Continuum simulations of acetylcholine diffusion with reaction-determined boundaries in neuromuscular junction models

The reaction-diffusion system of the neuromuscular junction has been modeled in 3D using the finite element package FEtk. The numerical solution of the dynamics of acetylcholine with the detailed reaction processes of acetylcholinesterases and nicotinic acetylcholine receptors has been discussed with the reaction-determined boundary conditions. The simulation results describe the detailed acetylcholine hydrolysis process, and reveal the time-dependent interconversion of the closed and open states of the acetylcholine receptors as well as the percentages of unliganded/monoliganded/diliganded states during the neuro-transmission. The finite element method has demonstrated its flexibility and robustness in modeling large biological systems.     

An ultrasensitive high-throughput electrochemiluminescence immunoassay for the Cdc42-associated protein tyrosine kinase ACK1

Several drugs inhibiting protein kinases have been launched successfully, demonstrating the attractiveness of protein kinases as therapeutic targets. Functional genomics research within both academia and industry has led to the identification of many more kinases as potential drug targets. Although a number of well-known formats are used for measuring protein kinase activity, some less well-characterized protein kinases identified through functional genomics present particular challenges for existing assay formats when there is limited knowledge of the endogenous substrates or activation mechanisms for these novel kinase targets. This is especially the case when a very sensitive assay is required to differentiate often highly potent inhibitors developed by late-stage medicinal chemistry programs. ACK1 is a non-receptor tyrosine kinase that has been shown to be involved in tumorigenesis and metastasis. Here we describe the development of an extremely sensitive high-throughput assay for ACK1 capable of detecting 240 fmol per well of the kinase reaction product employing a BV-tag-based electrochemiluminescence assay. This assay is universally applicable to protein tyrosine kinases using a BV-tag-labeled monoclonal antibody against phosphotyrosine. Furthermore, this assay can be extended to the evaluation of Ser/Thr kinases in those cases where an antibody recognizing the phospho-product is available.     

Sensitivity of Meloidogyne incognita and Rotylenchulus reniformis to Fluopyram

Fluopyram is a succinate dehydrogenase inhibitor (SDHI) fungicide that is being evaluated as a seed treatment and in-furrow spray at planting on row crops for management of fungal diseases and its effect on plant-parasitic nematodes. Currently, there are no data on nematode toxicity, nematode recovery, or effects on nematode infection for Meloidogyne incognita or Rotylenchulus reniformis after exposure to low concentrations of fluopyram. Nematode toxicity and recovery experiments were conducted in aqueous solutions of fluopyram, while root infection assays were conducted on tomato. Nematode paralysis was observed after 2 hr of exposure at 1.0 µg/ml fluopyram for both nematode species. Using an assay of nematode motility, 2-hr EC₅₀ values of 5.18 and 12.99 µg/ml fluopyram were calculated for M. incognita and R. reniformis, respectively. Nematode recovery in motility was greater than 50% for M. incognita and R. reniformis 24 hr after nematodes were rinsed and removed from a 1-hr treatment of 5.18 and 12.99 µg/ml fluopyram, respectively. Nematode infection of tomato roots was reduced and inversely proportional to 1-hr treatments with water solutions of fluopyram at low concentrations, which ranged from 1.3 to 5.2 µg/ml for M. incognita and 3.3 to 13.0 µg/ml for R. reniformis. Though fluopyram is nematistatic, low concentrations of the fungicide were effective at reducing the ability of both nematode species to infect tomato roots.     

The red death meets the abdominal bristle: Polygenic mutation for susceptibility to a bacterial pathogen in Caenorhabditis elegans

Understanding the genetic basis of susceptibility to pathogens is an important goal of medicine and of evolutionary biology. A key first step toward understanding the genetics and evolution of any phenotypic trait is characterizing the role of mutation. However, the rate at which mutation introduces genetic variance for pathogen susceptibility in any organism is essentially unknown. Here, we quantify the per‐generation input of genetic variance by mutation (VM) for susceptibility of Caenorhabditis elegans to the pathogenic bacterium Pseudomonas aeruginosa (defined as the median time of death, LT50). VM for LT50 is slightly less than VM for a variety of life‐history and morphological traits in this strain of C. elegans, but is well within the range of reported values in a variety of organisms. Mean LT50 did not change significantly over 250 generations of mutation accumulation. Comparison of VM to the standing genetic variance (VG) implies a strength of selection against new mutations of a few tenths of a percent. These results suggest that the substantial standing genetic variation for susceptibility of C. elegans to P. aeruginosa can be explained by polygenic mutation coupled with purifying selection.     

