The cardiac sodium channel displays differential distribution in the conduction system and transmural heterogeneity in the murine ventricular myocardium

Cardiac sodium channels are responsible for conduction in the normal and diseased heart. We aimed to investigate regional and transmural distribution of sodium channel expression and function in the myocardium. Sodium channel Scn5a mRNA and Na(v)1.5 protein distribution was investigated in adult and embryonic mouse heart through immunohistochemistry and in situ hybridization. Functional sodium channel availability in subepicardial and subendocardial myocytes was assessed using patch-clamp technique. Adult and embryonic (ED14.5) mouse heart sections showed low expression of Na(v)1.5 in the HCN4-positive sinoatrial and atrioventricular nodes. In contrast, high expression levels of Na(v)1.5 were observed in the HCN4-positive and Cx43-negative AV or His bundle, bundle branches and Purkinje fibers. In both ventricles, a transmural gradient was observed, with a low Na(v)1.5 labeling intensity in the subepicardium as compared to the subendocardium. Similar Scn5a mRNA expression patterns were observed on in situ hybridization of embryonic and adult tissue. Maximal action potential upstroke velocity was significantly lower in subepicardial myocytes (mean +/- SEM 309 +/- 32 V/s; n = 14) compared to subendocardial myocytes (394 +/- 32 V/s; n = 11; P < 0.05), indicating decreased sodium channel availability in subepicardium compared to subendocardium. Scn5a and Na(v)1.5 show heterogeneous distribution patterns within the cardiac conduction system and across the ventricular wall. This differential distribution of the cardiac sodium channel may have profound consequences for conduction disease phenotypes and arrhythmogenesis in the setting of sodium channel diseas

Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data

Despite the central role of quantitative PCR (qPCR) in the quantification of mRNA transcripts, most analyses of qPCR data are still delegated to the software that comes with the qPCR apparatus. This is especially true for the handling of the fluorescence baseline. This article shows that baseline estimation errors are directly reflected in the observed PCR efficiency values and are thus propagated exponentially in the estimated starting concentrations as well as ‘fold-difference’ results. Because of the unknown origin and kinetics of the baseline fluorescence, the fluorescence values monitored in the initial cycles of the PCR reaction cannot be used to estimate a useful baseline value. An algorithm that estimates the baseline by reconstructing the log-linear phase downward from the early plateau phase of the PCR reaction was developed and shown to lead to very reproducible PCR efficiency values. PCR efficiency values were determined per sample by fitting a regression line to a subset of data points in the log-linear phase. The variability, as well as the bias, in qPCR results was significantly reduced when the mean of these PCR efficiencies per amplicon was used in the calculation of an estimate of the starting concentration per sample

Comprehensive Gene-Expression Survey Identifies Wif1 as a Modulator of Cardiomyocyte Differentiation

During chicken cardiac development the proepicardium (PE) forms the epicardium (Epi), which contributes to several non-myocardial lineages within the heart. In contrast to Epi-explant cultures, PE explants can differentiate into a cardiomyocyte phenotype. By temporal microarray expression profiles of PE-explant cultures and maturing Epi cells, we identified genes specifically associated with differentiation towards either of these lineages and genes that are associated with the Epi-lineage restriction. We found a central role for Wnt signaling in the determination of the different cell lineages. Immunofluorescent staining after recombinant-protein incubation in PE-explant cultures indicated that the early upregulated Wnt inhibitory factor-1 (Wif1), stimulates cardiomyocyte differentiation in a similar manner as Wnt stimulation. Concordingly, in the mouse pluripotent embryogenic carcinoma cell line p19cl6, early and late Wif1 exposure enhances and attenuates differentiation, respectively. In ovo exposure of the HH12 chicken embryonic heart to Wif1 increases the Tbx18-positive cardiac progenitor pool. These data indicate that Wif1 enhances cardiomyogenesis

Photon vs. proton radiochemotherapy: Effects on brain tissue volume and perfusion

Neuro Imaging Researc

EORTC Early Clinical Studies Group early phase II trial of S-1 in patients with advanced or metastatic colorectal cancer

