Turing's diffusion-driven instability for the standard two species reaction-diffusion system is only achievable under well-known and rather restrictive conditions on both the diffusion rates and the kinetic parameters, which necessitates the pairing of a self-activator with a self-inhibitor. In this study we generalize the standard two-species model by considering the case where the reactants can bind to an immobile substrate, for instance extra-cellular matrix, and investigate the influence of this dynamics on Turing's diffusion-driven instability. Such systems have been previously studied on the grounds that binding of the self-activator to a substrate may effectively reduce its diffusion rate and thus induce a Turing instability for species with equal diffusion coefficients, as originally demonstrated by Lengyel and Epstein (1992) under the assumption that the bound state dynamics occurs on a fast timescale. We, however, analyse the full system without any separation of timescales and demonstrate that the full system also allows a relaxation of the standard constraints on the reaction kinetics for the Turing instability, increasing the type of interactions that could give rise to spatial patterning. In particular, we show that two self-activators can undertake a diffusively driven instability in the presence of a binding immobile substrate, highlighting that the interactions required of a putative biological Turing instability need not be associated with a self-activator-self-inhibitor morphogen pair.

• Keywords
• Morphogen, Non-diffusive substrate, Pattern formation, Turing instability,
• MeSH
• Models, Biological * MeSH
• Diffusion MeSH
• Kinetics MeSH
• Numerical Analysis, Computer-Assisted MeSH
• Substrate Specificity MeSH
• Publication type
• Journal Article MeSH

The 3'-5', 3'-5' cyclic dinucleotides (3'3'CDNs) are bacterial second messengers that can also bind to the stimulator of interferon genes (STING) adaptor protein in vertebrates and activate the host innate immunity. Here, we profiled the substrate specificity of four bacterial dinucleotide synthases from Vibrio cholerae (DncV), Bacillus thuringiensis (btDisA), Escherichia coli (dgcZ), and Thermotoga maritima (tDGC) using a library of 33 nucleoside-5'-triphosphate analogues and then employed these enzymes to synthesize 24 3'3'CDNs. The STING affinity of CDNs was evaluated in cell-based and biochemical assays, and their ability to induce cytokines was determined by employing human peripheral blood mononuclear cells. Interestingly, the prepared heterodimeric 3'3'CDNs bound to the STING much better than their homodimeric counterparts and showed similar or better potency than bacterial 3'3'CDNs. We also rationalized the experimental findings by in-depth STING-CDN structure-activity correlations by dissecting computed interaction free energies into a set of well-defined and intuitive terms. To this aim, we employed state-of-the-art methods of computational chemistry, such as quantum mechanics/molecular mechanics (QM/MM) calculations, and complemented the computed results with the {STING:3'3'c-di-ara-AMP} X-ray crystallographic structure. QM/MM identified three outliers (mostly homodimers) for which we have no clear explanation of their impaired binding with respect to their heterodimeric counterparts, whereas the R2 = 0.7 correlation between the computed ΔG'int_rel and experimental ΔTm's for the remaining ligands has been very encouraging.

• MeSH
• Bacillus thuringiensis enzymology   ultrastructure   MeSH
• Cytokines chemistry   genetics   MeSH
• Escherichia coli enzymology   ultrastructure   MeSH
• Crystallography, X-Ray MeSH
• Quantum Theory MeSH
• Leukocytes, Mononuclear chemistry   enzymology   MeSH
• Humans MeSH
• Membrane Proteins chemistry   genetics   ultrastructure   MeSH
• Nucleotides biosynthesis   chemistry   genetics   MeSH
• Immunity, Innate genetics   MeSH
• Substrate Specificity MeSH
• Thermotoga maritima enzymology   ultrastructure   MeSH
• Vibrio cholerae enzymology   ultrastructure   MeSH
• Structure-Activity Relationship * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cytokines MeSH
• Membrane Proteins MeSH
• Nucleotides MeSH
• STING1 protein, human MeSH Browser

