Structural Identification and Kinetic Analysis of the in Vitro Products Formed by Reaction of Bisphenol A‑3,4-quinone with N‑Acetylcysteine and Glutathione

Bisphenol A (BPA) has received considerable attention as an endocrine disrupting chemical and a possible substrate for genotoxic metabolites. BPA metabolism leads to formation of electrophilic o-quinones cable of binding to DNA and other endogenous nucleophiles. We have structurally identified the products resulting from the reaction of bisphenol A-3,4-quinone (BPAQ) with N-acetylcysteine (NAC) and glutathione (GSH). The major and minor isomers are both the result of 1,6-conjugate addition and are produced almost instantly in high yield. Reactions using 1.3 equiv of GSH showed the presence of a bis-glutathionyl adduct which was not observed using higher GSH concentration relative to BPAQ. NAC reactions with BPAQ showed no bis-N-acetylcysteinyl adducts. Stopped-flow kinetic analysis reveals the 1,6-conjugate additions to be reversible with a forward free energy of activation of 9.2 and 7.8 kcal/mol for the NAC and GSH reactions, respectively. The bimolecular forward rate constant at 19.4 °C was approximately three time faster for GSH compared to NAC, 1547 vs 496 M−1 s−1. The free energy of activation for the reverse reactions were similar, 11.7 and 11.2 kcal/mol for NAC and GSH, respectively. We plan to use this model system to further explore the mechanism of adduct formation between sulfur nucleophiles and o-quinones and the resulting chemical properties of both NAC and GSH adducts

Structure of the 5′ Untranslated Region of Enteroviral Genomic RNA

Enteroviral RNA genomes share a long, highly structured 5= untranslated region (5= UTR) containing a type I internal ribosome entry site (IRES). The 5= UTR is composed of stably folded RNA domains connected by unstructured RNA regions. Proper folding and functioning of the 5= UTR underlies the efficiency of viral replication and also determines viral virulence. We have characterized the structure of 5= UTR genomic RNA from coxsackievirus B3 using selective 2=-hydroxyl acylation analyzed by primer extension (SHAPE) and base-specific chemical probes in solution. Our results revealed novel structural features, including realignment of major domains, newly identified long-range interactions, and an intrinsically disordered connecting region. Together, these newly identified features contribute to a model for enteroviral 5= UTRs with type I IRES elements that links structure to function during the hierarchical processes directed by genomic RNA during viral infection. IMPORTANCE: Enterovirus infections are responsible for human diseases, including myocarditis, pancreatitis, acute flaccid paralysis, and poliomyelitis. The virulence of these viruses depends on efficient recognition of the RNA genome by a large family of host proteins and protein synthesis factors, which in turn relies on the threedimensional folding of the first 750 nucleotides of the molecule. Structural information about this region of the genome, called the 5= untranslated region (5= UTR), is needed to assist in the process of vaccine and antiviral development. This work presents a model for the structure of the enteroviral 5= UTR. The model includes an RNA element called an intrinsically disordered RNA region (IDRR). Intrinsically disordered proteins (IDPs) are well known, but correlates in RNA have not been proposed. The proposed IDRR is a 20-nucleotide region, long known for its functional importance, where structural flexibility helps explain recognition by factors controlling multiple functional states

Epidemiology of plasmid lineages mediating the spread of extended-spectrum beta-lactamases among clinical Escherichia coli

The prevalence of extended-spectrum beta-lactamases (ESBLs) among clinical isolates of Escherichia coli has been increasing, with this spread driven by ESBL-encoding plasmids. However, the epidemiology of ESBL-disseminating plasmids remains understudied, obscuring the roles of individual plasmid lineages in ESBL spread. To address this, we performed an in-depth genomic investigation of 149 clinical ESBL-like E. coli isolates from a tertiary care hospital. We obtained high-quality assemblies for 446 plasmids, revealing an extensive map of plasmid sharing that crosses time, space, and bacterial sequence type boundaries. Through a sequence-based network, we identified specific plasmid lineages that are responsible for the dissemination of major ESBLs. Notably, we demonstrate that IncF plasmids separate into 2 distinct lineages that are enriched for different ESBLs and occupy distinct host ranges. Our work provides a detailed picture of plasmid-mediated spread of ESBLs, demonstrating the extensive sequence diversity within identified lineages, while highlighting the genetic elements that underlie the persistence of these plasmids within the clinical E. coli population

Mathematical modelling of entropy generation in magnetized micropolar flow between co-rotating cylinders with internal heat generation

