Overlap Arrhythmia Syndromes Resulting from Multiple Genetic Variations Studied in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are used for genetic models of cardiac diseases. We report an arrhythmia syndrome consisting of Early Repolarization Syndrome (ERS) and Short QT Syndrome (SQTS). The index patient (MMRL1215) developed arrhythmia-mediated syncope after electrocution and was found to carry six mutations. Functional alterations resulting from these mutations were examined in patient-derived hiPSC-CMs. Electrophysiological recordings were made in hiPSC-CMs from MMRL1215 and healthy controls. ECG analysis of the index patient showed slurring of the QRS complex and QTc = 326 ms. Action potential (AP) recordings from MMRL1215 myocytes showed slower spontaneous activity and AP duration was shorter. Field potential recordings from MMRL1215 hiPSC-CMs lack a “pseudo” QRS complex suggesting reduced inward current(s). Voltage clamp analysis of I_Ca showed no difference in the magnitude of current. Measurements of I_Na reveal a 60% reduction in I_Na density in MMRL1215 hiPSC-CMs. Steady inactivation and recovery of I_Na was unaffected. mRNA analysis revealed ANK2 and SCN5A are significantly reduced in hiPSC-CM derived from MMRL1215, consistent with electrophysiological recordings. The polygenic cause of ERS/SQTS phenotype is likely due to a loss of I_Na due to a mutation in PKP2 coupled with and a gain of function in I_K,ATP due to a mutation in ABCC9.     

Glucuronidation of zearalenone,zeranol and four metabolites in vitro: Formation of glucuronides by various microsomes and human UDP‐glucuronosyltransferase isoforms

Glucuronidation constitutes an important pathway in the phase II metabolism of the mycotoxin zearalenone (ZEN) and the growth promotor α‐zearalanol (α‐ZAL, zeranol), but the enzymology of their formation is yet unknown. In the present study, ZEN, α‐ZAL and four of their major phase I metabolites were glucuronidated in vitro using hepatic microsomes from steer, pig, rat and human, intestinal microsomes from humans, and eleven recombinant human UDP‐glucuronosyltransferases (UGTs). After assigning chemical structures to the various glucuronides by using previously published information, the enzymatic activities of the various microsomes and UGT isoforms were determined together with the patterns of glucuronides generated. All six compounds were good substrates for all microsomes studied. With very few exceptions, glucuronidation occurred preferentially at the sterically unhindered phenolic 14‐hydroxyl group. UGT1A1, 1A3 and 1A8 had the highest activities and gave rise to the phenolic glucuronide, whereas glucuronidation of the aliphatic hydroxyl group was mostly mediated by UGT2B7 with low activity. Based on these in vitro data, ZEN, α‐ZAL and their metabolites must be expected to be readily glucuronidated both in the liver and intestine as well as in other extrahepatic organs of humans and various animal species.     

Effective Approaches for Estimating the Functionalization Pattern of Carboxymethyl Starch of Different Origin

Three types of differently prepared carboxymethyl starches were analyzed by HPLC and ¹H-NMR spectroscopy after chain degradation. In derivatives obtained in the conventional manner with values of the degree of substitution (DS) up to 0.85; glucose, mono- and di-O-carboxymethyl glucose were detected as building units. Comparison with statistic calculations revealed an even distribution of functional groups along the chain. ¹H-NMR studies confirmed a preferred substitution at the 2 position of the repeating unit. Comparable results were obtained for the carboxymethyl ether of amylose, amylopectin and β-cyclodextrin. The analysis of carboxymethylated starch samples prepared using a new synthesis concept via a reactive microstructure revealed a high DS achieved in a one-step synthesis as well as a non-statistic distribution of carboxymethyl groups along the chain. A significant amount of 2,3,4,6-tetra-O-functionalization, caused by the branched structure of starch, was found. Moreover, carboxymethylation of 6-O-triphenylmethyl starch and subsequent detritylation yields a regioselectively functionalized polymer consisting not only of the expected mono- and di-O-carboxymethylated repeating units but also containing a significant amount of 2,3,4-tri-O-functionalized anhydro-glucose units.     

Studies on the synthesis and characterization of carboxymethylcellulose

Carboxymethylcellulose (CMC) was prepared in a completely heterogeneous procedure in an isopropanol/water slurry and the influence of the reaction conditions on the pattern of functionalization within the anhydroglucose repeating unit as well as due to the formation of the four main repeating units (i. e. unfunctionalized, mono-, di-, and tricarboxymethylated) was checked. The concentration of aqueous sodium hydroxide solution, which is used to activate the cellulose, was varied in the range from 8 to 30% at reaction times from 2 to 6 h at 55°C. The reaction with sodium monochloroacetate leads to the highest degree of substitution (DS_CMC) of 1.24 at a NaOH concentration of 15% and 5 h reaction time. Using a lye concentration of 30%, no influence of the reaction time between 2 h and 6 h was found. From ¹H NMR spectroscopical studies it was concluded that both the concentration of the alkali hydroxide solution and the reaction time influence the partial DS_CMC at positions O-2 and O-6, while position O-3 shows the lowest reactivity in any case, independent of the reaction conditions used. With increasing lye concentration, carboxymethylation occurs preferably at O-6. Information about the content of the differently functionalized repeating units can be obtained by chromatographic analysis after complete depolymerization of the polymer chains. Surprisingly, in any product synthesized a statistic content of the building units of the polymer was found even at low activation of cellulose.     

