Insights into the binding of thiosemicarbazone derivatives with human serum albumin: spectroscopy and molecular modelling studies

4-[(1Z)-1-(2-carbamothioylhydrazinylidene)ethyl]phenyl acetate [Ace semi],4-[(1Z)-1-(2-carbamothioylhydrazinylidene)ethyl]phenyl propanoate [Pro semi] from the family of thiosemicarbazones derivative has been newly synthesized. It has good anticancer activity as well as antibacterial and it is also less toxic in nature, its binding characteristics are therefore of huge interest for understanding pharmacokinetic mechanism of the drug. The binding of thiosemicarbazone derivative to human serum albumin (HSA) has been investigated by studying its quenching mechanism, binding kinetics and the molecular distance (r) between donor (HSA) and acceptor (thiosemicarbazone derivative) was estimated according to Forster’s theory of non-radiative energy transfer using fluorescence spectroscopy. The binding dynamics has been elaborated using synchronous fluorescence spectroscopy, and the feature of thiosemicarbazone derivative induced structural changes of HSA has been studied by circular dichorism, Fourier transform infrared spectroscopy. Molecular modelling simulations explore the hydrophobic interaction and hydrogen bonding which stabilizes the interaction.     

Molecular insights into substrate binding mechanism of undecaprenyl pyrophosphate with membrane integrated phosphatidyl glycerophosphate phosphatase B (PgpB) using molecular dynamics simulation approach

Undecaprenyl phosphate (C55-P) acts as carrier lipid in the synthesis of peptidoglycan, which is de novo synthesized from dephosphorylation of undecaprenyl pyrophosphate (C55-PP). The phosphatidylglycerol phosphate phosphatase B (PgpB) catalyzes the dephosphorylation of C55-PP and forms C55-P. As no structural study has been made regarding the binding of C55-PP to PgpB, in the current study, in silico molecular docking, followed by 150 ns molecular dynamics simulation of the putative binding complex in membrane/solvent environment has been performed to understand conformational dynamics. Results are compared with simulated apo form and PE inhibitor-bound form. Analysis of correlated residual fluctuation network in apo form, C55-PP bound and PE inhibitor-bound form suggests that difference in dynamic coupling between TM domain and α2 and α3 helix of periplasmic domain provides ligand binding to facilitate catalysis or to show inhibitory activity. Distance distribution in catalytic residual pair, H207-R104; H207-R201 and H207-D211 which stabilizes phosphate-enzyme intermediate shows a narrow peak in 2.4–3.6 Å in substrate-bound compared to apo form. Binding interactions and binding free energy analyses complement the partial inhibition of PE where PE has less binding free energy compared to the C55-PP substrate as well as the difference in binding interaction with catalytic pocket. Thus, the present study provides how substrate binding couples the movement in TM domain and periplasmic domain which might help in the understanding of active site communication in PgpB. C55-PP phosphatase interactions with a catalytic pocket of PgpB provide new insight for designing drugs against bacterial infection.     

A Multi-Variant,Viral Dynamic Model of Genotype 1 HCV to Assess the in vivo Evolution of Protease-Inhibitor Resistant Variants

Variants resistant to compounds specifically targeting HCV are observed in clinical trials. A multi-variant viral dynamic model was developed to quantify the evolution and in vivo fitness of variants in subjects dosed with monotherapy of an HCV protease inhibitor, telaprevir. Variant fitness was estimated using a model in which variants were selected by competition for shared limited replication space. Fitness was represented in the absence of telaprevir by different variant production rate constants and in the presence of telaprevir by additional antiviral blockage by telaprevir. Model parameters, including rate constants for viral production, clearance, and effective telaprevir concentration, were estimated from 1) plasma HCV RNA levels of subjects before, during, and after dosing, 2) post-dosing prevalence of plasma variants from subjects, and 3) sensitivity of variants to telaprevir in the HCV replicon. The model provided a good fit to plasma HCV RNA levels observed both during and after telaprevir dosing, as well as to variant prevalence observed after telaprevir dosing. After an initial sharp decline in HCV RNA levels during dosing with telaprevir, HCV RNA levels increased in some subjects. The model predicted this increase to be caused by pre-existing variants with sufficient fitness to expand once available replication space increased due to rapid clearance of wild-type (WT) virus. The average replicative fitness estimates in the absence of telaprevir ranged from 1% to 68% of WT fitness. Compared to the relative fitness method, the in vivo estimates from the viral dynamic model corresponded more closely to in vitro replicon data, as well as to qualitative behaviors observed in both on-dosing and long-term post-dosing clinical data. The modeling fitness estimates were robust in sensitivity analyses in which the restoration dynamics of replication space and assumptions of HCV mutation rates were varied.     

