TRPC1 channels underlie stretch-modulated sarcoplasmic reticulum calcium leak in cardiomyocytes.

Transient receptor potential canonical 1 (TRPC1) channels are Ca2+-permeable ion channels expressed in cardiomyocytes. An involvement of TRPC1 channels in cardiac diseases is widely established. However, the physiological role of TRPC1 channels and the mechanisms through which they contribute to disease development are still under investigation. Our prior work suggested that TRPC1 forms Ca2+ leak channels located in the sarcoplasmic reticulum (SR) membrane. Prior studies suggested that TRPC1 channels in the cell membrane are mechanosensitive, but this was not yet investigated in cardiomyocytes or for SR localized TRPC1 channels. We applied adenoviral transfection to overexpress or suppress TRPC1 expression in neonatal rat ventricular myocytes (NRVMs). Transfections were evaluated with RT-qPCR, western blot, and fluorescent imaging. Single-molecule localization microscopy revealed high colocalization of exogenously expressed TRPC1 and the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA2). To test our hypothesis that TRPC1 channels contribute to mechanosensitive Ca2+ SR leak, we directly measured SR Ca2+ concentration ([Ca2+]SR) using adenoviral transfection with a novel ratiometric genetically encoded SR-targeting Ca2+ sensor. We performed fluorescence imaging to quantitatively assess [Ca2+]SR and leak through TRPC1 channels of NRVMs cultured on stretchable silicone membranes. [Ca2+]SR was increased in cells with suppressed TRPC1 expression vs. control and Transient receptor potential canonical 1-overexpressing cells. We also detected a significant reduction in [Ca2+]SR in cells with Transient receptor potential canonical 1 overexpression when 10% uniaxial stretch was applied. These findings indicate that TRPC1 channels underlie the mechanosensitive modulation of [Ca2+]SR. Our findings are critical for understanding the physiological role of TRPC1 channels and support the development of pharmacological therapies for cardiac diseases.

Modulation of Calcium Transients in Cardiomyocytes by Transient Receptor Potential Canonical 6 Channels.

Transient receptor potential canonical 6 (TRPC6) channels are non-selective cation channels that are thought to underlie mechano-modulation of calcium signaling in cardiomyocytes. TRPC6 channels are involved in development of cardiac hypertrophy and related calcineurin-nuclear factor of activated T cells (NFAT) signaling. However, the exact location and roles of TRPC6 channels remain ill-defined in cardiomyocytes. We used an expression system based on neonatal rat ventricular myocytes (NRVMs) to investigate the location of TRPC6 channels and their role in calcium signaling. NRVMs isolated from 1- to 2-day-old animals were cultured and infected with an adenoviral vector to express enhanced-green fluorescent protein (eGFP) or TRPC6-eGFP. After 3 days, NRVMs were fixed, immunolabeled, and imaged with confocal and super-resolution microscopy to determine TRPC6 localization. Cytosolic calcium transients at 0.5 and 1 Hz pacing rates were recorded in NRVMs using indo-1, a ratio-metric calcium dye. Confocal and super-resolution microscopy suggested that TRPC6-eGFP localized to the sarcolemma. NRVMs infected with TRPC6-eGFP exhibited higher diastolic and systolic cytosolic calcium concentration as well as increased sarcoplasmic reticulum (SR) calcium load compared to eGFP infected cells. We applied a computer model comprising sarcolemmal TRPC6 current to explain our experimental findings. Altogether, our studies indicate that TRPC6 channels play a role in sarcolemmal and intracellular calcium signaling in cardiomyocytes. Our findings support the hypothesis that upregulation or activation of TRPC6 channels, e.g., in disease, leads to sustained elevation of the cytosolic calcium concentration, which is thought to activate calcineurin-NFAT signaling and cardiac hypertrophic remodeling. Also, our findings support the hypothesis that mechanosensitivity of TRPC6 channels modulates cytosolic calcium transients and SR calcium load.

