Sucrose Nonfermenting-Related Kinase Enzyme-Mediated Rho-Associated Kinase Signaling is Responsible for Cardiac Function.

BACKGROUND: Cardiac metabolism is critical for the functioning of the heart, and disturbance in this homeostasis is likely to influence cardiac disorders or cardiomyopathy. Our laboratory has previously shown that SNRK (sucrose nonfermenting related kinase) enzyme, which belongs to the AMPK (adenosine monophosphate-activated kinase) family, was essential for cardiac metabolism in mammals. Snrk global homozygous knockout (KO) mice die at postnatal day 0, and conditional deletion of Snrk in cardiomyocytes (Snrk cmcKO) leads to cardiac failure and death by 8 to 10 months. METHODS AND RESULTS: We performed additional cardiac functional studies using echocardiography and identified further cardiac functional deficits in Snrk cmcKO mice. Nuclear magnetic resonance-based metabolomics analysis identified key metabolic pathway deficits in SNRK knockdown cardiomyocytes in vitro. Specifically, metabolites involved in lipid metabolism and oxidative phosphorylation are altered, and perturbations in these pathways can result in cardiac function deficits and heart failure. A phosphopeptide-based proteomic screen identified ROCK (Rho-associated kinase) as a putative substrate for SNRK, and mass spec-based fragment analysis confirmed key amino acid residues on ROCK that are phosphorylated by SNRK. Western blot analysis on heart lysates from Snrk cmcKO adult mice and SNRK knockdown cardiomyocytes showed increased ROCK activity. In addition, in vivo inhibition of ROCK partially rescued the in vivo Snrk cmcKO cardiac function deficits. CONCLUSIONS: Collectively, our data suggest that SNRK in cardiomyocytes is responsible for maintaining cardiac metabolic homeostasis, which is mediated in part by ROCK, and alteration of this homeostasis influences cardiac function in the adult heart

Attachment of Salmonella strains to a plant cell wall model is modulated by surface characteristics and not by specific carbohydrate interactions

Background: Processing of fresh produce exposes cut surfaces of plant cell walls that then become vulnerable to human foodborne pathogen attachment and contamination, particularly by Salmonella enterica. Plant cell walls are mainly composed of the polysaccharides cellulose, pectin and hemicelluloses (predominantly xyloglucan). Our previous work used bacterial cellulose-based plant cell wall models to study the interaction between Salmonella and the various plant cell wall components. We demonstrated that Salmonella attachment was favoured in the presence of pectin while xyloglucan had no effect on its attachment. Xyloglucan significantly increased the attachment of Salmonella cells to the plant cell wall model only when it was in association with pectin. In this study, we investigate whether the plant cell wall polysaccharides mediate Salmonella attachment to the bacterial cellulose-based plant cell wall models through specific carbohydrate interactions or through the effects of carbohydrates on the physical characteristics of the attachment surface. Results: We found that none of the monosaccharides that make up the plant cell wall polysaccharides specifically inhibit Salmonella attachment to the bacterial cellulose-based plant cell wall models. Confocal laser scanning microscopy showed that Salmonella cells can penetrate and attach within the tightly arranged bacterial cellulose network. Analysis of images obtained from atomic force microscopy revealed that the bacterial cellulose-pectin-xyloglucan composite with 0.3 % (w/v) xyloglucan, previously shown to have the highest number of Salmonella cells attached to it, had significantly thicker cellulose fibrils compared to other composites. Scanning electron microscopy images also showed that the bacterial cellulose and bacterial cellulose-xyloglucan composites were more porous when compared to the other composites containing pectin. Conclusions: Our study found that the attachment of Salmonella cells to cut plant cell walls was not mediated by specific carbohydrate interactions. This suggests that the attachment of Salmonella strains to the plant cell wall models were more dependent on the structural characteristics of the attachment surface. Pectin reduces the porosity and space between cellulose fibrils, which then forms a matrix that is able to retain Salmonella cells within the bacterial cellulose network. When present with pectin, xyloglucan provides a greater surface for Salmonella cells to attach through the thickening of cellulose fibrils

Endothelial Cell Surface Expressed Chemotaxis and Apoptosis Regulator (ECSCR) Regulates Lipolysis in White Adipocytes via the PTEN/AKT Signaling Pathway.

Elevated plasma triglycerides are associated with increased susceptibility to heart disease and stroke, but the mechanisms behind this relationship are unclear. A clearer understanding of gene products which influence plasma triglycerides might help identify new therapeutic targets for these diseases. The Endothelial Cell Surface expressed Chemotaxis and apoptosis Regulator (ECSCR) was initially studied as an endothelial cell marker, but has recently been identified in white adipocytes, the primary storage cell type for triglycerides. Here we confirm ECSCR expression in white adipocytes and show that Ecscr knockout mice show elevated fasting plasma triglycerides. At a cellular level, cultured 3T3-L1 adipocytes silenced for Ecscr show a blunted Akt phosphorylation response. Additionally we show that the phosphatase and tensin homology containing (PTEN) lipid phosphatase association with ECSCR is increased by insulin stimulation. These data suggest a scenario by which ECSCR contributes to control of white adipocyte lipolysis. In this scenario, white adipocytes lacking Ecscr display elevated PTEN activity, thereby reducing AKT activation and impairing insulin-mediated suppression of lipolysis. Collectively, these results suggest that ECSCR plays a critical function in regulating lipolysis in white adipose tissue