Cancer of the colon and rectum is one of the most frequent malignancies both in the US and Europe. Standard palliative therapy is based on 5-fluorouracil/folinic acid combinations, with or without oxaliplatin or irinotecan, given intravenously. Oral medication has the advantage of greater patient convenience and acceptance and potential cost savings. S-1 is a new oral fluorinated pyrimidine derivative. In a nonrandomized phase II study, patients with advanced/metastatic colorectal cancer were treated with S-1 at 40 mg m-2 b.i.d. for 28 consecutive days, repeated every 5 weeks, but by amendment the dose was reduced to 35 mg m-2 during the study because of a higher than expected number of severe adverse drug reactions. In total 47 patients with colorectal cancer were included. In the 37 evaluable patients there were nine partial responses (24%), 17 stable diseases (46%) and 11 patients had progressive disease (30%). Diarrhoea occurred frequently and was often severe: in the 40 and 35 mg m-2 group, respectively, 38 and 35% of the patients experienced grade 3-4 diarrhoea. The other toxicities were limited and manageable. S-1 is active in advanced colorectal cancer, but in order to establish a safer dose the drug should be subject to further investigations

Disparate requirements for the Walker A and B ATPase motifs of human RAD51D in homologous recombination

In vertebrates, homologous recombinational repair (HRR) requires RAD51 and five RAD51 paralogs (XRCC2, XRCC3, RAD51B, RAD51C and RAD51D) that all contain conserved Walker A and B ATPase motifs. In human RAD51D we examined the requirement for these motifs in interactions with XRCC2 and RAD51C, and for survival of cells in response to DNA interstrand crosslinks (ICLs). Ectopic expression of wild-type human RAD51D or mutants having a non-functional A or B motif was used to test for complementation of a rad51d knockout hamster CHO cell line. Although A-motif mutants complement very efficiently, B-motif mutants do not. Consistent with these results, experiments using the yeast two- and three-hybrid systems show that the interactions between RAD51D and its XRCC2 and RAD51C partners also require a functional RAD51D B motif, but not motif A. Similarly, hamster Xrcc2 is unable to bind to the non-complementing human RAD51D B-motif mutants in co-immunoprecipitation assays. We conclude that a functional Walker B motif, but not A motif, is necessary for RAD51D's interactions with other paralogs and for efficient HRR. We present a model in which ATPase sites are formed in a bipartite manner between RAD51D and other RAD51 paralogs

The BMP Antagonist Follistatin-Like 1 Is Required for Skeletal and Lung Organogenesis

Follistatin-like 1 (Fstl1) is a secreted protein of the BMP inhibitor class. During development, expression of Fstl1 is already found in cleavage stage embryos and becomes gradually restricted to mesenchymal elements of most organs during subsequent development. Knock down experiments in chicken and zebrafish demonstrated a role as a BMP antagonist in early development. To investigate the role of Fstl1 during mouse development, a conditional Fstl1 KO allele as well as a Fstl1-GFP reporter mouse were created. KO mice die at birth from respiratory distress and show multiple defects in lung development. Also, skeletal development is affected. Endochondral bone development, limb patterning as well as patterning of the axial skeleton are perturbed in the absence of Fstl1. Taken together, these observations show that Fstl1 is a crucial regulator in BMP signalling during mouse development

Transanal minimally invasive surgery (TAMIS) versus endoscopic submucosal dissection (ESD) for resection of non-pedunculated rectal lesions (TRIASSIC study):study protocol of a European multicenter randomised controlled trial