Location and function of transient receptor potential canonical channel 1 in ventricular myocytes.

Transient receptor potential canonical 1 (TRPC1) protein is abundantly expressed in cardiomyocytes. While TRPC1 is supposed to be critically involved in cardiac hypertrophy, its physiological role in cardiomyocytes is poorly understood. We investigated the subcellular location of TRPC1 and its contribution to Ca2+ signaling in mammalian ventricular myocytes. Immunolabeling, three-dimensional scanning confocal microscopy and quantitative colocalization analysis revealed an abundant intracellular location of TRPC1 in neonatal rat ventricular myocytes (NRVMs) and adult rabbit ventricular myocytes. TRPC1 was colocalized with intracellular proteins including sarco/endoplasmic reticulum Ca2+ ATPase 2 in the sarcoplasmic reticulum (SR). Colocalization with wheat germ agglutinin, which labels the glycocalyx and thus marks the sarcolemma including the transverse tubular system, was low. Super-resolution and immunoelectron microscopy supported the intracellular location of TRPC1. We investigated Ca2+ signaling in NRVMs after adenoviral TRPC1 overexpression or silencing. In NRVMs bathed in Na+ and Ca2+ free solution, TRPC1 overexpression and silencing was associated with a decreased and increased SR Ca2+ content, respectively. In isolated rabbit cardiomyocytes bathed in Na+ and Ca2+ free solution, we found an increased decay of the cytosolic Ca2+ concentration [Ca2+]i and increased SR Ca2+ content in the presence of the TRPC channel blocker SKF-96365. In a computational model of rabbit ventricular myocytes at physiological pacing rates, Ca2+ leak through SR TRPC channels increased the systolic and diastolic [Ca2+]i with only minor effects on the action potential and SR Ca2+ content. Our studies suggest that TRPC1 channels are localized in the SR, and not present in the sarcolemma of ventricular myocytes. The studies provide evidence for a role of TRPC1 as a contributor to SR Ca2+ leak in cardiomyocytes, which was previously explained by ryanodine receptors only. We propose that the findings will guide us to an understanding of TRPC1 channels as modulators of [Ca2+]i and contractility in cardiomyocytes.

A slow transcription rate causes embryonic lethality and perturbs kinetic coupling of neuronal genes.

The rate of RNA polymerase II (RNAPII) elongation has an important role in the control of alternative splicing (AS); however, the in vivo consequences of an altered elongation rate are unknown. Here, we generated mouse embryonic stem cells (ESCs) knocked in for a slow elongating form of RNAPII We show that a reduced transcriptional elongation rate results in early embryonic lethality in mice. Focusing on neuronal differentiation as a model, we observed that slow elongation impairs development of the neural lineage from ESCs, which is accompanied by changes in AS and in gene expression along this pathway. In particular, we found a crucial role for RNAPII elongation rate in transcription and splicing of long neuronal genes involved in synapse signaling. The impact of the kinetic coupling of RNAPII elongation rate with AS is greater in ESC-differentiated neurons than in pluripotent cells. Our results demonstrate the requirement for an appropriate transcriptional elongation rate to ensure proper gene expression and to regulate AS during development.

IL-27p28 Production by XCR1+ Dendritic Cells and Monocytes Effectively Predicts Adjuvant-Elicited CD8+ T Cell Responses.

It is well accepted that the innate response is a necessary prerequisite to the formation of the adaptive response. This is true for T cell responses against infections or adjuvanted subunit vaccination. However, specific innate parameters with predictive value for the magnitude of an adjuvant-elicited T cell response have yet to be identified. We previously reported how T cell responses induced by subunit vaccination were dependent on the cytokine IL-27. These findings were unexpected, given that T cell responses to an infection typically increase in the absence of IL-27. Using a novel IL-27p28-eGFP reporter mouse, we now show that the degree to which an adjuvant induces IL-27p28 production from dendritic cells and monocytes directly predicts the magnitude of the T cell response elicited. To our knowledge, these data are the first to identify a concrete innate correlate of vaccine-elicited cellular immunity, and they have significant practical and mechanistic implications for subunit vaccine biology.

