Biomarkers of toxicity in Clarias gariepinus exposed to sublethal concentrations of polycyclic aromatic hydrocarbons

Physiological, biochemical and histological indices in Clarias gariepinus broodstock, and teratogenic indices in embryos exposed to sublethal concentrations of naphthalene, phenanthrene and pyrene were investigated in 2014 using a static-renewal bioassay protocol. Phenanthrene (1.41 mg l^?1) was the most toxic, followed by pyrene (1.53 mg l^?1) and naphthalene (7.21 mg l^?1), based on 96 h LC50 values. Hepatosomatic indices were significantly higher in naphthalene- and pyrene-treated males compared with solvent controls, whereas fecundity in females was significantly lower by factors of 2.4 (naphthalene), 2.8 (phenanthrene) and 2.4 (pyrene), compared with controls. Catalase levels were lower in female phenanthrene-treated fish compared with controls. Histological alterations observed in PAH-treated fish include oedema, inflammatory cells, epithelial lifting and hyperplasia in the gills, vacuolation, haemosiderin pigments and sinusoidal congestion in the liver, and degenerated zona radiata in the ovary. Teratogenic effects were not observed, as evidenced by the lack of histological alterations in embryos spawned from pre-exposed broodstock. Sex-specific responses and the utility of biomarkers at cellular and individual levels of organisation are therefore demonstrated for holistic evaluations of polycyclic aromatic hydrocarbons in ecotoxicological studies.     

Investigation of the impact of MIG1 and MIG2 on the physiology of Saccharomyces cerevisiae

The gene functions of MIG1 and MIG2 are well known for their role in glucose control in Saccharomyces cerevisiae. A prototrophic mig1 disruptant (T468) and mig1mig2 double disruptant (T475) as well as their congenic wild-type strain (CEN.PK 113-7D) were analysed for changes in their peripheral metabolism (batch cultivations on sugar mixtures) and central metabolism (batch and continuous cultivations as well as acceleratostats). Sucrose metabolism was alleviated of glucose control in the mig1 disruptant, and even more so in the mig1mig2 disruptant compared with their wild-type strain. The lag phase in a batch cultivation grown on a glucose-galactose mixture was reduced by 50% in either disruptant, i.e. additional disruption of MIG2 in a mig1 background did not further alleviate galactose metabolism from glucose control. In contrast, both disruptants exhibited a more stringent glucose control of maltose metabolism compared with the wild-type strain. Growing on glucose, the mig1mig2 double disruptant exhibited a 12% higher specific growth rate than the wild-type strain, as well as a significantly higher respiratory capacity.     

Signaling from beta1- and beta2-adrenergic receptors is defined by differential interactions with PDE4

Beta1- and beta2-adrenergic receptors (betaARs) are highly homologous, yet they play clearly distinct roles in cardiac physiology and pathology. Myocyte contraction, for instance, is readily stimulated by beta1AR but not beta2AR signaling, and chronic stimulation of the two receptors has opposing effects on myocyte apoptosis and cell survival. Differences in the assembly of macromolecular signaling complexes may explain the distinct biological outcomes. Here, we demonstrate that beta1AR forms a signaling complex with a cAMP-specific phosphodiesterase (PDE) in a manner inherently different from a beta2AR/beta-arrestin/PDE complex reported previously. The beta1AR binds a PDE variant, PDE4D8, in a direct manner, and occupancy of the receptor by an agonist causes dissociation of this complex. Conversely, agonist binding to the beta2AR is a prerequisite for the recruitment of a complex consisting of beta-arrestin and the PDE4D variant, PDE4D5, to the receptor. We propose that the distinct modes of interaction with PDEs result in divergent cAMP signals in the vicinity of the two receptors, thus, providing an additional layer of complexity to enforce the specificity of beta1- and beta2-adrenoceptor signaling.     

Viral polypeptides detected by a complement-dependent neutralizing murine monoclonal antibody to human cytomegalovirus.

Murine monoclonal antibodies were produced which coimmunoprecipitated, under reducing conditions, 130,000- and 55,000-dalton (Da) polypeptides from cells infected with human cytomegalovirus (CMV) strain AD169. A 92,000-Da species, possibly a biosynthetic intermediate, was also detectable. One of the monoclonal antibodies, 15D8, neutralized CMV AD169 only in the presence of guinea pig complement. A second monoclonal antibody, 14E10, coimmunoprecipitated the 130,000- and 55,000-Da polypeptides but did not neutralize viral infectivity. By sequential immunoprecipitation, both monoclonal antibodies have been shown to recognize the same polypeptides. Monoclonal antibody 15D8 detected the 130,000- and 55,000-Da polypeptides in five of six clinical strains and three laboratory strains tested. The 14E10 monoclonal antibody detected the 130,000-Da protein in four of six CMV clinical isolates and in strain AD169 but did not immunoprecipitate any polypeptides from extracts of cells infected with either Towne or Davis laboratory strains. In kinetic studies, the synthesis of the 130,000-Da polypeptide preceded the appearance of the 55,000-Da polypeptide. In infected cells radiolabeled with a pulse of L-[35S]methionine, the isotope was initially detected in the 130,000-Da polypeptide but could be chased into the 55,000-Da polypeptide. These polypeptides exist in the intracellular and extracellular virus as disulfide-linked multimers. Extracellular virus contained a high-molecular-weight (greater than 200,000 Da) multimer composed entirely of 55,000-Da polypeptides. In extracts from infected cells an additional high-molecular-weight multimer was detected consisting of disulfide-linked 130,000-Da polypeptides.     