Organic dye-tagged lipid analogs are essential for many fluorescence-based investigations of complex membrane structures, especially when using advanced microscopy approaches. However, lipid analogs may interfere with membrane structure and dynamics, and it is not obvious that the properties of lipid analogs would match those of non-labeled host lipids. In this work, we bridged atomistic simulations with super-resolution imaging experiments and biomimetic membranes to assess the performance of commonly used sphingomyelin-based lipid analogs. The objective was to compare, on equal footing, the relative strengths and weaknesses of acyl chain labeling, headgroup labeling, and labeling based on poly-ethyl-glycol (PEG) linkers in determining biomembrane properties. We observed that the most appropriate strategy to minimize dye-induced membrane perturbations and to allow consideration of Brownian-like diffusion in liquid-ordered membrane environments is to decouple the dye from a membrane by a PEG linker attached to a lipid headgroup. Yet, while the use of PEG linkers may sound a rational and even an obvious approach to explore membrane dynamics, the results also suggest that the dyes exploiting PEG linkers interfere with molecular interactions and their dynamics. Overall, the results highlight the great care needed when using fluorescent lipid analogs, in particular accurate controls.

• Keywords
• Atomistic simulation, Fluorescent probe, Lipid membrane, Molecular dynamics simulation, PEG linker, Super-resolution microscopy,
• MeSH
• Diffusion MeSH
• Fluorescent Dyes chemistry   metabolism   MeSH
• Phosphatidylcholines chemistry   MeSH
• Lipid Bilayers chemistry   metabolism   MeSH
• Polyethylene Glycols chemistry   MeSH
• Molecular Dynamics Simulation MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 1,2-oleoylphosphatidylcholine MeSH Browser
• Fluorescent Dyes MeSH
• Phosphatidylcholines MeSH
• Lipid Bilayers MeSH
• Polyethylene Glycols MeSH

Catheter ablation (CA) has become an established treatment strategy for managing recurrent ventricular tachycardias (VTs) in patients with structural heart disease. In recent years, percutaneous mechanical circulatory support (PMCS) devices have been increasingly used intra-operatively to improve the ablation outcome. One indication would be rescue therapy for patients who develop haemodynamic deterioration during the ablation. However, more efforts are focused on identifying subjects who are at high risk of such deterioration and could benefit from the pre-emptive use of the PMCS. The third reason to use PMCS could be the inability to identify diffuse substrate, especially in non-ischaemic cardiomyopathy. This paper reviews available experiences using various types of PMCS in different clinical scenarios. Although PMCS allows mapping during VT, it does not significantly influence acute outcomes and not convincingly long-term outcomes. On the contrary, the complication rate appears to be higher in PMCS cohorts. Our data suggest that even in patients with severe left ventricular dysfunction, the substrate modification can be performed without the need for general anaesthesia and risk of haemodynamic decompensation. In end-stage heart failure associated with the electrical storm, implantation of a left ventricular assist device (or PMCS with a transition to the left ventricular assist device) might be the preferred strategy before CA. In high-risk patients who are not potential candidates for these treatment options, radiotherapy could be considered as a bail-out treatment of recurrent VTs. These approaches should be studied in prospective trials.

• Keywords
• Catheter ablation, Percutaneous mechanical support, Stereotactic arrhythmia radiotherapy, Substrate modification, Ventricular tachycardia,
• MeSH
• Hemodynamics MeSH
• Catheter Ablation * methods   adverse effects   MeSH
• Tachycardia, Ventricular * surgery   physiopathology   MeSH
• Humans MeSH
• Heart-Assist Devices * MeSH
• Risk Factors MeSH
• Heart Failure physiopathology   therapy   MeSH
• Treatment Outcome MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Review MeSH

The effect of non-ionic detergents on baclofen (GABAB-R agonist)-stimulated G-protein activity was measured as a [(35)S]GTPgammaS binding assay in the plasma membranes (PM) isolated from the brain tissue. The effect was clearly biphasic--a decrease in the activity was followed by an activation maximum and finally, at high concentrations, drastic inhibition of the G-protein activity was noticed. Contrarily, specific radioligand binding to GABAB-receptor was inhibited in the whole range of detergent concentrations step by step, i.e. it was strictly monophasic. The magnitude of both detergent effects was decreased in the same order of potency: Brij58>Triton X-100>Digitonin. The identical order was found when comparing detergents ability to alter fluorescence anisotropy of the membrane probe 1,6-diphenyl-1,3,5-hexatriene (rDPH) incorporated into the hydrophobic PM interior. Decrease of rDPH, in the order of Brij58>Triton X-100>Digitonin, was reflected as decrease of the S-order parameter and rotation correlation time phi paralleled by an increase of diffusion wobbling constant Dw (analysis by time-resolved fluorescence according to "wobble-in-cone" model). The influence of the detergents on the membrane organization at the polar headgroup region was characterized by Laurdan generalized polarization (GP). As before, the effect of detergents on GP parameters proceeded in the order: Brij58>Triton X-100>Digitonin.