Physiological and pathophysiological role of transient receptor potential canonical channels in cardiac myocytes.

Transient receptor potential canonical (TRPC) channels constitute a family of seven Ca2+ permeable ion channels, named TRPC1 to 7. These channels are abundantly expressed in the mammalian heart, yet mechanisms underlying activation of TRPC channels and their precise role in cardiac physiology remain poorly understood. In this review, we perused original literature regarding TRPC channels in cardiomyocytes. We first reviewed studies on TRPC channel assembly and sub-cellular localization across multiple species and cell types. Our review indicates that TRPC localization in cardiac cells is still a topic of controversy. We then examined common molecular biology tools used to infer on location and physiological roles of TRPC channels in the heart. We subsequently reviewed pharmacological tools used to modulate TRPC activity in both cardiac and non-cardiac cells. Suggested physiological roles in the heart include modulation of heart rate and sensing of mechanical strain. We examined studies on the contribution of TRPC to cardiac pathophysiology, mainly hypertrophic signaling. Several TRPC channels, particularly TRPC1, 3 and 6 were proposed to play a crucial role in hypertrophic signaling. Finally, we discussed gaps in our understanding of the location and physiological role of TRPC channels in cardiomyocytes. Closing these gaps will be crucial to gain a full understanding of the role of TRPC channels in cardiac pathophysiology and to further explore these channels as targets for treatments for cardiac diseases, in particular, hypertrophy.

MSK1 regulates transcriptional induction of Arc/Arg3.1 in response to neurotrophins.

The immediate early gene activity-regulated cytoskeletal protein (Arc)/Arg3.1 and the neurotrophin brain-derived neurotrophic factor (BDNF) play important roles in synaptic plasticity and learning and memory in the mammalian brain. However, the mechanisms by which BDNF regulates the expression of Arc/Arg3.1 are unclear. In this study, we show that BDNF acts via the ERK1/2 pathway to activate the nuclear kinase mitogen- and stress-activated protein kinase 1 (MSK1). MSK1 then induces Arc/Arg3.1 expression via the phosphorylation of histone H3 at the Arc/Arg3.1 promoter. MSK1 can also phosphorylate the transcription factor cyclic-AMP response element-binding protein (CREB) on Ser133. However, this is not required for BDNF-induced Arc.Arg3.1 transcription as a Ser133Ala knockin mutation had no effect on Arc/Arg3.1 induction. In parallel, ERK1/2 directly activates Arc/Arg3.1 mRNA transcription via at least one serum response element on the promoter, which bind a complex of the Serum Response Factor (SRF) and a Ternary Complex Factor (TCF).

A meta-analysis comparing the risk of metastases in patients with rectal cancer and MRI-detected extramural vascular invasion (mrEMVI) vs mrEMVI-negative cases.

BACKGROUND: Pathological extramural vascular invasion (EMVI) is an independent prognostic factor in rectal cancer, but can also be identified on MRI-detected extramural vascular invasion (mrEMVI). We perform a meta-analysis to determine the risk of metastatic disease at presentation and after surgery in mrEMVI-positive patients compared with negative tumours. METHODS: Electronic databases were searched from January 1980 to March 2016. Conventional meta-analytical techniques were used to provide a summative outcome. Quality assessment of the studies was performed. RESULTS: Six articles reported on mrEMVI in 1262 patients. There were 403 patients in the mrEMVI-positive group and 859 patients in the mrEMVI-negative group. The combined prevalence of mrEMVI-positive tumours was 0.346(range=0.198-0.574). Patients with mrEMVI-positive tumours presented more frequently with metastases compared to mrEMVI-negative tumours (fixed effects model: odds ratio (OR)=5.68, 95% confidence interval (CI) (3.75, 8.61), z=8.21, df=2, P<0.001). Patients who were mrEMVI-positive developed metastases more frequently during follow-up (random effects model: OR=3.91, 95% CI (2.61, 5.86), z=6.63, df=5, P<0.001). CONCLUSIONS: MRI-detected extramural vascular invasion is prevalent in one-third of patients with rectal cancer. MRI-detected extramural vascular invasion is a poor prognostic factor as evidenced by the five-fold increased rate of synchronous metastases, and almost four-fold ongoing risk of developing metastases in follow-up after surgery.