Highly Specific Contractions of a Single CAG/CTG Trinucleotide Repeat by TALEN in Yeast

Trinucleotide repeat expansions are responsible for more than two dozens severe neurological disorders in humans. A double-strand break between two short CAG/CTG trinucleotide repeats was formerly shown to induce a high frequency of repeat contractions in yeast. Here, using a dedicated TALEN, we show that induction of a double-strand break into a CAG/CTG trinucleotide repeat in heterozygous yeast diploid cells results in gene conversion of the repeat tract with near 100% efficacy, deleting the repeat tract. Induction of the same TALEN in homozygous yeast diploids leads to contractions of both repeats to a final length of 3–13 triplets, with 100% efficacy in cells that survived the double-strand breaks. Whole-genome sequencing of surviving yeast cells shows that the TALEN does not increase mutation rate. No other CAG/CTG repeat of the yeast genome showed any length alteration or mutation. No large genomic rearrangement such as aneuploidy, segmental duplication or translocation was detected. It is the first demonstration that induction of a TALEN in an eukaryotic cell leads to shortening of trinucleotide repeat tracts to lengths below pathological thresholds in humans, with 100% efficacy and very high specificity.     

microRNA-210 is upregulated in hypoxic cardiomyocytes through Akt- and p53-dependent pathways and exerts cytoprotective effects

microRNA-210 (miR-210) is upregulated in hypoxia, but its function in cardiomyocytes and its regulation in response to hypoxia are not well characterized. The purpose of this study was to identify upstream regulators of miR-210, as well as to characterize miR-210's function in cardiomyocytes. We first showed miR-210 is upregulated through both hypoxia-inducible factor (HIF)-dependent and -independent pathways, since aryl hydrocarbon nuclear translocator (ARNT) knockout mouse embryonic fibroblasts (MEF), lacking intact HIF signaling, still displayed increased miR-210 levels in hypoxia. To determine the mechanism for HIF-independent regulation of miR-210, we focused on p53 and protein kinase B (Akt). Overexpression of p53 in wild-type MEFs induced miR-210, whereas p53 overexpression in ARNT knockout MEFs did not, suggesting p53 regulates miR-210 in a HIF-dependent mechanism. Akt inhibition reduced miR-210 induction by hypoxia, whereas Akt overexpression increased miR-210 levels in both wild-type and ARNT knockout MEFs, indicating Akt regulation of miR-210 is HIF-independent. We then studied the effects of miR-210 in cardiomyocytes. Overexpression of miR-210 reduced cell death in response to oxidative stress and reduced reactive oxygen species (ROS) production both at baseline and after treatment with antimycin A. Furthermore, downregulation of miR-210 increased ROS after hypoxia-reoxygenation. To determine a mechanism for the cytoprotective effects of miR-210, we focused on the predicted target, apoptosis-inducing factor, mitochondrion-associated 3 (AIFM3), known to induce cell death. Although miR-210 reduced AIFM3 levels, overexpression of AIFM3 in the presence of miR-210 overexpression did not reduce cellular viability either at baseline or after hydrogen peroxide treatment, suggesting AIFM3 does not mediate miR-210's cytoprotective effects. Furthermore, HIF-3α, a negative regulator of HIF signaling, is targeted by miR-210, but miR-210 does not modulate HIF activity. In conclusion, we demonstrate a novel role for p53 and Akt in regulating miR-210 and demonstrate that, in cardiomyocytes, miR-210 exerts cytoprotective effects, potentially by reducing mitochondrial ROS production.     

Quantitative characterization of the influence of the nanoscale morphology of nanostructured surfaces on bacterial adhesion and biofilm formation

Bacterial infection of implants and prosthetic devices is one of the most common causes of implant failure. The nanostructured surface of biocompatible materials strongly influences the adhesion and proliferation of mammalian cells on solid substrates. The observation of this phenomenon has led to an increased effort to develop new strategies to prevent bacterial adhesion and biofilm formation, primarily through nanoengineering the topology of the materials used in implantable devices. While several studies have demonstrated the influence of nanoscale surface morphology on prokaryotic cell attachment, none have provided a quantitative understanding of this phenomenon. Using supersonic cluster beam deposition, we produced nanostructured titania thin films with controlled and reproducible nanoscale morphology respectively. We characterized the surface morphology; composition and wettability by means of atomic force microscopy, X-ray photoemission spectroscopy and contact angle measurements. We studied how protein adsorption is influenced by the physico-chemical surface parameters. Lastly, we characterized Escherichia coli and Staphylococcus aureus adhesion on nanostructured titania surfaces. Our results show that the increase in surface pore aspect ratio and volume, related to the increase of surface roughness, improves protein adsorption, which in turn downplays bacterial adhesion and biofilm formation. As roughness increases up to about 20 nm, bacterial adhesion and biofilm formation are enhanced; the further increase of roughness causes a significant decrease of bacterial adhesion and inhibits biofilm formation. We interpret the observed trend in bacterial adhesion as the combined effect of passivation and flattening effects induced by morphology-dependent protein adsorption. Our findings demonstrate that bacterial adhesion and biofilm formation on nanostructured titanium oxide surfaces are significantly influenced by nanoscale morphological features. The quantitative information, provided by this study about the relation between surface nanoscale morphology and bacterial adhesion points towards the rational design of implant surfaces that control or inhibit bacterial adhesion and biofilm formation.