Optimized Protocol for Isolation of Small Extracellular Vesicles from Human and Murine Lymphoid Tissues

Small extracellular vesicles (sEVs) are nanoparticles responsible for cell-to-cell communication released by healthy and cancer cells. Different roles have been described for sEVs in physiological and pathological contexts, including acceleration of tissue regeneration, modulation of tumor microenvironment, or premetastatic niche formation, and they are discussed as promising biomarkers for diagnosis and prognosis in body fluids. Although efforts have been made to standardize techniques for isolation and characterization of sEVs, current protocols often result in co-isolation of soluble protein or lipid complexes and of other extracellular vesicles. The risk of contaminated preparations is particularly high when isolating sEVs from tissues. As a consequence, the interpretation of data aiming at understanding the functional role of sEVs remains challenging and inconsistent. Here, we report an optimized protocol for isolation of sEVs from human and murine lymphoid tissues. sEVs from freshly resected human lymph nodes and murine spleens were isolated comparing two different approaches—(1) ultracentrifugation on a sucrose density cushion and (2) combined ultracentrifugation with size-exclusion chromatography. The purity of sEV preparations was analyzed using state-of-the-art techniques, including immunoblots, nanoparticle tracking analysis, and electron microscopy. Our results clearly demonstrate the superiority of size-exclusion chromatography, which resulted in a higher yield and purity of sEVs, and we show that their functionality alters significantly between the two isolation protocols

Dual Specificity Phosphatase 5 Is Essential for T Cell Survival

<div><p>The mitogen-activated protein kinase (MAPK) pathway regulates many key cellular processes such as differentiation, apoptosis, and survival. The final proteins in this pathway, ERK1/2, are regulated by dual specificity phosphatase 5 (DUSP5). DUSP5 is a nuclear, inducible phosphatase with high affinity and fidelity for ERK1/2. By regulating the final step in the MAPK signaling cascade, DUSP5 exerts strong regulatory control over a central cellular pathway. Like other DUSPs, DUSP5 plays an important role in immune function. In this study, we have utilized new knockout mouse reagents to explore its function further. We demonstrate that global loss of DUSP5 does not result in any gross phenotypic changes. However, loss of DUSP5 affects memory/effector CD8⁺ T cell populations in response to acute viral infection. Specifically, <i>Dusp5</i>^<i>-/-</i> mice have decreased proportions of short-lived effector cells (SLECs) and increased proportions of memory precursor effector cells (MPECs) in response to infection. Further, we show that this phenotype is T cell intrinsic; a bone marrow chimera model restricting loss of DUSP5 to the CD8⁺ T cell compartment displays a similar phenotype. <i>Dusp5</i>^<i>-/-</i> T cells also display increased proliferation, increased apoptosis, and altered metabolic profiles, suggesting that DUSP5 is a pro-survival protein in T cells.</p></div

Tissue expression of ECSCR protein in mouse and human white adipocytes.

<p><b>A.</b> Anti-ECSCR western blot of adult male mouse tissues. Results are representative of lysates from three independent animals. WAT, epididymal white adipose tissue. Tub, tubulin loading control. <b>B.</b> ECSCR protein in cultured 3T3-L1 cells during differentiation to adipocytes. ECSCR protein is compared to reference lipid droplet marker perilipin, with GAPDH as loading control. <b>C.</b> Fractionation of freshly resected human white adipose tissue into mature adipocytes (buoyant fraction) and stromal vascular fraction (SVF). ECSCR is prominent on lipid-bearing, buoyant white adipocytes. SVF antigen CD34 is detected only in the pelleted fraction. <b>D.</b> Confocal micrographs of human muscle and WAT tissue sections stained with UEA-1 lectin (Red) to show vasculature and, anti-ECSCR antiserum (Green). Top: Skeletal muscle. ECSCR is present on the capillaries and on an unidentified resident cell type. Striated muscle fibers are negative for ECSCR. Bottom: White adipose tissue. Adipocyte profiles, immune reactive for ECSCR but negative for UEA lectin, are indicated with (*).</p

<i>Ecscr</i> silenced 3T3-L1 adipocytes and <i>ex vivo</i> differentiated <i>Ecscr</i>^-/- adipocyte cells show reduced Akt phosphorylation.

<p><b>A-C.</b> 3T3-L1 pre-adipocytes were transduced with lentiviral control shRNA or <i>Ecscr</i>-targeting shRNA and allowed to differentiate after puromycin selection. Adipocytes were then stimulated with insulin or isoproterenol and analyzed for lipolysis pathways (<b>A and B</b>), and glycerol release (<b>C</b>). Results are presented as mean +/- SEM of three independent experiments. Similar results were obtained with a second, independent, <i>Ecscr</i> targeting shRNA (not shown). <b>D and E.</b> Stromal vascular fraction cells obtained from wild-type (left) or <i>Ecscr</i>^-/- mice (right) were differentiated into mature adipocytes, stimulated as indicated, then analyzed for lipolysis relevant phospho-epitopes. <b>D.</b> Ex-vivo adipocytes from <i>Ecscr</i>^-/-mice show deficient insulin-dependent AKT activation. <b>E.</b> Densitometry quantitation of pAKT (s473) blots (mean +/- SEM of three independent experiments. For panels <b>B,C</b> and <b>E, *, p<0.05, **, p<0.01, Student’s T-test.</b></p