Local staging and assessment of colon cancer with 1.5-T magnetic resonance imaging.

OBJECTIVE:: The aim of this study was to assess the accuracy of 1.5-T MRI in the pre-operative local T and N staging of colon cancer and identification of extramural vascular invasion (EMVI). METHODS:: Between 2010 and 2012, 60 patients with adenocarcinoma of the colon were prospectively recruited at 2 centres. 55 patients were included for final analysis. Patients received pre-operative 1.5-T MRI with high-resolution T2 weighted, gadolinium-enhanced T1 weighted and diffusion-weighted images. These were blindly assessed by two expert radiologists. Accuracy of the T-stage, N-stage and EMVI assessment was evaluated using post-operative histology as the gold standard. RESULTS:: Results are reported for two readers. Identification of T3 disease demonstrated an accuracy of 71% and 51%, sensitivity of 74% and 42% and specificity of 74% and 83%. Identification of N1 disease demonstrated an accuracy of 57% for both readers, sensitivity of 26% and 35% and specificity of 81% and 74%. Identification of EMVI demonstrated an accuracy of 74% and 69%, sensitivity 63% and 26% and specificity 80% and 91%. CONCLUSION:: 1.5-T MRI achieved a moderate accuracy in the local evaluation of colon cancer, but cannot be recommended to replace CT on the basis of this study. ADVANCES IN KNOWLEDGE:: This study confirms that MRI is a viable alternative to CT for the local assessment of colon cancer, but this study does not reproduce the very high accuracy reported in the only other study to assess the accuracy of MRI in colon cancer staging.

Pre-operative staging of rectal cancer: a review of imaging techniques.

INTRODUCTION: The treatment of rectal cancer has diversified in recent years, presenting the clinician and patient with increasingly challenging management decisions. At the heart of this decision-making process are two competing interests; more radical but more morbid treatments which optimize oncological outcome, and less radical treatments which preserve organs and function but may pose a greater risk of disease recurrence. AREAS COVERED: Imaging plays a vital role informing this decision-making process, both by providing prognostic details about the cancer before the start of treatment and by updating this picture as the cancer responds or fails to respond to treatment. There is a range of available imaging modalities, each with its strengths and weaknesses. Optimizing rectal cancer treatment requires a clear understanding of the important questions that imaging needs to answer and the optimum imaging strategy. Expert Commentary: This article provides an evidence-based review of the available imaging techniques and an expert commentary on the best imaging strategy.

The E3 ubiquitin ligase activity of RING1B is not essential for early mouse development.

Polycomb-repressive complex 1 (PRC1) and PRC2 maintain repression at many developmental genes in mouse embryonic stem cells and are required for early development. However, it is still unclear how they are targeted and how they function. We show that the ability of RING1B, a core component of PRC1, to ubiquitinate histone H2A is dispensable for early mouse embryonic development and much of the gene repression activity of PRC1. Our data support a model in which PRC1 and PRC2 reinforce each other's binding but suggest that the key functions of PRC1 lie beyond the enzymatic capabilities of RING1B.

Bismuth incorporation and the role of ordering in GaAsBi/GaAs structures.