• MeSH
• 2-Naphthylamine analogs & derivatives   MeSH
• Cell Membrane drug effects   metabolism   MeSH
• Cetomacrogol pharmacology   MeSH
• Diphenylhexatriene * MeSH
• Diffusion MeSH
• Fluorescent Dyes MeSH
• Spectrometry, Fluorescence MeSH
• Rats MeSH
• Laurates * MeSH
• Brain drug effects   metabolism   MeSH
• Octoxynol pharmacology   MeSH
• GTP-Binding Proteins metabolism   MeSH
• Receptors, GABA-B metabolism   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 2-Naphthylamine MeSH
• Cetomacrogol MeSH
• Diphenylhexatriene * MeSH
• Fluorescent Dyes MeSH
• Laurates * MeSH
• laurdan MeSH Browser
• Octoxynol MeSH
• GTP-Binding Proteins MeSH
• Receptors, GABA-B MeSH

Ribosomes synthesizing proteins containing consecutive proline residues become stalled and require rescue via the action of uniquely modified translation elongation factors, EF-P in bacteria, or archaeal/eukaryotic a/eIF5A. To date, no structures exist of EF-P or eIF5A in complex with translating ribosomes stalled at polyproline stretches, and thus structural insight into how EF-P/eIF5A rescue these arrested ribosomes has been lacking. Here we present cryo-EM structures of ribosomes stalled on proline stretches, without and with modified EF-P. The structures suggest that the favored conformation of the polyproline-containing nascent chain is incompatible with the peptide exit tunnel of the ribosome and leads to destabilization of the peptidyl-tRNA. Binding of EF-P stabilizes the P-site tRNA, particularly via interactions between its modification and the CCA end, thereby enforcing an alternative conformation of the polyproline-containing nascent chain, which allows a favorable substrate geometry for peptide bond formation.

• Keywords
• EF-P, RNA, a-IF5A, eIF5A, nascent chain, prolines, ribosome, single particle cryo-EM, stalling, translation elongation,
• MeSH
• Cryoelectron Microscopy MeSH
• Peptide Elongation Factors chemistry   genetics   metabolism   ultrastructure   MeSH
• Escherichia coli genetics   metabolism   MeSH
• Eukaryotic Translation Initiation Factor 5A MeSH
• Peptide Initiation Factors chemistry   metabolism   MeSH
• Nucleic Acid Conformation MeSH
• Protein Conformation MeSH
• RNA, Messenger chemistry   genetics   metabolism   MeSH
• Mutation MeSH
• Peptides chemistry   metabolism   MeSH
• RNA-Binding Proteins chemistry   metabolism   MeSH
• Escherichia coli Proteins chemistry   genetics   metabolism   ultrastructure   MeSH
• Protein Biosynthesis MeSH
• Ribosomes chemistry   metabolism   ultrastructure   MeSH
• RNA, Transfer chemistry   genetics   metabolism   MeSH
• Molecular Dynamics Simulation MeSH
• Molecular Docking Simulation MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Structure-Activity Relationship MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Peptide Elongation Factors MeSH
• factor EF-P MeSH Browser
• Peptide Initiation Factors MeSH
• RNA, Messenger MeSH
• Peptides MeSH
• polyproline MeSH Browser
• RNA-Binding Proteins MeSH
• Escherichia coli Proteins MeSH
• RNA, Transfer MeSH

