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Cardiac resynchronization therapy (CRT), the application of biventricular stimulation to correct discoordinate contraction, is the only heart failure treatment that enhances acute and chronic systolic function, increases cardiac work, and reduces mortality. Resting myocyte function also increases after CRT despite only modest improvement in calcium transients, suggesting that CRT may enhance myofilament calcium responsiveness. To test this hypothesis, we examined adult dogs subjected to tachypacing-induced heart failure for 6 weeks, concurrent with ventricular dyssynchrony ([HF.sub.dys]) or CRT. Myofilament force-calcium relationships were measured in skinned trabeculae and/or myocytes. Compared with control, maximal calcium-activated force and calcium sensitivity declined globally in HFdys; however, CRT restored both. Phosphatase PP1 induced calcium desensitization in control and CRT-treated cells, while [HF.sub.dys] cells were unaffected, implying that CRT enhances myofilament phosphorylation. Proteomics revealed phosphorylation sites on Z-disk and M-band proteins, which were predicted to be targets of glycogen synthase kinase-3β (GSK-3β ). We found that GSK-3β was deactivated in [HF.sub.dys] and reactivated by CRT. Mass spectrometry of myofilament proteins from [HF.sub.dys] animals incubated with GSK-3β confirmed GSK-3β -dependent phosphorylation at many of the same sites observed with CRT. GSK-3β restored calcium sensitivity in HFdys, but did not affect control or CRT cells. These data indicate that CRT improves calcium responsiveness of myofilaments following [HF.sub.dys] through GSK-3β reactivation, identifying a therapeutic approach to enhancing contractile function., Introduction For the past 30 years or more, the quest to develop treatments that directly improve systolic performance of the failing heart while also conferring long-term survival benefits has been [...]

Research has been conducted on cellulose binding and hydrolysis by cellulases. The cellulase steady-state and pre-steady-state kinetics have been investigated via the study of the prepared 4-methyl-7-thioumbelliferyl-beta-D-cellobioside and the results demonstrate that it is a ground-state substrate analogue for 4-methylumbelliferyl-beta-D-cellobioside.

Duchenne muscular dystrophy (DMD) is an X-linked disorder with dystrophin loss that results in skeletal and cardiacmuscleweakening and early death. Loss of the dystrophin-sarcoglycan complex delocalizes nitric oxide synthase (NOS) to alter its signaling, and augments mechanosensitive intracellular Ca2+ influx. The latter has been coupled to hyperactivation of the nonselective cation channel, transient receptor potential canonical channel 6 (Trpc6), in isolated myocytes. As Ca2+ also activates NOS, we hypothesized that Trpc6 would help to mediate nitric oxide (NO) dysregulation and that this would be manifest in increased myocardial S-nitrosylation, a posttranslational modification increasingly implicated in neurodegenerative, inflammatory, and muscle disease. Using a recently developed dual-labeling proteomic strategy, we identified 1,276 S-nitrosylated cysteine residues [S-nitrosothiol (SNO)] on 491 proteins in resting hearts from a mouse model of DMD (dmdmdx:utrn+/-). These largely consisted of mitochondrial proteins, metabolic regulators, and sarcomeric proteins, with 80%of them also modified in wild type (WT). S-nitrosylation levels, however, were increased in DMD. Genetic deletion of Trpc6 in this model (dmdmdx:utrn+/-:trpc6-/-) reversed ∼70% of these changes. Trpc6 deletion also ameliorated left ventricular dilation, improved cardiac function, and tended to reduce fibrosis. Furthermore, under catecholamine stimulation, which also increases NO synthesis and intracellular Ca2+ along with cardiac workload, the hypernitrosylated state remained as it did at baseline. However, the impact of Trpc6 deletion on the SNO proteome became less marked. These findings reveal a role for Trpc6-mediated hypernitrosylation in dmdmdx:utrn+/- mice and support accumulating evidence that implicates nitrosative stress in cardiac and muscle disease. [ABSTRACT FROM AUTHOR]