The structure and composition of single GaAsBi/GaAs epilayers grown by molecular beam epitaxy were investigated by optical and transmission electron microscopy techniques. Firstly, the GaAsBi layers exhibit two distinct regions and a varying Bi composition profile in the growth direction. In the lower (25 nm) region, the Bi content decays exponentially from an initial maximum value, while the upper region comprises an almost constant Bi content until the end of the layer. Secondly, despite the relatively low Bi content, CuPtB-type ordering was observed both in electron diffraction patterns and in fast Fourier transform reconstructions from high-resolution transmission electron microscopy images. The estimation of the long-range ordering parameter and the development of ordering maps by using geometrical phase algorithms indicate a direct connection between the solubility of Bi and the amount of ordering. The occurrence of both phase separation and atomic ordering has a significant effect on the optical properties of these layers. PACS: 78.55.Cr III-V semiconductors; 68.55.Nq composition and phase identification; 68.55.Ln defects and impurities: doping, implantation, distribution, concentration, etc; 64.75.St phase separation and segregation in.

Biodiversity research in the "big data" era: GigaScience and Pensoft work together to publish the most data-rich species description.

With the publication of the first eukaryotic species description, combining transcriptomic, DNA barcoding, and micro-CT imaging data, GigaScience and Pensoft demonstrate how classical taxonomic description of a new species can be enhanced by applying new generation molecular methods, and novel computing and imaging technologies. This 'holistic' approach in taxonomic description of a new species of cave-dwelling centipede is published in the Biodiversity Data Journal (BDJ), with coordinated data release in the GigaScience GigaDB database.

Metagenomic analysis: the challenge of the data bonanza.

Several thousand metagenomes have already been sequenced, and this number is set to grow rapidly in the forthcoming years as the uptake of high-throughput sequencing technologies continues. Hand-in-hand with this data bonanza comes the computationally overwhelming task of analysis. Herein, we describe some of the bioinformatic approaches currently used by metagenomics researchers to analyze their data, the issues they face and the steps that could be taken to help overcome these challenges.

Adverse features on rectal MRI identify a high-risk group that may benefit from more intensive preoperative staging and treatment.

BACKGROUND: Magnetic resonance imaging (MRI) is highly accurate in local staging of rectal cancer. It can identify features known to be associated with increased risk of metastatic disease. We evaluated the incidence of synchronous metastatic disease on fludeoxyglucose-positron emission tomography/computed tomography (FDG-PET/CT) and contrast-enhanced multiple-row detector computed tomography (ceMDCT) in MRI-stratified high- and low-risk rectal cancers. The aim was to determine the incidence of synchronous metastatic disease according to MRI risk features. METHODS: A total of 236 patients with rectal cancer were recruited to a study evaluating FDG-PET/CT. All patients underwent MRI staging and were stratified into high and low risk (high risk: extramural venous invasion, extramural spread of >5 mm or T4, involved circumferential resection margin or intersphincteric plane involved for low rectal tumors). Confirmed metastases were those identified on FDG-PET/CT and ceMDCT. RESULTS: Imaging data were available for 230 (97.5%) of 236 patients. Incidence of confirmed distant metastases was significantly greater in the MRI high-risk group, with 28 (20.7%) of 135 (95% confidence interval [CI] 14.8-28.3), versus the low-risk group, with 4 (4.2%) of 95 (95% CI 1.7-10.3) (odds ratio 6.0, 95% CI 2.0-17.6, P<0.001). CONCLUSIONS: Adverse features found on rectal MRI identify patients at increased risk of synchronous metastatic disease. This group may benefit from additional preoperative investigation for synchronous metastases such as FDG-PET/CT or liver MRI and from alternative neoadjuvant chemotherapy regimens including induction chemotherapy.

Targeted disruption of TgPhIL1 in Toxoplasma gondii results in altered parasite morphology and fitness.