Pentacycloundecane lactam vs lactone norstatine type protease HIV inhibitors: binding energy calculations and DFT study

Background

Novel pentacycloundecane (PCU)-lactone-CO-EAIS peptide inhibitors were designed, synthesized, and evaluated against wild-type C-South African (C-SA) HIV-1 protease. Three compounds are reported herein, two of which displayed IC₅₀ values of less than 1.00 μM. A comparative MM-PB(GB)SA binding free energy of solvation values of PCU-lactam and lactone models and their enantiomers as well as the PCU-lactam-NH-EAIS and lactone-CO-EAIS peptide inhibitors and their corresponding diastereomers complexed with South African HIV protease (C-SA) was performed. This will enable us to rationalize the considerable difference between inhibitory concentration (IC₅₀) of PCU-lactam-NH-EAIS and PCU-lactone-CO-EAIS peptides.

Results

The PCU-lactam model exhibited more negative calculated binding free energies of solvation than the PCU-lactone model. The same trend was observed for the PCU-peptide inhibitors, which correspond to the experimental activities for the PCU-lactam-NH-EAIS peptide (IC₅₀ = 0.076 μM) and the PCU-lactone-CO-EAIS peptide inhibitors (IC₅₀ = 0.850 μM). Furthermore, a density functional theory (DFT) study on the natural atomic charges of the nitrogen and oxygen atoms of the three PCU-lactam, PCU-lactim and PCU-lactone models were performed using natural bond orbital (NBO) analysis. Electrostatic potential maps were also used to visualize the electron density around electron-rich regions. The asymmetry parameter (η) and quadrupole coupling constant (χ) values of the nitrogen and oxygen nuclei of the model compounds were calculated at the same level of theory. Electronic molecular properties including polarizability and electric dipole moments were also calculated and compared. The Gibbs theoretical free solvation energies of solvation (∆G_solv) were also considered.

Conclusions

A general trend is observed that the lactam species appears to have a larger negative charge distribution around the heteroatoms, larger quadrupole constant, dipole moment and better solvation energy, in comparison to the PCU-lactone model. It can be argued that these characteristics will ensure better eletronic interaction between the lactam and the receptor, corresponding to the observed HIV protease activities in terms of experimental IC₅₀ data.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-015-0115-5) contains supplementary material, which is available to authorized users.     

A simple real-time assay for in vitro translation

A high-throughput assay for real-time measurement of translation rates in cell-free protein synthesis (SNAP assay) is described. The SNAP assay enables quantitative, real-time measurement of overall translation rates in vitro via the synthesis of O⁶-alkylguanine DNA O⁶-alkyltransferase (SNAP). SNAP production is continuously detected by fluorescence produced by the reaction of SNAP with a range of quenched fluorogenic substrates. The capabilities of the assay are exemplified by measurements of the activities of Escherichia coli MRE600 ribosomes and fluorescently labeled E. coli mutant ribosomes in the PURExpress translation system and by determination of the 50% inhibitory concentrations (IC₅₀) of three common macrolide antibiotics.     

Black soybean extract can attenuate thrombosis through inhibition of collagen-induced platelet activation

Many clinical trials have demonstrated the beneficial effects of soybean (Glycine max) on general cardiovascular health. Among a variety of soybeans, black soybean is known to display diverse biological activities superior to those of yellow and green soybeans, such as in antioxidant, anti-inflammatory and anticancer activities. However, few studies have been directed on the effect of black soybean on cardiovascular function. In this study, we aimed to investigate the effect of black soybean extract (BB) on platelet activation, a key contributor to thrombotic diseases. In freshly isolated human platelets, BB has shown potent inhibitory activity on collagen-induced platelet aggregation, while yellow soybean extract had marginal activity only. BB also attenuated serotonin secretion and P-selectin expression, which are important factors for the platelet–tissue interaction along with thromboxane A₂ formation. These in vitro results were further confirmed in an ex vivo platelet aggregation measurement and in vivo venous thrombosis model where oral administration of BB reduced collagen-induced platelet aggregation and FeCl₃-induced thrombus formation significantly. A potential active ingredient for antiplatelet effects of BB was isolated and identified to be adenosine through bioassay-directed fractionation and NMR and ESI-MS analyses. These results indicate that black soybean can be a novel dietary supplement for the prevention of cardiovascular risks and the improvement of blood circulation.     

