SarcTrack.

RATIONALE: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in combination with CRISPR/Cas9 genome editing provide unparalleled opportunities to study cardiac biology and disease. However, sarcomeres, the fundamental units of myocyte contraction, are immature and nonlinear in hiPSC-CMs, which technically challenge accurate functional interrogation of contractile parameters in beating cells. Furthermore, existing analysis methods are relatively low-throughput, indirectly assess contractility, or only assess well-aligned sarcomeres found in mature cardiac tissues. OBJECTIVE: We aimed to develop an analysis platform that directly, rapidly, and automatically tracks sarcomeres in beating cardiomyocytes. The platform should assess sarcomere content, contraction and relaxation parameters, and beat rate. METHODS AND RESULTS: We developed SarcTrack, a MatLab software that monitors fluorescently tagged sarcomeres in hiPSC-CMs. The algorithm determines sarcomere content, sarcomere length, and returns rates of sarcomere contraction and relaxation. By rapid measurement of hundreds of sarcomeres in each hiPSC-CM, SarcTrack provides large data sets for robust statistical analyses of multiple contractile parameters. We validated SarcTrack by analyzing drug-treated hiPSC-CMs, confirming the contractility effects of compounds that directly activate (CK-1827452) or inhibit (MYK-461) myosin molecules or indirectly alter contractility (verapamil and propranolol). SarcTrack analysis of hiPSC-CMs carrying a heterozygous truncation variant in the myosin-binding protein C ( MYBPC3) gene, which causes hypertrophic cardiomyopathy, recapitulated seminal disease phenotypes including cardiac hypercontractility and diminished relaxation, abnormalities that normalized with MYK-461 treatment. CONCLUSIONS: SarcTrack provides a direct and efficient method to quantitatively assess sarcomere function. By improving existing contractility analysis methods and overcoming technical challenges associated with functional evaluation of hiPSC-CMs, SarcTrack enhances translational prospects for sarcomere-regulating therapeutics and accelerates interrogation of human cardiac genetic variants.

Vitamin E pretreatment prevents histopathological effects in tilapia (Oreochromis niloticus) acutely exposed to cylindrospermopsin.

Cylindrospermopsin (CYN) is a cyanotoxin frequently involved in blooms with a predominantly extracellular availability, which makes it easily taken up by a variety of aquatic organisms. CYN is a potent protein and glutathione synthesis inhibitor, and also induces genotoxicity, oxidative stress and several histopathological lesions. The present study investigates the protective role of a vitamin E pretreatment (700 mg vit E/kg fish bw/day, for 7 days) on the histopathological alterations induced in different organs of tilapia (Oreochromis niloticus) acutely exposed to a single oral dose of 400 µg pure CYN/kg bw fish. The major histological changes observed were degenerative glucogenic process and loss of the hepatic structure in the liver, glomerulopathy and tubular tumefaction in the kidney, myofibrolysis and edema in the heart, catarrhal enteritis and necrosis in the gastrointestinal tract, hyperemic processes in the gill lamellae, and high basophilia, degeneration and tumefaction of granular neurons in the brain. Vitamin E pretreatment was effective in preventing or ameliorating the abovementioned alterations induced by CYN. In addition, a morphometric study indicated that the average nuclear diameter of hepatocytes, and cross-sections of proximal and distal convoluted tubules, together with the cardiac fiber and capillaries diameters represent a useful tool to evaluate the damage induced by CYN. This is the first study reporting vitamin E prevention of histopathological damage in tissues (liver, kidney, heart, gastrointestinal tract, gills and brain) of fish intoxicated with CYN. Therefore, vitamin E can be considered a useful chemoprotectant in the treatment of histopathological changes induced in CYN-intoxicated fish. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1469-1485, 2016.

Alogliptin, a dipeptidyl peptidase-4 inhibitor, regulates the atrial arrhythmogenic substrate in rabbits.

BACKGROUND: Dipeptidyl peptidase-4 (DPP-4) inhibitors were recently reported to have cardioprotective effects via amelioration of ventricular function. However, the role of DPP-4 inhibition in atrial remodeling, especially of the arrhythmogenic substrate, remains unclear. OBJECTIVE: We investigated the effects of a DPP-4 inhibitor, alogliptin, on atrial fibrillation (AF) in a rabbit model of heart failure caused by ventricular tachypacing (VTP). METHODS: Rabbits subjected to VTP at 380 bpm for 1 or 3 weeks, with or without alogliptin treatment, were assessed using echocardiography, electrophysiology, histology, and immunoblotting and compared with nonpaced animals. RESULTS: VTP rabbits exhibited increased duration of atrial burst pacing-induced AF, whereas administration of alogliptin shortened this duration by 73%. The extent of atrial fibrosis after VTP was reduced by 39% in the alogliptin-treated group. VTP rabbits treated with alogliptin displayed a 1.6-fold increase in left atrial myocardial capillary density compared with nontreated rabbits. A 2-fold increase in endothelial nitric oxide synthase (eNOS) phosphorylation was observed in the left atrium of alogliptin-treated rabbits compared with nontreated rabbits. Moreover, a nitric oxide synthase inhibitor (N(ω)-nitro-l-arginine methyl ester) blocked the beneficial effects of alogliptin on AF duration, fibrosis, and capillary density. CONCLUSION: Alogliptin shortened the duration of AF caused by VTP-induced fibrotic atrial tissue by augmenting atrial angiogenesis and activating eNOS. Our findings suggest that DPP-4 inhibitors may be useful in the prevention of heart failure-induced AF.

