Regulation of skeletal muscle creatine kinase from a hibernating mammal

Control over skeletal muscle energetics is critical in hibernation to sustain viability over weeks of cold torpor and to support shivering thermogenesis during arousal. Creatine kinase (CK)1Abbreviations used: CK, creatine kinase; Cr, creatine; PCr, phosphocreatine; PKC, protein kinase C.1 has a key role in muscle energetics and this study analyzes muscle CK from ground squirrels, Spermophilus richardsonii. CK activity was ∼20% lower during hibernation than in euthermia, as was CK protein whereas CK mRNA was reduced by ∼70%. Hibernator CK showed reduced affinity for ATP and creatine, compared with euthermic CK. Incubations that promoted endogenous protein kinase or phosphatase action, coupled with ion exchange chromatography to separate high and low phosphate forms, showed that soluble CK from euthermic squirrels was a mix of phosphorylated and dephosphorylated forms whereas only phospho-CK was detected in hibernating animals. High and low phosphate CK forms had different affinities for ATP and creatine substrates but did not differ in stability to urea denaturation. About 20-25% of CK was bound to the insoluble fraction of muscle and bound CK showed different kinetic responses to kinase and phosphatase treatments

Suppression of MAPKAPK2 during mammalian hibernation

Metabolic signaling coordinates the transition by hibernating mammals from euthermia into profound torpor. Organ-specific responses by activated p38 mitogen activated protein kinase (MAPK) are known to contribute to this transition. Therefore, we hypothesized that the MAPK-activated protein kinase-2 (MAPKAPK2), a downstream target of p38 MAPK, would also be active in establishing the torpid state. Kinetic parameters of MAPKAPK2 from skeletal muscle of Richardson's ground squirrels, Spermophilus richardsonii, were analyzed using a fluorescence assay. MAPKAPK2 activity was 27.4±1.27pmol/min/mg in muscle from euthermic squirrels and decreased by ∼63% during cold torpor, while total protein levels were unchanged (as assessed by immunoblotting). In vitro treatment of MAPKAPK2 via stimulation of endogenous phosphatases and addition of commercial alkaline phosphatase decreased enzyme activity to only ∼3-5% of its original value in muscle extracts from both euthermic and hibernating squirrels suggesting that posttranslational modification suppresses MAPKAPK2 during the transition from euthermic to torpid states. Enzyme S0.5 and nH values for ATP and peptide substrates changed significantly between euthermia and torpor, and also between assays at 22 versus 10°C but, kinetic parameters were actually closely conserved when values for the euthermic enzyme at 22°C were directly compared with the hibernator enzyme at 10°C. Arrhenius plots showed significantly different activation energies of 40.8±0.7 and 54.3±2.7kJ/mol for the muscle enzyme from euthermic versus torpid animals, respectively but MAPKAPK2 from the two physiological states showed no difference in sensitivity to urea denaturation. Overall, the results show that total activity of MAPKAPK2 is in fact reduced, despite previous findings of p38 MAPK activation, and kinetic parameters are altered when ground squirrels enter torpor but protein stability is not apparently changed. The data suggest that MAPKAPK2 suppression may have a significant role in the differential regulation of muscle target proteins when ground squirrels enter torpor

Development and Evaluation of Novel Aptamers Specific for Human PD1 Using Hybrid Systematic Evolution of Ligands by Exponential Enrichment Approach

Background: Programmed cell death protein 1 (PD1; also known as CD279) is an inhibitory receptor on T lymphocytes interacting with PD1-ligand 1 and PD1-ligand 2 in the synapse of T cells and antigen presenting cells (APC) resulting in the suppression of T cell activity. Systematic evolution of ligands by exponential enrichment (SELEX) is a method for generating aptamers which can bind specifically to the target of interest. PD-1 antagonistic aptamers could introduce an attractive alternative over the antibody-based treatments due to the distinguished advantages of aptamers including small size and efficient tissue penetration, low cost, lack of immunogenicity, and ease of manufacturing. Methods: Here, we developed single-stranded DNA aptamers which bind specifically to the human extracellular domain of PD-1. We performed hybrid SELEX, a combination of targeting of recombinant proteins and cell membrane expressed PD1 to select and identify specific aptamers and for the first time, homology of aptamer sequences selected from protein and cell SELEX pool have been evaluated in this study. Results: C42�aptamer, one of the selected aptamers, could specifically bind to human PD1 with dissociation constant in the nanomolar range. Although the developed aptamer inhibited binding of PD1 to PD-L1 but it was not able to restore the cell proliferation and cytokine production of the CD8+ CD279+ T cells. Conclusion: Further studies are required to assess the therapeutic potential of C42 aptamer and other aptamers developed in this study. The introduced PD1 specific aptamers can be used for specific detection of PD1 in diagnostic assay such as immunohistochemistry and targeted drug delivery to PD+ T cells. © 2020, © 2020 Taylor & Francis Group, LLC