Thermodynamic and biological evaluation of a thrombin binding aptamer modified with several unlocked nucleic acid (UNA) monomers and a 2'-C-piperazino-UNA monomer

Thrombin binding aptamer is a DNA 15-mer which forms a G-quadruplex structure and possess promising anticoagulant properties due to specific interactions with thrombin. Herein we present the influence of a single 2'-C-piperazino-UNA residue and UNA residues incorporated in several positions on thermodynamics, kinetics and biological properties of the aptamer. 2'-C-Piperazino-UNA is characterized by more efficient stabilization of quadruplex structure in comparison to regular UNA and increases thermodynamic stability of TBA by 0.28-0.44 kcal/mol in a position depending manner with retained quadruplex topology and molecularity. The presence of UNA-U in positions U3, U7, and U12 results in the highest stabilization of G-quadruplex structure (ΔΔG(37)(°)=-1.03kcal/mol). On the contrary, the largest destabilization mounting to 1.79 kcal/mol was observed when UNA residues were placed in positions U7, G8, and U9. Kinetic studies indicate no strict correlation between thermodynamic stability of modified variants and their binding affinity to thrombin. Most of the studied variants bind thrombin, albeit with decreased affinity in reference to unmodified TBA. Thrombin time assay studies indicate three variants as being as potent as TBA in fibrin clotting inhibition.     

Beta-Lactamase Producing Escherichia coli Isolates in Imported and Locally Produced Chicken Meat from Ghana

The use of antibiotics in food animals is of public health concern, because resistant zoonotic pathogens can be transmitted to humans. Furthermore, global trade with food may rapidly spread multi-resistant pathogens between countries and even continents. The purpose of the study was to investigate whether imported chicken meat and meat from locally reared chicken are potential sources for human exposure to multi resistant Escherichia coli isolates. 188 samples from imported and locally produced chicken meat were sampled and analyzed. 153 bacteria isolates were successfully cultured and identified as E. coli using MALDI-ToF. Of these 109 isolates were from meat whereas the remaining 44 were isolated from the cloaca of locally reared live chickens. Antimicrobial susceptibility test was done on the identified E. coli isolates. Additionally, beta-lactamases production (ESBL and/or AmpC) were phenotypically confirmed on all isolates showing resistance to cefpodoxime. Beta-lactamase producing (BLP) E. coli meat isolates were further genotyped. Antimicrobial resistance to four antibiotic markers with highest resistance was detected more frequently in isolates from local chickens compared to imported chickens (tetracycline 88.9% vs. 57.5%, sulphonamide 75.0% vs. 46.6%, ampicillin 69.4% vs. 61.6% and trimethoprim 66.7% vs. 38.4%). Beta-lactamase production was found in 29 E. coli meat isolates, with 56.9% of them being multiple drug resistant (≥ 3). The predominant phylogroup identified was B1 followed by A and D, with similar distribution among the isolates from meat of locally reared chickens and imported chickens. Beta-lactamase producing genotype bla _CTX-M-15 (50%; 10/20) was the most frequently drug resistant gene detected. More BLP E. coli isolates were found in imported chicken meat compared to locally reared chickens, demonstrating that these isolates may be spreading through food trade. In conclusion, both imported and locally produced chicken meats are potential sources for human exposure to BLP E. coli.     

Characterization of a Highly Conserved Binding Site of Mlh1 Required for Exonuclease I-Dependent Mismatch Repair

Mlh1 is an essential factor of mismatch repair (MMR) and meiotic recombination. It interacts through its C-terminal region with MutL homologs and proteins involved in DNA repair and replication. In this study, we identified the site of yeast Mlh1 critical for the interaction with Exo1, Ntg2, and Sgs1 proteins, designated as site S2 by reference to the Mlh1/Pms1 heterodimerization site S1. We show that site S2 is also involved in the interaction between human MLH1 and EXO1 or BLM. Binding at this site involves a common motif on Mlh1 partners that we called the MIP-box for the Mlh1 interacting protein box. Direct and specific interactions between yeast Mlh1 and peptides derived from Exo1, Ntg2, and Sgs1 and between human MLH1 and peptide derived from EXO1 and BLM were measured with K_d values ranging from 8.1 to 17.4 μM. In Saccharomyces cerevisiae, a mutant of Mlh1 targeted at site S2 (Mlh1-E682A) behaves as a hypomorphic form of Exo1. The site S2 in Mlh1 mediates Exo1 recruitment in order to optimize MMR-dependent mutation avoidance. Given the conservation of Mlh1 and Exo1 interaction, it may readily impact Mlh1-dependent functions such as cancer prevention in higher eukaryotes.     

Prediction of the articular eminence shape in a patient with unilateral hypoplasia of the right mandibular ramus before and after distraction osteogenesis—A simulation study

The aim of this work was to predict the shape of the articular eminence in a patient with unilateral hypoplasia of the right mandibular ramus before and after distraction osteogenesis (DO). Using a patient-specific musculoskeletal model of the mandible the hypothesis that the observed differences in this patient in the left and right articular eminence inclinations were consistent with minimisation of joint loads was tested. Moreover, a prediction was made of the final shape of the articular eminence after DO when the expected remodelling has reached a steady state. The individual muscle forces and the average TMJ loading were computed for each combination of articular eminence angles both before and after DO. This exhaustive parameter study provides a full overview of average TMJ loading depending on the angles of the articular eminences. Before DO the parameter study resulted in different articular eminence inclinations between left and right sides consistent with patient data obtained from CT scans, indicating that in this patient the articular eminence shapes result from minimisation of joint loads. The simulation model predicts development of almost equal articular eminence shapes after DO. The same tendency was observed in cone beam CT scans (NewTom) of the patient taken 6.5 years after surgery.