Comparative characterisation of two nitroreductases from Giardia lamblia as potential activators of nitro compounds

Giardia lamblia is a protozoan parasite that causes giardiasis, a diarrhoeal disease affecting humans and various animal species. Nitro drugs such as the nitroimidazole metronidazole and the nitrothiazolide nitazoxanide are used for treatment of giardiasis. Nitroreductases such as GlNR1 and GlNR2 may play a role in activation or inactivation of these drugs. The aim of this work is to characterise these two enzymes using functional assays. For respective analyses recombinant analogues from GlNR1 and GlNR2 were produced in Escherichia coli. E. coli expressing GlNR1 and GlNR2 alone or together were grown in the presence of nitro compounds. Furthermore, pull-down assays were performed using HA-tagged GlNR1 and GlNR2 as baits. As expected, E. coli expressing GlNR1 were more susceptible to metronidazole under aerobic and semi-aerobic and to nitazoxanide under semi-aerobic growth conditions whereas E. coli expressing GlNR2 were susceptible to neither drug. Interestingly, expression of both nitroreductases gave the same results as expression of GlNR2 alone. In functional assays, both nitroreductases had their strongest activities on the quinone menadione (vitamin K3) and FAD, but reduction of nitro compounds including the nitro drugs metronidazole and nitazoxanidewas clearly detected. Full reduction of 7-nitrocoumarin to 7-aminocoumarin was preferentially achieved with GlNR2. Pull-down assays revealed that GlNR1 and GlNR2 interacted in vivo forming a multienzyme complex. These findings suggest that both nitroreductases are multifunctional. Their main biological role may reside in the reduction of vitamin K analogues and FAD. Activation by GlNR1 or inactivation by GlNR2 of nitro drugs may be the consequence of a secondary enzymatic activity either yielding (GlNR1) or eliminating (GlNR2) toxic intermediates after reduction of these compounds. © 2015 The Authors. Published by Elsevier Ltd on behalf of Australian Society for Parasitology. This is an open access article under the CC BY-NC-ND license

Identification and characterisation of ncRNAs in dormant bacteria

Recent years have led to increasing interest and appreciation of the possible importance of single cell heterogeneity in various biological processes. One of the examples of phenotypic heterogeneity in bacterial populations is antibiotic tolerant persister cells. Such an antibiotic tolerance phenotype is of considerable clinical relevance since dormant bacteria can re-establish infections rapidly after the antibiotic treatment has been terminated. Up to now mechanisms for establishing the persistence phenomenon in bacteria have remained largely enigmatic. Persisters are cells considered to be in a dormant state with down regulated gene expression. Only recently small regulatory RNAs (sRNAs) have been appreciated as important regulators of gene expression in response to environmental stimuli and several theoretical studies have suggested a possible involvement of sRNAs in the mechanisms of regulated heterogeneity in bacteria. We have experimentally addressed this potential link between sRNAs and persistence/dormancy in E. coli as an example of heterogeneity. Beside classical sRNAs we are focusing also on sRNAs directly associating with and possibly regulating the ribosome, the central enzyme of gene expression. The persister and dormant cell specific sRNA profile is studied by the comparative analysis of sRNA profile changes of the whole bacterial population after antibiotic killing. From RNA-Seq data ~ 25 000 potentially stable RNA fragments were identified and initial analysis predicted ~300 of them to be dormant/persister cell specific. After further evaluation the most prominent dormant/persister cell specific sRNAs are functionally characterized and their potential role in the persistence/dormancy will be evaluated by applying genetic, molecular and biochemical tools. The potential results of this project will provide a better understanding on the molecular mechanism of bacterial persistence/dormancy and on the role of ribosome-bound sRNA molecules in fine-tuning gene expression.