Protein kinase C-δ (PKCδ) is a signalling kinase that regulates many cellular responses.Although most studies focus on allosteric mechanisms that activate PKCδ at membranes, PKCδ also is controlled via multi-site phosphorylation [Gong et al. (2015) Mol. Cell. Biol. 35, 1727-1740]. The present study uses MS-based methods to identify PKCδ phosphorylation at Thr50 and Ser645 (in resting and PMA-treated cardiomyocytes) as well as Thr37, Thr38, Ser130, Thr164, Thr211, Thr215, Ser218, Thr295, Ser299 and Thr656 (as sites that increase with PMA). We focused on the consequences of phosphorylation at Ser130 and Thr141 (sites just N-terminal to the pseudosubstrate domain).We show that S130D and T141E substitutions co-operate to increase PKCδ's basal lipid-independent activity and that Ser130/Thr141 di-phosphorylation influences PKCδ's substrate specificity. We recently reported that PKCδ preferentially phosphorylates substrates with a phosphoacceptor serine residue and that this is due to constitutive phosphorylation at Ser357, an ATP-positioning G-loop site that limits PKCδ's threonine kinase activity [Gong et al. (2015) Mol. Cell. Biol. 35, 1727-1740]. The present study shows that S130D and T141E substitutions increase PKCδ's threonine kinase activity indirectly by decreasing G loop phosphorylation at Ser357. A S130F substitution [that mimics a S130F single-nt polymorphism (SNP) identified in some human populations] also increases PKCδ's maximal lipid-dependent catalytic activity and confers threonine kinase activity. Finally, we show that Ser130/Thr141 phosphorylations relieve auto-inhibitory constraints that limit PKCδ's activity and substrate specificity in a cell-based context. Since phosphorylation sites map to similar positions relative to the pseudosubstrate domains of other PKCs, our results suggest that phosphorylation in this region of the enzyme may constitute a general mechanism to control PKC isoform activity. [ABSTRACT FROM AUTHOR]

Transfer RNA (tRNA) modifications are crucial for protein synthesis, but their position-specific physiological roles remain poorly understood. Here we investigate the impact of N4-acetylcytidine (ac 4 C), a highly conserved tRNA modification, using a Thumpd1 knockout mouse model. We find that loss of Thumpd1-dependent tRNA acetylation leads to reduced levels of tRNA Leu , increased ribosome stalling, and activation of eIF2α phosphorylation. Thumpd1 knockout mice exhibit growth defects and sterility. Remarkably, concurrent knockout of Thumpd1 and the stress-sensing kinase Gcn2 causes penetrant postnatal lethality, indicating a critical genetic interaction. Our findings demonstrate that a modification restricted to a single position within type II cytosolic tRNAs can regulate ribosome-mediated stress signaling in mammalian organisms, with implications for our understanding of translation control as well as therapeutic interventions., Competing Interests: DECLARATION OF INTERESTS The authors have no positions or financial interests to declare.

The tumor microenvironment consists of resident tumor cells organized within a compositionally diverse, three-dimensional (3D) extracellular matrix (ECM) network that cannot be replicated in vitro using bottom-up synthesis. We report a new self-assembly system to engineer ECM-rich 3D MatriSpheres wherein tumor cells actively organize and concentrate microgram quantities of decellularized ECM dispersions which modulate cell phenotype. 3D colorectal cancer (CRC) MatriSpheres were created using decellularized small intestine submucosa (SIS) as an orthotopic ECM source that had greater proteomic homology to CRC tumor ECM than traditional ECM formulations such as Matrigel. SIS ECM was rapidly concentrated from its environment and assembled into ECM-rich 3D stroma-like regions by mouse and human CRC cell lines within 4-5 days via a mechanism that was rheologically distinct from bulk hydrogel formation. Both ECM organization and transcriptional regulation by 3D ECM cues affected programs of malignancy, lipid metabolism, and immunoregulation that corresponded with an in vivo MC38 tumor cell subpopulation identified via single cell RNA sequencing. This 3D modeling approach stimulates tumor specific tissue morphogenesis that incorporates the complexities of both cancer cell and ECM compartments in a scalable, spontaneous assembly process that may further facilitate precision medicine., Competing Interests: Competing interests: None

Data independent acquisition (DIA also termed SWATH) is an emerging technology in the field of mass spectrometry based proteomics. Although the concept of DIA has been around for over a decade, the recent advancements, in particular the speed of acquisition, of mass analyzers have pushed the technique into the spotlight and allowed for high-quality DIA data to be routinely acquired by proteomics labs. In this chapter we will discuss the protocols used for DIA acquisition using the Sciex TripleTOF mass spectrometers and data analysis using the Sciex processing software.