Aryl uracil inhibitors of hepatitis C virus NS5B polymerase: synthesis and characterization of analogs with a fused 5,6-bicyclic ring motif.

The synthesis and structure-activity relationships of a novel aryl uracil series which contains a fused 5,6-bicyclic ring unit for HCV NS5B inhibition is described. Several analogs display replicon cell culture potencies in the low nanomolar range along with excellent rat pharmacokinetic values.

Substrate/Product-targeted NMR monitoring of pyrimidine catabolism and its inhibition by a clinical drug.

We report the application of one-dimensional triple-resonance NMR to metabolic analysis and thereon-based evaluation of drug activity. Doubly (13)C/(15)N-labeled uracil ([(15)N1,(13)C6]-uracil) was prepared. Its catabolic (degradative) conversion to [(13)C3,(15)N4]-ß-alanine and inhibition thereof by gimeracil, a clinical co-drug used with the antitumor agent 5-fluorouracil, in mouse liver lysates were monitored specifically using one-dimensional triple-resonance ((1)H-{(13)C-(15)N}) NMR, but not double-resonance ((1)H-{(13)C}) NMR, in a ratiometric manner. The administration of labeled uracil to a mouse resulted in its non-selective distribution in various organs, with efficient catabolism to labeled ß-alanine exclusively in the liver. The co-administration of gimeracil inhibited the catabolic conversion of uracil in the liver. In marked contrast to in vitro results, however, gimeracil had practically no effect on the level of uracil in the liver. The potentiality of triple-resonance NMR in the analysis of in vivo pharmaceutical activity of drugs targeting particular metabolic reactions is discussed.

Role of the Escherichia coli and human DNA glycosylases that remove 5-formyluracil from DNA in the prevention of mutations.

Ionizing radiation induces a wide variety of modifications to purine and pyrimidine residues. The exocyclic methyl group of thymine does not escape oxidative damage. Any 5-hydroperoxymethyluracil produced is spontaneously decomposed to form 5-formyluracil (5-foU) and 5-hydroxymethyluracil. The yield of 5-foU by ionizing radiation is roughly the same as that of 8-oxoguanine. 5-foU is a potential mutagenic damage in vitro and in vivo. Mammalian cells have an activity that removes 5-foU from X-irradiated DNA. Furthermore, the Nth, Nei and MutM proteins of E. coli have DNA glycosylase/AP lyase activities that recognize and remove 5-foU in DNA. The mutation frequency of 5-foU-containing plasmid increases when replicated in E. coli nthneimutMalkA. Single mutations in the nth, nei or mutM gene do not affect the mutation frequency. Therefore, these gene products are likely backup enzymes used to repair 5-foU in DNA. Furthermore, the human hNTH1 enzyme, a homologue of E. coli Nth, is found to have similar DNA glycosylase activity to that of the Nth protein.

A highly selective, high-affinity transporter for uracil in Trypanosoma brucei brucei: evidence for proton-dependent transport.

The presence of an uptake mechanism for uracil in procyclic forms of the protozoan parasite Trypanosoma brucei brucei was investigated. Uptake of [3H]uracil at 22 degrees C was rapid and saturable and appeared to be mediated by a single high-affinity transporter, designated U1, with an apparent Km of 0.46 +/- 0.09 microM and a Vmax of 0.65 +/- 0.08 pmol x (10(7) cells)(-1) x s(-1). [3H]Uracil uptake was not inhibited by a broad range of purine and pyrimidine nucleosides and nucleobases (concentrations up to 1 mM), with the exception of uridine, which acted as an apparent weak inhibitor (Ki value of 48 +/- 15 microM). Similarly, most chemical analogues of uracil, such as 5-chlorouracil, 3-deazauracil, and 2-thiouracil, had little or no affinity for the U1 carrier. Only 5-fluorouracil was found to be a relatively potent inhibitor of uracil uptake (Ki = 3.2 +/- 0.4 microM). Transport of uracil was independent of extracellular sodium and potassium gradients, as replacement of NaCl in the assay buffer by N-methyl-D-glucamine, KCl, LiCl, CsCl, or RbCl did not affect initial rates of transport. However, the proton ionophore carbonyl cyanide chlorophenylhydrazone inhibited up to 70% of [3H]uracil flux. These data show that uracil uptake in T. b. brucei procyclics is mediated by a single high-affinity transporter with high substrate selectivity and are consistent with a nucleobase-H+-symporter model for this carrier.

Antagonistic effect of nucleic acid precursors on the anticoccidial activity of 6-azauracil.