Identification and characterisation of ncRNAs in dormant bacteria

Recent years have led to increasing interest and appreciation of the possible importance of single cell heterogeneity in various biological processes. One of the examples of phenotypic heterogeneity in bacterial populations is antibiotic tolerant persister cells. Such an antibiotic tolerance phenotype is of considerable clinical relevance since dormant bacteria can re-establish infections rapidly after the antibiotic treatment has been terminated. Up to now mechanisms for establishing the persistence phenomenon in bacteria have remained largely enigmatic. Persisters are cells considered to be in a dormant state with down regulated gene expression. Only recently small regulatory RNAs (sRNAs) have been appreciated as important regulators of gene expression in response to environmental stimuli and several theoretical studies have suggested a possible involvement of sRNAs in the mechanisms of regulated heterogeneity in bacteria. We have experimentally addressed this potential link between sRNAs and persistence/dormancy in E. coli as an example of heterogeneity. Beside classical sRNAs we are focusing also on sRNAs directly associating with and possibly regulating the ribosome, the central enzyme of gene expression. The persister and dormant cell specific sRNA profile is studied by the comparative analysis of sRNA profile changes of the whole bacterial population after antibiotic killing. From RNA-Seq data ~ 25 000 potentially stable RNA fragments were identified and initial analysis predicted ~300 of them to be dormant/persister cell specific. After further evaluation the most prominent dormant/persister cell specific sRNAs are functionally characterized and their potential role in the persistence/dormancy will be evaluated by applying genetic, molecular and biochemical tools. The potential results of this project will provide a better understanding on the molecular mechanism of bacterial persistence/dormancy and on the role of ribosome-bound sRNA molecules in fine-tuning gene expression.

Identification and characterisation of ncRNAs in dormant bacteria

Recent years have led to increasing interest and appreciation of the possible importance of single cell heterogeneity in various biological processes. One of the examples of phenotypic heterogeneity in bacterial populations is antibiotic tolerant persister cells. Such an antibiotic tolerance phenotype is of considerable clinical relevance since dormant bacteria can re-establish infections rapidly after the antibiotic treatment has been terminated. Up to now mechanisms for establishing the persistence phenomenon in bacteria have remained largely enigmatic. Persisters are cells considered to be in a dormant state with down regulated gene expression. Only recently small regulatory RNAs (sRNAs) have been appreciated as important regulators of gene expression in response to environmental stimuli and several theoretical studies have suggested a possible involvement of sRNAs in the mechanisms of regulated heterogeneity in bacteria. We have experimentally addressed this potential link between sRNAs and persistence/dormancy in E. coli as an example of heterogeneity. Beside classical sRNAs we are focusing also on sRNAs directly associating with and possibly regulating the ribosome, the central enzyme of gene expression. The persister and dormant cell specific sRNA profile is studied by the comparative analysis of sRNA profile changes of the whole bacterial population after antibiotic killing. From RNA-Seq data ~ 25 000 potentially stable RNA fragments were identified and initial analysis predicted ~300 of them to be dormant/persister cell specific. After further evaluation the most prominent dormant/persister cell specific sRNAs are functionally characterized and their potential role in the persistence/dormancy will be evaluated by applying genetic, molecular and biochemical tools. The potential results of this project will provide a better understanding on the molecular mechanism of bacterial persistence/dormancy and on the role of ribosome-bound sRNA molecules in fine-tuning gene expression.

Production and characterisation of monoclonal antibodies specific for bovine interleukin-4

Genetic immunisation is a simple method for producing polyclonal antibodies in mice. By this method, we produced antibodies against bovine interleukin-4 (BoIL-4). After a final injection with a recombinant BoIL-4 protein, nine stable hybridoma cell lines were established which secreted monoclonal antibodies (MAbs) against this cytokine. Specific binding of each of the MAbs to recombinant BoIL-4 produced by Escherichia coli, baculovirus, and Trypanosoma brucei was demonstrated in an indirect ELISA and/or in Western blotting. These MAbs recognise the same antigenic region localised in the first 47 amino acids of the mature protein. None of them was able to neutralise the biological activity of the BoIL-4 under the conditions tested but one allowed the detection of BoIL-4 by flow cytometry.

Characterisation of gp34, a GPI-anchored protein expressed by schizonts of Theileria parva and T. annulata

Using bioinformatics tools, we searched the predicted Theileria annulata and T. parva proteomes for putative schizont surface proteins. This led to the identification of gp34, a GPI-anchored protein that is stage-specifically expressed by schizonts of both Theileria species and is downregulated upon induction of merogony. Transfection experiments in HeLa cells showed that the gp34 signal peptide and GPI anchor signal are also functional in higher eukaryotes. Epitope-tagged Tp-gp34, but not Ta-gp34, expressed in the cytosol of COS-7 cells was found to localise to the central spindle and midbody. Overexpression of Tp-gp34 and Ta-gp34 induced cytokinetic defects and resulted in accumulation of binucleated cells. These findings suggest that gp34 could contribute to important parasite-host interactions during host cell division.