BACKGROUND: In the recent years two innovative approaches have become available for minimally invasive en bloc resections of large non-pedunculated rectal lesions (polyps and early cancers). One is Transanal Minimally Invasive Surgery (TAMIS), the other is Endoscopic Submucosal Dissection (ESD). Both techniques are standard of care, but a direct randomised comparison is lacking. The choice between either of these procedures is dependent on local expertise or availability rather than evidence-based. The European Society for Endoscopy has recommended that a comparison between ESD and local surgical resection is needed to guide decision making for the optimal approach for the removal of large rectal lesions in Western countries. The aim of this study is to directly compare both procedures in a randomised setting with regard to effectiveness, safety and perceived patient burden. METHODS: Multicenter randomised trial in 15 hospitals in the Netherlands. Patients with non-pedunculated lesions > 2 cm, where the bulk of the lesion is below 15 cm from the anal verge, will be randomised between either a TAMIS or an ESD procedure. Lesions judged to be deeply invasive by an expert panel will be excluded. The primary endpoint is the cumulative local recurrence rate at follow-up rectoscopy at 12 months. Secondary endpoints are: 1) Radical (R0-) resection rate; 2) Perceived burden and quality of life; 3) Cost effectiveness at 12 months; 4) Surgical referral rate at 12 months; 5) Complication rate; 6) Local recurrence rate at 6 months. For this non-inferiority trial, the total sample size of 198 is based on an expected local recurrence rate of 3% in the ESD group, 6% in the TAMIS group and considering a difference of less than 6% to be non-inferior. DISCUSSION: This is the first European randomised controlled trial comparing the effectiveness and safety of TAMIS and ESD for the en bloc resection of large non-pedunculated rectal lesions. This is important as the detection rate of these adenomas is expected to further increase with the introduction of colorectal screening programs throughout Europe. This study will therefore support an optimal use of healthcare resources in the future. TRIAL REGISTRATION: Netherlands Trial Register, NL7083 , 06 July 2018

Cardiac regeneration: different cells same goal

Cardiovascular diseases are the leading cause of mortality, morbidity, hospitalization and impaired quality of life. In most, if not all, pathologic cardiac ischemia ensues triggering a succession of events leading to massive death of cardiomyocytes, fibroblast and extracellular matrix accumulation, cardiomyocyte hypertrophy which culminates in heart failure and eventually death. Though current pharmacological treatment is able to delay the succession of events and as a consequence the development of heart failure, the only currently available and effective treatment of end-stage heart failure is heart transplantation. However, donor heart availability and immunorejection upon transplantation seriously limit the applicability. Cardiac regeneration could provide a solution, making real a dream of both scientist and clinician in the previous century and ending an ongoing challenge for this century. In this review, we present a basic overview of the various cell types that have been used in both the clinical and research setting with respect to myocardial differentiation

Regulation of IL-2 gene expression by Siva and FOXP3 in human T cells

<p>Abstract</p> <p>Background</p> <p>Severe autoinflammatory diseases are associated with mutations in the <it>Foxp3 </it>locus in both mice and humans. <it>Foxp3 </it>is required for the development, function, and maintenance of regulatory T cells (T_regs), a subset of CD4 cells that suppress T cell activation and inflammatory processes. <it>Siva </it>is a pro-apoptotic gene that is expressed across a range of tissues, including CD4 T cells. Siva interacts with three tumor necrosis factor receptor (TNFR) family members that are constitutively expressed on T_regcells: CD27, GITR, and OX40.</p> <p>Results</p> <p>Here we report a biophysical interaction between FOXP3 and Siva. We mapped the interaction domains to Siva's C-terminus and to a central region of FOXP3. We showed that <it>Siva </it>repressed IL-2 induction by suppressing <it>IL-2 </it>promoter activity during T cell activation. Siva-1's repressive effect on <it>IL-2 </it>gene expression appears to be mediated by inhibition of NFkappaB, whereas FOXP3 repressed both NFkappaB and NFAT activity.</p> <p>Conclusions</p> <p>In summary, our data suggest that both <it>FOXP3 </it>and <it>Siva </it>function as negative regulators of IL-2 gene expression in T_regcells, via suppression of NFAT by <it>FOXP3 </it>and of NFkappaB by both <it>FOXP3 </it>and <it>Siva</it>. Our work contributes evidence for <it>Siva's </it>role as a T cell signalling mediator in addition to its known pro-apoptotic function. Though further investigations are needed, evidence for the biophysical interaction between FOXP3 and Siva invites the possibility that Siva may be important for proper T_regcell function.</p