The antagonistic phenomena to anticoccidial activities of 6-azauracil (AzU) induced by certain nucleic acid precursors were examined in battery experiments. Each nucleic acid precursor, orotate, orotidine, uracil, uridine, adenine and adenosine was mixed in feed in combination with effective levels of AzU. The test feed was provided through the test term to White Leghorn cockerels which were infected with Eimeria tenella, E. necatrix or E. acervulina. In vitro tests were also conducted for antagonistic phenomena similarly to in vivo test using E. tenella cultured in chick kidney (CK) cells. Orotate and uridine reduced dose-dependently the anticoccidial activity of AzU in E. tenella infection in chickens. Uracil and orotidine revealed similar effect. Adenine and adenosine did not show any antagonistic effect to AzU. In E. necatrix infection, orotate and uridine had a similar tendency to that of E. tenella, whereas uracil and orotidine was less pronounced. The activity of AzU against E. acervulina was also blocked by the treatment with uridine and orotate. AzU inhibited the schizont's development of E. tenella in the CK cells, and this effect was reduced by uridine, but not by orotate and orotidine. The antagonistic effect of the pyrimidine nucleic acid precursors on AzU activity suggested that the anticoccidial effect of AzU to E. tenella, E. necatrix and E. acervulina is derived from inhibition of the metabolism of pyrimidine nucleotide synthesis in these parasites.

Investigation for the characteristic anticoccidial activity of diclazuril in battery trials.

To clarify the character of the anticoccidial activity of diclazuril a series of battery trials was conducted. Diclazuril showed excellent anticoccidial activity in the infection of chickens with Eimeria tenella, E. necatrix or E. acervulina at the feeding level of 0.1 ppm. When diclazuril was administered in combination with a nucleic acid precursor, uracil, uridine, orotate or orotidine, the reduction of the activity of diclazuril to the infections induced by above species was not observed. While, bloody droppings with severe cecal lesions were resulted, when diclazuril was administered in combination with uridine 5(1)-diphosphoglucose (UDPG) or its N-acetyl amine (UDPGNAC) to chickens infected with E. tenella. While, body weight gain of the birds and oocyst output was not affected by these combination-treatment. Results demonstrated that the antagonistic effect of UDPG and UDPGNAC to diclazuril was partial. The possibility of the cross resistance between diclazuril and 6-azauracil (AzU) in E. tenella was investigated using two populations induced resistance to AzU or diclazuril. The results demonstrated that the cross resistance does not exist between AzU and diclazuril, indicating that the mode of action of each drug is different.

The biosynthetic origin of the pyridone ring of efrotomycin.

Nocardia lactamdurans has been shown to catabolyse uracil via the reductive pathway. The end product of this pathway, beta-alanine, is incorporated into the pyridone ring of efrotomycin. Support for this proposal includes: (1) reversal of thymine inhibition of efrotomycin biosynthesis by dihydrouracil and N-carbamoyl-beta-aline, two intermediates of the catabolic pathway; (2) incorporation of [5,6-3H]-uracil into efrotomycin with a relative molar specific activity of approximately 0.5, close to the theoretical maximum; and (3) 13C coupling at C4 and C5 of efrotomycin after feeding resting cells with [4,5-13C]-uracil. Our results do not rule out the possibility of an alternative source of beta-alanine or the coexistence of uracil catabolism via oxidative reactions.

[Action of heliomycin on the incorporation of labelled protein precursors and nucleic acid in Staphylococcus aureus cells]. / Deistvie geliomitsina na vkliuchenie mechenykh predshestvennikov belka i nukleinovykh kislot v kletki Staphylococcus aureus

In concentrations of 0.05-0.10 gamma/ml geliomycin suppresses inclusion of C14-uracyl into the acid-soluble fractions of the staphylococcal cells. Inclusion of C14-leucine at the same concentrations of the antibiotics was suppressed to a lesser extent. The above concentrations of geliomycin had practically no effect on inclusion of C14-thimine. Suppression of C14-uracyl inclusion was observed in 5 minutes of the cell incubation with the antibiotic, a significant decrease in C14-leucine inclusion being observed only in 30 minutes of the incubation. The effect of geliomycin on the staphylococcal cells within the concentrations inhibiting the microbial growth and suppressing inclusion of labeled uracyl and leucine into the cells was of the bacteriostatic nature. It was supposed that the antibacterial effect of geliomycin was based on suppression of RNA synthesis.

Inhibition of a DNA polymerase from Bacillus subtilis by hydroxyphenylazopyrimidines.

The antimicrobial agent, 6-(p-hydroxyphenylazo)-uracil, specifically inhibits DNA polymerase III of Bacillus subtilis with a K(i) of less than 1 muM. The inhibition requires prior reduction of the drug, is reversible, and is competitive with dGTP. High amounts of dATP prevent inhibition by the closely related drug, 6-(p-hydroxyphenylazo)-isocytosine. A model is presented in which the inhibitors base-pair with the template while part of a ternary complex with the enzyme. These results imply that DNA polymerase III of B. subtilis is necessary for chromosomal replication.