Comparison of biological stability and metabolism of CCK2 receptor targeting peptides, a collaborative project under COST BM0607

Stability of radiolabelled cholecystokinin 2 (CCK2) receptor targeting peptides has been a major limitation in the use of such radiopharmaceuticals especially for targeted radionuclide therapy applications, e.g. for treatment of medullary thyroid carcinoma (MTC). The purpose of this study was to compare the in vitro stability of a series of peptides binding to the CCK2 receptor [selected as part of the COST Action on Targeted Radionuclide Therapy (BM0607)] and to identify major cleavage sites.

Characterisation of extreme winter precipitation in Mediterranean coastal sites and associated anomalous atmospheric circulation patterns

We present an analysis of daily extreme precipitation events for the extended winter season (October–March) at 20 Mediterranean coastal sites covering the period 1950–2006. The heavy tailed behaviour of precipitation extremes and estimated return levels, including associated uncertainties, are derived applying a procedure based on the Generalized Pareto Distribution, in combination with recently developed methods. Precipitation extremes have an important contribution to make seasonal totals (approximately 60% for all series). Three stations (one in the western Mediterranean and the others in the eastern basin) have a 5-year return level above 100 mm, while the lowest value (estimated for two Italian series) is equal to 58 mm. As for the 50-year return level, an Italian station (Genoa) has the highest value of 264 mm, while the other values range from 82 to 200 mm. Furthermore, six series (from stations located in France, Italy, Greece, and Cyprus) show a significant negative tendency in the probability of observing an extreme event. The relationship between extreme precipitation events and the large scale atmospheric circulation at the upper, mid and low troposphere is investigated by using NCEP/NCAR reanalysis data. A 2-step classification procedure identifies three significant anomaly patterns both for the western-central and eastern part of the Mediterranean basin. In the western Mediterranean, the anomalous southwesterly surface to mid-tropospheric flow is connected with enhanced moisture transport from the Atlantic. During ≥5-year return level events, the subtropical jet stream axis is aligned with the African coastline and interacts with the eddy-driven jet stream. This is connected with enhanced large scale ascending motions, instability and leads to the development of severe precipitation events. For the eastern Mediterranean extreme precipitation events, the identified anomaly patterns suggest warm air advection connected with anomalous ascent motions and an increase of the low- to mid-tropospheric moisture. Furthermore, the jet stream position (during ≥5-year return level events) supports the eastern basin being in a divergence area, where ascent motions are favoured. Our results contribute to an improved understanding of daily precipitation extremes in the cold season and associated large scale atmospheric features.

Molecular and biochemical characterisation of a novel mutation in POLG associated with Alpers syndrome

Background DNA polymerase γ (POLG) is the only known mitochondrial DNA (mtDNA) polymerase. It mediates mtDNA replication and base excision repair. Mutations in the POLG gene lead to reduction of functional mtDNA (mtDNA depletion and/or deletions) and are therefore predicted to result in defective oxidative phosphorylation (OXPHOS). Many mutations map to the polymerase and exonuclease domains of the enzyme and produce a broad clinical spectrum. The most frequent mutation p.A467T is localised in the linker region between these domains. In compound heterozygote patients the p.A467T mutation has been described to be associated amongst others with fatal childhood encephalopathy. These patients have a poorer survival rate compared to homozygotes. Methods mtDNA content in various tissues (fibroblasts, muscle and liver) was quantified using quantitative PCR (qPCR). OXPHOS activities in the same tissues were assessed using spectrophotometric methods and catalytic stain of BN-PAGE. Results We characterise a novel splice site mutation in POLG found in trans with the p.A467T mutation in a 3.5 years old boy with valproic acid induced acute liver failure (Alpers-Huttenlocher syndrome). These mutations result in a tissue specific depletion of the mtDNA which correlates with the OXPHOS-activities. Conclusions mtDNA depletion can be expressed in a high tissue-specific manner and confirms the need to analyse primary tissue. Furthermore, POLG analysis optimises clinical management in the early stages of disease and reinforces the need for its evaluation before starting valproic acid treatment.

Systematic review of the survival rate and the incidence of biological, technical, and aesthetic complications of single crowns on implants reported in longitudinal studies with a mean follow-up of 5 years

To assess the 5-year survival of implant-supported single crowns (SCs) and to describe the incidence of biological, technical, and aesthetic complications. The focused question was: What is the survival rate of implants supporting single crowns and implant-supported crowns with a mean follow-up of 5 years and to which extent do biological, technical, and aesthetic complications occur?