The present study investigates analytically the entropy generation in magnetized micropolar fluid flow in between two vertical concentric rotating cylinders of infinite length. The surface of the inner cylinder is heated while the surface of the outer cylinder is cooled. Internal heat generation (which arises in energy systems) is incorporated. The Eringen thermo-micropolar fluid model is used to simulate the micro-structural rheological flow characteristics in the annulus region. The flow is subjected to a constant, static, axial magnetic field. The surface of the inner cylinder is prescribed to be isothermal (constant temperature wall condition), whereas the surface of the outer cylinder was exposed to convection cooling. The conservation equations are normalized and closed-form solutions are obtained for the velocity, microrotation and temperature. These are thereafter utilized to derive the expressions for entropy generation number, Bejan number and total entropy generation rate. The effects of relevant thermo-physical parameters on the flow, heat and entropy generation rate are displayed graphically and interpreted at length. It is observed that the external magnetic force enhances the entropy production rate is minimum at the center point of the channel and maximum in the proximity of the inner cylinder. This causes more wear and tear at the surface of the inner cylinder. Greater Hartmann number also elevates microrotation values in the entire annulus region. The study is relevant to optimization of chemical engineering processes, nuclear engineering cooling systems and propulsion systems utilizing non-Newtonian fluids and magnetohydrodynamics

Computation of entropy generation in dissipative transient natural convective viscoelastic flow

Entropy generation is an important aspect of modern thermal polymer processing optimization. Many polymers exhibit strongly non-Newtonian effects and dissipation effects in thermal processing. Motivated by these aspects in this article a numerical analysis of the entropy generation with viscous dissipation effect in an unsteady flow of viscoelastic fluid from a vertical cylinder is presented. The Reiner-Rivlin physical model of grade two (second grade fluid) is employed which can envisage normal stress variations in polymeric flow-fields. Viscosity variation is included. The obtained governing equations are resolved using implicit finite difference method of Crank-Nicolson type with well imposed initial and boundary conditions. Key control parameters are the second-grade viscoelastic fluid parameter (β), viscosity variation parameter (γ) and viscous dissipation parameter (ε). Also, group parameter (BrΩ-1), Grashof number (Gr) and Prandtl number (Pr) are examined. Numerical solutions are presented for steady-state flow variables, temperature, time histories of friction, wall heat transfer rate, entropy and Bejan curves for distinct values of control parameters. The results specify that entropy generation decreases with augmenting values of β, γ and Gr. The converse trend is noticed with increasing Pr and BrΩ-1. Furthermore, the computations reveal that entropy and Bejan lines only occur close to the hot cylinder wall

Effects of temperature-dependent viscosity variation on entropy generation, heat and fluid flow through a porous-saturated duct of rectangular cross-section

Effect of temperature-dependent viscosity on fully developed forced convection in a duct of rectangular cross-section occupied by a fluid-saturated porous medium is investigated analytically. The Darcy flow model is applied and the viscosity-temperature relation is assumed to be an inverse-linear one. The case of uniform heat flux on the walls, i.e. the H boundary condition in the terminology of Kays and Crawford, is treated. For the case of a fluid whose viscosity decreases with temperature, it is found that the effect of the variation is to increase the Nusselt number for heated walls. Having found the velocity and the temperature distribution, the second law of thermodynamics is invoked to find the local and average entropy generation rate. Expressions for the entropy generation rate, the Bejan number, the heat transfer irreversibility, and the fluid flow irreversibility are presented in terms of the Brinkman number, the Péclet number, the viscosity variation number, the dimensionless wall heat flux, and the aspect ratio (width to height ratio). These expressions let a parametric study of the problem based on which it is observed that the entropy generated due to flow in a duct of square cross-section is more than those of rectangular counterparts while increasing the aspect ratio decreases the entropy generation rate similar to what previously reported for the clear flow case

Probing RNA Structure in the 5\u27 Untranslated Region of Coxsackievirus B3 Genomic RNA

Coxsackievirus B3 (CVB3) is a cardiovirulent enterovirus that utilizes a 5’ untranslated region (5’UTR) to complete critical viral processes. This includes an internal ribosome entry site (IRES) responsible for cap-independent translation. Ample evidence supports the hypothesis that the 5’UTR with its IRES is an important virulence determinant for the virus. We are investigating the structure of the 5’UTR from RNA genomes derived from naturally occurring virulent and avirulent viruses. Chimeric constructs and site-directed mutations of these genomes are part of the analysis. Our structural studies include queries of the RNA itself as well as the RNA complexed with host proteins such as polyrC binding protein 2 (PCBP2). We explore RNA structure in solution using base-specific modifying agents such as dimethyl sulfate as well as backbone probes through selective 2\u27-hydroxyl acylation analyzed by primer extension (SHAPE). Our results have resulted in a detailed secondary structure model for the 5’UTR, including those domains involved in the IRES. Comparing the structure of 5’UTR sequences from virulent and avirulent genomes, including chimeric constructs, shows there are key structural differences that correlate to the virulence phenotype. These studies also identify a critical virulence determinant in domain II of the 5’UTR. Chemical probing of 5’UTR molecules with bound PCBP2 shows that regions in the cloverleaf (domain I) and domain IV are protected from modification, indicative of direct protein interaction in these regions. Interestingly, other regions, particularly in the capping stem-loops of domain IV, show increased accessibility to chemical probes, indicative of conformational changes in RNA structure in response to protein binding. These accessibility increases are concentration dependent. In summary, our structural studies are defining the structure and structural dynamics of the 5’UTR from an important enterovirus