexpressed connexin 43 was demonstrated by microinjection of a fluorescent dye showing enhanced gap-junctional intercellular transfer in connexin 43 transduced myoblasts compared with transfer in non-transduced myoblasts. Rat cardiac myocytes were cultured in multielectrode array culture dishes together with connexin 43/EGFP transduced skeletal myoblasts, control non-transduced skeletal myoblasts or alone. Extracellular field action potential activation rates in the co-cultures of connexin 43 transduced skeletal myoblasts with cardiac myocytes were significantly higher than in the co-cultures of non-transduced skeletal myoblasts with cardiac myocytes and similar to the rates in pure cultures of cardiac myocytes. CONCLUSION: The observed elevated field action potential activation rate in the co-cultures of cardiac myocytes with connexin 43 transduced skeletal myoblasts indicates enhanced cell-to-cell electrical coupling due to overexpression of connexin 43 in skeletal myoblasts. This study suggests that retroviral connexin 43 transduction can be employed to augment engineering of the electrocompetent cardiac grafts from patients own skeletal myoblasts

Supercooled confined water and the Mode Coupling crossover temperature

We present a Molecular Dynamics study of the single particle dynamics of supercooled water confined in a silica pore. Two dynamical regimes are found: close to the hydrophilic substrate molecules are below the Mode Coupling crossover temperature, $T_C$, already at ambient temperature. The water closer to the center of the pore (free water) approaches upon supercooling $T_C$ as predicted by Mode Coupling Theories. For free water the crossover temperature and crossover exponent $\gamma$ are extracted from power-law fits to both the diffusion coefficient and the relaxation time of the late $\alpha$ region.Comment: To be published, Phys. Rev. Lett., 4 pages, 3 figures, revTeX, minor changes in the figures, references added, changes in the tex

Manufacture, observation, and test of membranes with locatable single pores

A method for generating single pores down to 0.1 μm diameter in the center of a large circular foil is described, based on nuclear tracks. The foil is framed by a tension ring which enables one to handle the foils in a well‐defined precise way. The single pore has a lateral displacement of ±0.1 mm with respect to the tension ring center. The foils used are polycarbonate of the type Makrofol and have thicknesses between 2 and 10 μm. For calibration of the single pore diameters, multiple nuclear tracks between 0.1 and 3.5 μm diameter are etched and observed by microscopy. The microscopic observations are compared with gas‐flow measurements, using two alternative methods: multiple holes are tested under viscous flow conditions of N2 gas at normal temperature and pressure; single holes are tested under collisionless flow conditions of 4He gas at liquid‐nitrogen temperature, using a capacitance method.Peer reviewe

Viewpoint: A Pragmatic Approach to Constructing a Minimum Data Set for Care of Patients with HIV in Developing Countries

Providing quality health care requires access to continuous patient data that developing countries often lack. A panel of medical informatics specialists, clinical human immunodeficiency virus (HIV) specialists, and program managers suggests a minimum data set for supporting the management and monitoring of patients with HIV and their care programs in developing countries. The proposed minimum data set consists of data for registration and scheduling, monitoring and improving practice management, and describing clinical encounters and clinical care. Data should be numeric or coded using standard definitions and minimal free text. To enhance accuracy, efficiency, and availability, data should be recorded electronically by those generating them. Data elements must be sufficiently detailed to support clinical algorithms/guidelines and aggregation into broader categories for consumption by higher level users (e.g., national and international health care agencies). The proposed minimum data set will evolve over time as funding increases, care protocols change, and additional tests and treatments become available for HIV-infected patients in developing countrie

Prevalência e fatores de risco associados à Leptospira spp. em rebanhos bovinos da região centro-sul do estado do Paraná.

O objetivo deste trabalho foi determinar a prevalência de anticorpos anti-Leptospira spp. e os fatores de risco para infecção de Leptospira spp. na criação de rebanhos bovinos no centro-sul da região do Estado do Paraná

MicroRNA profile changes in human immunodeficiency virus type 1 (HIV-1) seropositive individuals

MicroRNAs (miRNAs) play diverse roles in regulating cellular and developmental functions. We have profiled the miRNA expression in peripheral blood mononuclear cells from 36 HIV-1 seropositive individuals and 12 normal controls. The HIV-1-positive individuals were categorized operationally into four classes based on their CD4+ T-cell counts and their viral loads. We report that specific miRNA signatures can be observed for each of the four classes

Morphology of supported polymer electrolyte ultra-thin films: a numerical study

Morphology of polymer electrolytes membranes (PEM), e.g., Nafion, inside PEM fuel cell catalyst layers has significant impact on the electrochemical activity and transport phenomena that determine cell performance. In those regions, Nafion can be found as an ultra-thin film, coating the catalyst and the catalyst support surfaces. The impact of the hydrophilic/hydrophobic character of these surfaces on the structural formation of the films has not been sufficiently explored yet. Here, we report about Molecular Dynamics simulation investigation of the substrate effects on the ionomer ultra-thin film morphology at different hydration levels. We use a mean-field-like model we introduced in previous publications for the interaction of the hydrated Nafion ionomer with a substrate, characterized by a tunable degree of hydrophilicity. We show that the affinity of the substrate with water plays a crucial role in the molecular rearrangement of the ionomer film, resulting in completely different morphologies. Detailed structural description in different regions of the film shows evidences of strongly heterogeneous behavior. A qualitative discussion of the implications of our observations on the PEMFC catalyst layer performance is finally proposed

Spectroscopic studies of Dy-168,170 using CLARA and PRISMA

Preliminary results from an experiment aiming at Dy-170. Submitted to the LNL Annual Report 2008.Comment: 2 pages, 4 figures, Submitted to the LNL Annual Report 200

Single-neutron transfer from 11Be gs via the (p,d) reaction with a radioactive beam

The 11Be(p,d)10Be reaction has been performed in inverse kinematics with a radioactive 11Be beam of E/A = 35.3 MeV. Angular distributions for the 0+ ground state, the 2+, 3.37 MeV state and the multiplet of states around 6 MeV in 10Be were measured at angles up to 16 deg CM by detecting the 10Be in a dispersion-matched spectrometer and the coincident deuterons in a silicon array. Distorted wave and coupled-channels calculations have been performed to investigate the amount of 2+ core excitation in 11Be gs. The use of "realistic" 11Be wave functions is emphasised and bound state form factors have been obtained by solving the particle-vibration coupling equations. This calculation gives a dominant 2s component in the 11Be gs wave function with a 16% [2+ x 1d] core excitation admixture. Cross sections calculated with these form factors are in good agreement with the present data. The Separation Energy prescription for the bound state wave function also gives satisfactory fits to the data, but leads to a significantly larger [2 x 1d] component in 11Be gs.Comment: 39 pages, 12 figures. Accepted for publication in Nuclear Physics A. Added minor corrections made in proof to pages 26 and 3