Striatin knock out induces a gain of function of INa and impaired Ca2+ handling in mESC-derived cardiomyocytes.

AIM: Striatin (Strn) is a scaffold protein expressed in cardiomyocytes (CMs) and alteration of its expression are described in various cardiac diseases. However, the alteration underlying its pathogenicity have been poorly investigated. METHODS: We studied the role(s) of cardiac Strn gene (STRN) by comparing the functional properties of CMs, generated from Strn-KO and isogenic WT mouse embryonic stem cell lines. RESULTS: The spontaneous beating rate of Strn-KO CMs was faster than WT cells, and this correlated with a larger fast INa conductance and no changes in If. Paced (2-8 Hz) Strn-KO CMs showed prolonged action potential (AP) duration in comparison with WT CMs and this was not associated with changes in ICaL and IKr. Motion video tracking analysis highlighted an altered contraction in Strn-KO CMs; this was associated with a global increase in intracellular Ca2+, caused by an enhanced late Na+ current density (INaL) and a reduced Na+/Ca2+ exchanger (NCX) activity and expression. Immunofluorescence analysis confirmed the higher Na+ channel expression and a more dynamic microtubule network in Strn-KO CMs than in WT. Indeed, incubation of Strn-KO CMs with the microtubule stabilizer taxol, induced a rescue (downregulation) of INa conductance toward WT levels. CONCLUSION: Loss of STRN alters CMs electrical and contractile profiles and affects cell functionality by a disarrangement of Strn-related multi-protein complexes. This leads to impaired microtubules dynamics and Na+ channels trafficking to the plasma membrane, causing a global Na+ and Ca2+ enhancement.

Digital PCR: a new technology for diagnosis of parasitic infections.

BACKGROUND: Parasitic infections are responsible for a significant burden of disease worldwide as a result of international travel and immigration. More accurate diagnostic tools are necessary in support to parasite control and elimination programmes in endemic regions as well as for rapid case detection in non-endemic areas. Digital PCR (dPCR) is a powerful technology with recent applications in parasitology. AIMS: This review provides for the first time an overview of dPCR as a novel technology applied to detection of parasitic infections, and highlights the most relevant potential benefits of this assay. SOURCES: Peer-reviewed literature pertinent to this review based on PubMed, Cochrane and Embase databases as well as laboratory experience of authors. CONTENT: Among the 86 studies retrieved, 17 used the dPCR applied to parasites belonging to protozoa (8), helminths (8) and arthropods (1) of clinical human interest. dPCR was adopted in four studies, respectively, for Plasmodium and Schistosoma japonicum. dPCR led to clear advantages over quantitative real-time PCR in P. falciparum and spp., and in S. japonicum showing higher sensitivity; and in Cryptosporidium with higher stability to inhibitors from stool. For all parasites, dPCR allows absolute quantitation without the need of a standard curve. Various dPCR platforms were used. A few critical factors need consideration: DNA load, choice of platform and reaction optimization. IMPLICATIONS: Owing to its sensitivity and quantitative characteristics, dPCR is a potential candidate to become an appealing new method among the molecular technologies for parasite detection and quantitative analysis in the future. In general, it has more applications than genomic DNA detection only, such as quantitation in mixed infections, gene expression and mutation analysis. dPCR should be considered in malaria screening and diagnosis as a complement to routine assays and in schistosomiasis elimination programmes. Standardized strategies and further studies are needed for the integration of dPCR in routine clinical laboratory.

Proteomic analysis of rat hippocampus after repeated psychosocial stress.