A model for the complex between E. coli RNase HI and the DNA/RNA hybrid (previously refined by molecular dynamics simulations) was used to determine the impact of the internucleotide linkage modifications (either 3-O-CH2-P-O-5' or 3-O-P-CH2-O-5) on the ability of the modified-DNA/RNA hybrid to create a complex with the protein. Modified internucleotide linkages were incorporated systematically at different positions close to the 3-end of the DNA strand to interfere with the DNA binding site of RNase H. Altogether, six trajectories were produced (length 1.5ns). Mutual hydrogen bonds connecting both strands of the nucleic acids hybrid, DNA with RNase H, RNA with RNase H, and the scissile bond with the Mg++. 4H2O chelate complex (bound in the active site) were analyzed in detaiL Many residues were involved in binding of the DNA (Arg88, Asn84, Trp85, Trp104, Tyr73, Lys99, Asn100, Thr43, and Asn 16) and RNA (Gln76, Gln72, Tyr73, Lys122, Glu48, Asn44, and Cys13) strand to the substrate-binding site of the RNase H enzyme. The most remarkable disturbance of the hydrogen bonding net was observed for structures with modified internucleotide linkages positioned in a way to interact with the Trp104, Tyr73, Lys99, and Asn100 residues (situated in the middle of the DNA binding site, where a cluster of Trp residues forms a rigid core of the protein structure).

• MeSH
• RNA, Bacterial chemistry   MeSH
• Models, Chemical MeSH
• DNA chemistry   MeSH
• Escherichia coli enzymology   MeSH
• Magnesium chemistry   MeSH
• Nucleic Acid Conformation MeSH
• Protein Conformation MeSH
• Molecular Conformation MeSH
• Models, Molecular MeSH
• Nucleotides chemistry   MeSH
• Organophosphonates chemistry   MeSH
• Computer Simulation MeSH
• Ribonuclease H chemistry   MeSH
• RNA chemistry   MeSH
• Tryptophan chemistry   MeSH
• Tyrosine chemistry   MeSH
• Binding Sites MeSH
• Hydrogen Bonding MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA, Bacterial MeSH
• DNA MeSH
• Magnesium MeSH
• Nucleotides MeSH
• Organophosphonates MeSH
• ribonuclease HI MeSH Browser
• Ribonuclease H MeSH
• RNA MeSH
• Tryptophan MeSH
• Tyrosine MeSH

An elevated level of fibroblast growth factor-2 (FGF-2) in peripheral blood is considered to play a role in regulating the growth of leukemia cells. Here, we show that the level of plasma FGF-2 is increased in 54% of B cell chronic lymphocytic leukemias (B-CLL) and in 44% of chronic myeloid leukemias (CML). Notably, white blood cells (WBCs) from B-CLL patients contain 18, 22 and 24 kDa isoforms of FGF-2 whereas WBCs from CML patients contain only the 24 kDa isoform. Furthermore, as cultured B-CLL WBCs release 18 kDa FGF-2 into the medium, they constitute a potential source of FGF-2 in the blood. In a receptor binding assay, 125I-FGF-2 binds weakly to B-CLL WBCs, whereas the ligand binds more strongly to CML WBCs. Correspondingly, FGF-2 is unable to activate mitogen-activated protein kinase kinase (MEK) and its substrate, extracellular signal-regulated kinase (ERK), in B-CLL cells, whereas phosphorylation of both these cell growth-related kinases increases following treatment of CML WBCs. We conclude that B-CLL WBCs secrete FGF-2 with no apparent autocrine actions. In contrast, WBCs in CML bind FGF-2 provided by other FGF-2-hyperproducing cells and activate the MEK/ERK kinase cascade, possibly to modulate cell growth.

• MeSH
• Leukemia, Lymphocytic, Chronic, B-Cell blood   MeSH
• Leukemia, Myelogenous, Chronic, BCR-ABL Positive blood   MeSH
• DNA Primers MeSH
• Adult MeSH
• Fibroblast Growth Factor 2 blood   chemistry   MeSH
• Culture Media, Conditioned MeSH
• Middle Aged MeSH
• Humans MeSH
• RNA, Messenger genetics   MeSH
• Molecular Weight MeSH
• Receptors, Fibroblast Growth Factor genetics   MeSH
• Base Sequence MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA Primers MeSH
• Fibroblast Growth Factor 2 MeSH
• Culture Media, Conditioned MeSH
• RNA, Messenger MeSH
• Receptors, Fibroblast Growth Factor MeSH