Metabolic conversion of dietary flavonoids alters their anti-inflammatory and antioxidant properties

The notion that dietary flavonoids exert beneficial health effects in humans is often based on in vitro studies using the glycoside or aglycone forms of these flavonoids. However, flavonoids are extensively metabolized in humans, resulting in the formation of glucuronide, methyl, and sulfate derivatives, which may have different properties than their parent compounds. The goal of this study was to investigate whether different chemical modifications of the same flavonoid molecule affect its biological and antioxidant activities. Hence, we studied the anti-inflammatory effects of several major human metabolites of quercetin and (-)-epigallocatechin-3-O-gallate (EGCG) by assessing their inhibitory effects on tumor necrosis factor α (TNFα)-induced protein expression of cellular adhesion molecules in human aortic endothelial cells (HAEC). HAEC were incubated with 1-30 μM quercetin, 3'- or 4'-O-methyl-quercetin, quercetin-3-O-glucuronide, and quercetin-3'-O-sulfate or 20-100 μM EGCG, 4'-O-methyl-EGCG, and 4',4'-di-O-methyl-EGCG, prior to coincubation with 100 U/ml of TNFα. 3'-O-Methyl-quercetin, 4'-O-methyl-quercetin, and their parent aglycone compound, quercetin, all effectively inhibited expression of intercellular adhesion molecule-1 (ICAM-1) with IC(50) values (concentration required for 50% inhibition) of 8.0, 5.0, and 4.4 μM, respectively; E-selectin expression was suppressed to a somewhat lesser but still significant degree by all three compounds, whereas vascular cell adhesion molecule-1 (VCAM-1) was not affected. In contrast, quercetin-3-O-glucuronide (20-100 μM), quercetin-3'-O-sulfate (10-30 μM), and phenolic acid metabolites of quercetin (20-100 μM) did not inhibit adhesion molecule expression. 4',4'-Di-O-methyl-EGCG selectively inhibited ICAM-1 expression with an IC(50) value of 94 μM, whereas EGCG (20-60 μM) and 4'-O-methyl-EGCG (20-100 μM) had no effect. The inhibitory effects of 3'-O-methyl-quercetin and 4',4'-di-O-methyl-EGCG on adhesion molecule expression were not related either to inhibition of NF-κB activation or to their antioxidant reducing capacity. Our data indicate that flavonoid metabolites have different biological and antioxidant properties than their parent compounds, and suggest that data from in vitro studies using nonmetabolites of flavonoids are of limited relevance in vivo.     

Cell death via mitochondrial apoptotic pathway due to activation of Bax by lysosomal photodamage

Lysosomal photosensitizers have been used in photodynamic therapy. The combination of such photosensitizers and light causes lysosomal photodamage, inducing cell death. Lysosomal disruption can lead to apoptosis but its signaling pathways remain to be elucidated. In this study, N-aspartyl chlorin e6 (NPe6), an effective photosensitizer that preferentially accumulates in lysosomes, was used to study the mechanism of apoptosis caused by lysosomal photodamage. Apoptosis in living human lung adenocarcinoma cells (ASTC-a-1) after NPe6-photodynamic treatment (NPe6-PDT) was studied using real-time single-cell analysis. Our results demonstrated that NPe6-PDT induced rapid generation of reactive oxygen species (ROS). The photodynamically produced ROS caused a rapid destruction of lysosomes, leading to release of cathepsins, and the ROS scavengers vitamin C and NAC prevent the effects. Then the following spatiotemporal sequence of cellular events was observed during cell apoptosis: Bcl-2-associated X protein (Bax) activation, cytochrome c release, and caspase-9/-3 activation. Importantly, the activation of Bax proved to be a crucial event in this apoptotic machinery, because suppressing the endogenous Bax using siRNA could significantly inhibit cytochrome c release and caspase-9/-3 activation and protect the cell from death. In conclusion, this study demonstrates that PDT with lysosomal photosensitizer induces Bax activation and subsequently initiates the mitochondrial apoptotic pathway.     

Design of a potent, soluble glucokinase activator with increased pharmacokinetic half-life

The continued optimization of a series of glucokinase activators is described, including attempts to understand the interplay between molecular structure and the composite parameter of unbound clearance. These studies resulted in the discovery of a new scaffold for glucokinase activators and further exploration of this scaffold led to the identification of GKA60. GKA60 maintains an excellent balance of potency and physical properties whilst possessing a significantly different, but complimentary, pre-clinical pharmacokinetic profile compared with the previously disclosed compound GKA50.