In Silico Drug Repositioning to Target the SARS-CoV-2 Main Protease as Covalent Inhibitors Employing a Combined Structure-Based Virtual Screening Strategy of Pharmacophore Models and Covalent Docking

The epidemic caused by the SARS-CoV-2 coronavirus, which has spread rapidly throughout the world, requires urgent and effective treatments considering that the appearance of viral variants limits the efficacy of vaccines. The main protease of SARS-CoV-2 (M^pro) is a highly conserved cysteine proteinase, fundamental for the replication of the coronavirus and with a specific cleavage mechanism that positions it as an attractive therapeutic target for the proposal of irreversible inhibitors. A structure-based strategy combining 3D pharmacophoric modeling, virtual screening, and covalent docking was employed to identify the interactions required for molecular recognition, as well as the spatial orientation of the electrophilic warhead, of various drugs, to achieve a covalent interaction with Cys145 of M^pro. The virtual screening on the structure-based pharmacophoric map of the SARS-CoV-2 M^pro in complex with an inhibitor N3 (reference compound) provided high efficiency by identifying 53 drugs (FDA and DrugBank databases) with probabilities of covalent binding, including N3 (Michael acceptor) and others with a variety of electrophilic warheads. Adding the energy contributions of affinity for non-covalent and covalent docking, 16 promising drugs were obtained. Our findings suggest that the FDA-approved drugs Vaborbactam, Cimetidine, Ixazomib, Scopolamine, and Bicalutamide, as well as the other investigational peptide-like drugs (DB04234, DB03456, DB07224, DB7252, and CMX-2043) are potential covalent inhibitors of SARS-CoV-2 M^pro.     

Polymer dissolution. I. On the dissolution behavior of copolymers of methyl methacrylate and methacrylic acid

Copolymers of methyl methacrylate with methacrylic acid [P(MMA/MA)] are interesting resist materials for microlithography. At moderate baking temperatures, these copolymers undergo an intramolecular cyclization, yielding terpolymers containing anhydride moieties. This process has striking consequences for the dissolution behavior and results in higher ketone solubilities. The dissolution rates in methyl ethyl ketone (MEK) and in mixtures with ethyl glycol (EG) correlate in an Arrhenius-like manner with the reciprocal baking temperatures. Two distinct temperature ranges with different slopes between 130–180°C and 180–230°C are found. This is in good agreement with other findings, indicating a different mechanism of anhydride formation in these temperature regions. The activation energies for the dissolution of P(MMA/MA) in EG or EG/MEK mixtures are about 17 kcal/mol and those of the thermally treated material in MEK or MEK/EG or mixtures of MEK with methyl isobutyl ketone (MIBK) are in the range of 20–30 kcal/mol. For the investigation of the M_n dependence, γ-irradiated probes of the copolymer were used. We obtained the usual exponential M_n dependence with an exponent of 0.7–0.8 in MIBK and 0.3–0.4 in EG. Our findings are in agreement with a “relaxation-controlled” dissolution behavior, especially for the anhydride-containing terpolymer. No residual layers or pronounced inhibition periods indicative for gel-layer formation, however, could be found. We suggest a normal dissolution process with a very small gel layer. For the copolymer in an alcohol-containing solvent, a stress-driven dissolution behaviour is more likely. © 1994 John Wiley & Sons, Inc.     

Molecular-Cling-Effect of Fluoroethylene Carbonate Characterized via Ethoxy(pentafluoro)cyclotriphosphazene on SiOx/C Anode Materials – A New Perspective for Formerly Sub-Sufficient SEI Forming Additive Compounds

Effective electrolyte compositions are of primary importance in raising the performance of lithium-ion batteries (LIBs). Recently, fluorinated cyclic phosphazenes in combination with fluoroethylene carbonate (FEC) have been introduced as promising electrolyte additives, which can decompose to form an effective dense, uniform, and thin protective layer on the surface of electrodes. Although the basic electrochemical aspects of cyclic fluorinated phosphazenes combined with FEC were introduced, it is still unclear how these two compounds interact constructively during operation. This study investigates the complementary effect of FEC and ethoxy(pentafluoro)cyclotriphosphazene (EtPFPN) in aprotic organic electrolyte in LiNi_0.5Co_0.2Mn_0.3O ∥ SiO_x/C full cells. The formation mechanism of lithium ethyl methyl carbonate (LEMC)-EtPFPN interphasial intermediate products and the reaction mechanism of lithium alkoxide with EtPFPN are proposed and supported by Density Functional Theory calculations. A novel property of FEC is also discussed here, called molecular-cling-effect (MCE). To the best knowledge, the MCE has not been reported in the literature, although FEC belongs to one of the most investigated electrolyte additives. The beneficial MCE of FEC toward the sub-sufficient solid-electrolyte interphase forming additive compound EtPFPN is investigated via gas chromatography-mass spectrometry, gas chromatography high resolution-accurate mass spectrometry, in situ shell-isolated nanoparticle-enhanced Raman spectroscopy, and scanning electron microscopy.