The patients with mantle cell lymphoma (MCL) have translocation t(11;14) associated with cyclin D1 overexpression. We observed that iron (an essential cofactor of dioxygenases including prolyl hydroxylases [PHDs]) depletion by deferoxamine blocked MCL cells' proliferation, increased expression of DNA damage marker γH2AX, induced cell cycle arrest and decreased cyclin D1 level. Treatment of MCL cell lines with dimethyloxalylglycine, which blocks dioxygenases involving PHDs by competing with their substrate 2-oxoglutarate, leads to their decreased proliferation and the decrease of cyclin D1 level. We then postulated that loss of EGLN2/PHD1 in MCL cells may lead to down-regulation of cyclin D1 by blocking the degradation of FOXO3A, a cyclin D1 suppressor. However, the CRISPR/Cas9-based loss-of-function of EGLN2/PHD1 did not affect cyclin D1 expression and the loss of FOXO3A did not restore cyclin D1 levels after iron chelation. These data suggest that expression of cyclin D1 in MCL is not controlled by ENGL2/PHD1-FOXO3A pathway and that chelation- and 2-oxoglutarate competition-mediated down-regulation of cyclin D1 in MCL cells is driven by yet unknown mechanism involving iron- and 2-oxoglutarate-dependent dioxygenases other than PHD1. These data support further exploration of the use of iron chelation and 2-oxoglutarate-dependent dioxygenase inhibitors as a novel therapy of MCL.

• Keywords
• 2-oxoglutarate-dependent enzymes, DNA damage, cell cycle, iron, mantle cell lymphoma, prolyl hydroxylases (EGLN/PHDs),
• MeSH
• Amino Acids, Dicarboxylic pharmacology   MeSH
• Iron Chelating Agents pharmacology   MeSH
• Cyclin D1 metabolism   MeSH
• Deferoxamine pharmacology   MeSH
• Iron Deficiencies MeSH
• Dioxygenases antagonists & inhibitors   metabolism   MeSH
• Down-Regulation drug effects   MeSH
• Hydroxylation MeSH
• Cell Hypoxia drug effects   MeSH
• Enzyme Inhibitors pharmacology   MeSH
• Ketoglutaric Acids pharmacology   MeSH
• Humans MeSH
• Lymphoma, Mantle-Cell enzymology   MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Cell Line, Tumor MeSH
• DNA Damage MeSH
• Hypoxia-Inducible Factor-Proline Dioxygenases metabolism   MeSH
• Forkhead Box Protein O3 genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Amino Acids, Dicarboxylic MeSH
• Iron Chelating Agents MeSH
• Cyclin D1 MeSH
• Deferoxamine MeSH
• Dioxygenases MeSH
• EGLN2 protein, human MeSH Browser
• FOXO3 protein, human MeSH Browser
• Enzyme Inhibitors MeSH
• Ketoglutaric Acids MeSH
• RNA, Messenger MeSH
• oxalylglycine MeSH Browser
• Hypoxia-Inducible Factor-Proline Dioxygenases MeSH
• Forkhead Box Protein O3 MeSH

A mathematical model of an enzymatic separating microreactor with the electro-osmotic control of reaction component transport rates is analysed. The micro-reactor is considered in a form of a thin channel filled with a gel containing an immobilised enzyme and an adsorbent where the enzyme reaction, the molecular diffusion, the electro-osmotic flux and the adsorption take place. The substrate inhibited enzyme reaction splitting a non-ionic substrate to two non-ionic products is considered. The reactor operates in a periodic regime, when the channel entry is exposed to the periodic substrate concentration pulses. A chromatographic separation of reaction components, therefore, proceeds in the channel. Effects of principal operational parameters of the reactor system-the reaction channel length, the electric current density, the substrate inlet concentration, the rate of adsorption, and the enzyme activity--on resolution of the products at reactor outlet are analysed. The existence of optimum parameter values (maximising the resolution of reaction products) is shown and a multiparametric optimisation of the reactor performance is accomplished.

• MeSH
• Models, Biological * MeSH
• Bioreactors * MeSH
• Enzymes, Immobilized metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Enzymes, Immobilized MeSH