Since stress plays a role in the onset and physiopathology of psychiatric diseases, animal models of chronic stress may offer insights into pathways operating in mood disorders. The aim of this study was to identify the molecular changes induced in rat hippocampus by repeated exposure to psychosocial stress with a proteomic technique. In the social defeat model, the experimental animal was defeated by a dominant male eight times. Additional groups of rats were submitted to a single defeat or placed in an empty cage (controls). The open field test was carried out on parallel animal groups. The day after the last exposure, levels of hippocampal proteins were compared between groups after separation by 2-D gel electrophoresis and image analysis. Spots showing significantly altered levels were submitted to peptide fingerprinting mass spectrometry for protein identification. The intensity of 69 spots was significantly modified by repeated stress and 21 proteins were unambiguously identified, belonging to different cellular functions, including protein folding, signal transduction, synaptic plasticity, cytoskeleton regulation and energy metabolism. This work identified molecular changes in protein levels caused by exposure to repeated psychosocial stress. The pattern of changes induced by repeated stress was quantitatively and qualitatively different from that observed after a single exposure. Several changed proteins have already been associated with stress-related responses; some of them are here described for the first time in relation to stress.

Evaluation of quality of life in patients with total or partial edentulism treated with computer-assisted implantology.

AIM: The study deals with a preliminary analysis that compares quality of life of a randomized sample of patients with total or partial edentulism rehabilitated through conventional implantology or computer-assisted implantology. METHODS: The first group was treated with conventional implantology, while the second group was treated with NobelGuide™ computer-assisted implantology. every patient has filled up a questionnaire about quality of life in presurgical period (sf-361), in postsurgical period (sf-361; tiq2) and about the gratification after prosthetic treatment. the questionnaire has evaluated physical, general and psycho-emotive health parameter. RESULTS: SF-36 has demonstrated an improvement in quality of life after computer-assisted surgery. tiq has revealed that patients symptoms in post-surgical week were inferior in quality and in quantity in NobelGuide™ technique. gratification questionnaire has demonstrated that quality of life improvement matches patient full satisfaction after the treatment. CONCLUSIONS: NobelGuide™ protocol improves physical health after implantology with positive reflections on psycho-emotive health. furthermore prefabricated temporary prostheses reduces treatment time and patient discomfort.

Regulation of cytoskeleton machinery, neurogenesis and energy metabolism pathways in a rat gene-environment model of depression revealed by proteomic analysis.

The development of major depression requires both genetic and environmental factors. A brain proteomic investigation on the genetic model of Flinders sensitive and resistant line (FSL-FRL) rats was performed. Maternal separation (MS) was also applied to identify protein networks affected by stress exposure, since early-life trauma is considered an important antecedent of depression. Hippocampus (HIP) and prefrontal/frontal cortex proteins were extracted and separated by 2-Dimensional (2-D) gel electrophoresis. After image analysis, significantly modulated proteins in the different conditions analysed were identified by mass spectrometry. The expression of proteins involved in energy metabolism, cellular localization and transport, cytoskeleton organization and apoptosis differed in the two lines. Maternal separation differently affected the genetic backgrounds, by modulating cytoskeleton and neuron morphogenesis proteins in FSL; energy metabolism, cellular localization, neuron differentiation and intracellular transport in FRL. The present work shows that different mechanisms could be involved in the pathophysiology of depression and the vulnerability to stress, suggesting possible new cellular pathways and key markers for the study of affective disorders.

Effect of experimental conditions on the analysis of sodium dodecyl sulphate polyacrylamide gel electrophoresis separated proteins by matrix-assisted laser desorption/ ionisation mass spectrometry.

Two mixtures of proteins having molecular weights in the range approximately 8-97 kDa were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and examined by delayed extraction matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS). Part of our aim in this study is to gain more insight into the influence of the various experimental conditions on the overall quality of the acquired mass spectral data. Different protein extraction procedures, two staining agents, and extraction times, were among the parameters assessed. In terms of the overall quality of the acquired mass spectra and the speed of protein recovery, ultrasonic assisted passive elution, into a solvent mixture containing formic acid/acetonitrile/2-isopropanol/water, was found to be more efficient than other elution procedures. The higher resolution associated with the delayed extraction mode allowed the identification of a number of protein modifications, including multiple formylation provoked by formic acid, cysteine alkylation caused by unpolymerised acrylamide monomers, and complexation with the staining reagents. The detection of these modifications, however, was limited to proteins under 30 kDa. Analysis of a ubiquitin tryptic digest by reflectron MALDI time-of-flight (TOF) allowed reliable identification of a number of the formylation sites.