Improved anticancer efficiency of Mitoxantrone by Curcumin loaded PLGA nanoparticles targeted with AS1411 aptamer

Objective(s): Mitoxantrone (MTX) is one of the most commonly used chemotherapeutic agents for treatment of different cancers. However, prolonged treatment with MTX results in unwanted side effects and drug resistant cancer cells. Combination therapies and exploiting of targeted nanoparticles have the potential of improving the efficiency of drug treatment as well as reducing the side effects. Curcumin (CUR) is a biological molecules with anticancer property. In this study, we investigated whether targeted PLGA (Poly Lactic-co-Glycolic Acid)-CUR nanoparticles (NPs) can reinforce the effect of MTX on breast cancer cells. Materials and Methods: PLGA NPs containing CUR targeted with AS1411 aptamer were prepared by single emulsion evaporation method. Physicochemical properties of NPs were investigated. The cytotoxicity of non-targeted and targeted NPs along with MTX was evaluated on MCF7, 4T1 and L929 cell lines. Results: The results showed that PLGA-CUR NPs were synthetized with an average encapsulation efficiency of 66 with a mean size of 18

Conformational switch of insulin-binding aptamer into G-quadruplex induced by K⁺ and Na⁺: an experimental and theoretical approach

<div><p>Guanine-rich sequences can form the G-quadruplex structure in the presence of specific metal ions. Here, circular dichroism, UV–vis absorption, fluorescence, and molecular dynamics simulation studies revealed that insulin-binding aptamer (IBA) could form an intramolecular G-quadruplex structure after binding K⁺. Circular dichroism (CD) spectra demonstrated that IBA could fold into a parallel G-quadruplex with a strong positive peak at 263 nm. Analysis of equilibrium titration data revealed that cation binding was cooperative with the Hill coefficient of 2.01 in K⁺ and 1.90 in Na⁺. Thermal denaturation assays indicated that K⁺-induced G-quadruplex is more stable than Na⁺-induced structure. Folding of IBA into G-quadruplex leading to the contact quenching occurs as a result of the formation of a nonfluorescent complex between donor and acceptor. Based on fluorescence quenching of IBA folding, a potassium-sensing aptasensor in the range of 0–1.4 mM was proposed. Since the quenching process was predominantly static, the binding constant and the number of binding sites were determined. In this research, based on the experimental data, the initial model of IBA G-quadruplex was constructed by molecular modeling method. The modeling structure of IBA is an intramolecular parallel-strand quadruplex conformation with two guanine tetrads. The extended molecular dynamics simulation for the model indicated that the G-quadruplex maintains its structure very well in aqueous solution in presence of K⁺ in the central cavity. In contrast, it was demonstrated that the G-quadruplex structure of model in the water collapses in absence of this cation.</p></div

Design and implementation of the morphoSys reconfigurable computing processor

Abstract. In this paper, we describe the implementation of MorphoSys, a reconfigurable processing system targeted at data-parallel and computation-intensive applications. The MorphoSys architecture consists of a reconfigurable component (an array of reconfigurable cells) combined with a RISC control processor and a high bandwidth memory interface. We briefly discuss the system-level model, array architecture, and control processor. Next, we present the detailed design implementation and the various aspects of physical layout of different subblocks of MorphoSys. The physical layout was constrained for 100 MHz operation, with low power consumption, and was implemented using 0.35 m, four metal layer CMOS (3.3 Volts) technology. We provide simulation results for the MorphoSys architecture (based on VHDL model) for some typical data-parallel applications (video compression and automatic target recognition). The results indicate that the MorphoSys system can achieve significantly better performance for most of these applications in comparison with other systems and processors. 1