The inner membrane complex (IMC), a series of flattened vesicles at the periphery of apicomplexan parasites, is thought to be important for parasite shape, motility and replication, but few of the IMC proteins that function in these processes have been identified. TgPhIL1, a Toxoplasma gondii protein that was previously identified through photosensitized labeling with 5-[(125)I] iodonapthaline-1-azide, associates with the IMC and/or underlying cytoskeleton and is concentrated at the apical end of the parasite. Orthologs of TgPhIL1 are found in other apicomplexans, but the function of this conserved protein family is unknown. As a first step towards determining the function of TgPhIL1 and its orthologs, we generated a T. gondii parasite line in which the single copy of TgPhIL1 was disrupted by homologous recombination. The TgPhIL1 knockout parasites have a distinctly different morphology than wild-type parasites, and normal shape is restored in the knockout background after complementation with the wild-type allele. The knockout parasites are outcompeted in culture by parasites expressing functional TgPhIL1, and they generate a reduced parasite load in the spleen and liver of infected mice. These findings demonstrate a role for TgPhIL1 in the morphology, growth and fitness of T. gondii tachyzoites.

Generation of a conditional CREB Ser133Ala knockin mouse.

Phosphorylation of Ser133 in the transcription factor CREB is an important mechanism for regulating its transcriptional activity, however recent work has suggested significant roles for other regulatory inputs into CREB. To allow study of this in vivo, we have generated a Ser133 to alanine knockin mutation in the mouse CREB locus. As CREB knockout is perinatal lethal, a minigene strategy was used to allow conditional knockin of the Ser133Ala mutation in adult mice using Cre recombinase. While some expression of the mutated protein was observed prior to Cre expression, following Cre expression in either T cells or neurons only the mutated CREB protein was detected.

Subunit 3 of the COP9 signalosome is poised to facilitate communication between the extracellular matrix and the nucleus through the muscle-specific beta1D integrin.

Yeast two-hybrid analysis (Fields and Song, 1989, Nature, 340:245-246) was used to screen a human heart library to isolate proteins interacting with the adult muscle-specific beta1D integrin but not with beta1A integrin. In addition to previously identified interactions (RACK 1(Liliental and Chang, 1998, Journal of Biological Chemistry, 273:2379-2383) and alpha-actinin (Otey et al., 1990, Journal of Cell Biology, 111:721-729), the authors isolated several novel candidates. These include subunit 3 (CSN3/Sgn3) of the COP9 signalosome complex, cyclins D1, D2, and D3, RanBPM, and a recently identified protein COG8/DOR1. These protein interactions were specific for beta1D integrin, as no binding to beta1A integrin cytoplasmic domain was measurable by two-hybrid analysis. This paper presents the initial characterization of the interaction of CSN3 with beta1D integrin, the localization of CSN3 and the other COP9 signalosome subunits in embryonic and adult cardiac myocytes and their response to muscle cell differentiation.

Mutation analysis of 24 known cancer genes in the NCI-60 cell line set.

The panel of 60 human cancer cell lines (the NCI-60) assembled by the National Cancer Institute for anticancer drug discovery is a widely used resource. The NCI-60 has been characterized pharmacologically and at the molecular level more extensively than any other set of cell lines. However, no systematic mutation analysis of genes causally implicated in oncogenesis has been reported. This study reports the sequence analysis of 24 known cancer genes in the NCI-60 and an assessment of 4 of the 24 genes for homozygous deletions. One hundred thirty-seven oncogenic mutations were identified in 14 (APC, BRAF, CDKN2, CTNNB1, HRAS, KRAS, NRAS, SMAD4, PIK3CA, PTEN, RB1, STK11, TP53, and VHL) of the 24 genes. All lines have at least one mutation among the cancer genes examined, with most lines (73%) having more than one. Identification of those cancer genes mutated in the NCI-60, in combination with pharmacologic and molecular profiles of the cells, will allow for more informed interpretation of anticancer agent screening and will enhance the use of the NCI-60 cell lines for molecularly targeted screens.