Metallo-beta-lactamases of Pseudomonas aeruginosa--a novel mechanism resistance to beta-lactam antibiotics

Since about twenty years, following the introduction into therapeutic of news beta-lactam antibiotics (broad-spectrum cephalosporins, monobactams and carbapenems), a very significant number of new beta-lactamases appeared. These enzymes confer to the bacteria which put them, the means of resisting new molecules. The genetic events involved in this evolution are of two types: evolution of old enzymes by mutation and especially appearance of new genes coming for some, from bacteria of the environment. Numerous mechanisms of enzymatic resistance to the carbapenems have been described in Pseudomonas aeruginosa. The important mechanism of inactivation carbapenems is production variety of b-lactam hydrolysing enzymes associated to carbapenemases. The metallo-beta-enzymes (IMP, VIM, SPM, GIM types) are the most clinically significant carbapenemases. P. aeruginosa posses MBLs and seem to have acquired them through transmissible genetic elements (plasmids or transposons associated with integron) and can be transmission to other bacteria. They have reported worldwide but mostly from South East Asia and Europe. The enzymes, belonging to the molecular class B family, are the most worrisome of all beta-lactamases because they confer resistance to carbapenems and all the beta-lactams (with the exception of aztreonam) and usually to aminoglycosides and quinolones. The dissemination of MBLs genes is thought to be driven by regional consumption of extended--spectrum antibiotics (e.g. cephalosporins and carbapenems), and therefore care must be taken that these drugs are not used unnecessarily.     

O-phospho-L-serine: a modulator of bone healing in calcium-phosphate cements

Bone substitution materials are seen as an alternative to autogenous bone transplants in the reconstruction of human bone structures. The aim of the present animal study was to evaluate the clinical handling and the conditions of bone healing after the application of a phosphoserine and collagen-I-modified calcium-phosphate cement (Biozement D). The application of phosphoserine is supposed to influence the texture of the extracellular matrix. Standardised bone defects were created in the lower jaw of 10 adult minipigs. These defects were reconstructed with a pasty calcium-phosphate cement mixture. After a healing time of 4 months, the animals were sacrificed. The mandibles of all animals were resected and non-decalcified histological sections of the areas of interest were prepared. The experiment was evaluated by means of qualitative histology and histomorphometry. The hydroxyapatite cement entirely hardened intraoperatively. Modelling and handling of the cement was facile and the margin fit to the host bone was excellent. Histology showed that resorption started in the periphery and proceeded exceptionally fast. The bony substitution, especially in phosphoserine-endowed cements, was very promising. After a healing period of 4 months, phosphoserine cements showed a bone regeneration of nearly two-thirds of the defect sizes. In the applied animal experiment, the newly developed hydroxyapatite collagen-I cement is well suited for bone substitution due to its easy handling, its excellent integration and good resorption characteristics. The addition of phosphoserine is very promising in terms of influencing resorption features and bone regeneration.     

Titrating Theileria parva: single stocks against combination of stocks

Theileria parva is the causative agent of East Coast fever (ECF), an important cattle disease in East and Central Africa. One of the methods for control of ECF is 'infection and treatment', a procedure in which an animal is infected with the live parasite and at the same time treated with a long-acting oxytetracycline formulation, restraining the infection and allowing a protective cellular immune response to develop. Optimal immunizing doses were estimated using models of trichotomous response: dysimmunization (death or severe reaction during immunization), immunization failure (death or severe reaction during lethal challenge) and successful immunization (neither dysimmunization nor immunization failure). In this paper we present methods of interpreting immunization trials and apply these methods to previously unpublished data from two such trials: one with a mixture of three T. parva stocks and one with a single T. parva stock. We explain why titration trials conducted with a cocktail of antigens could predict a suboptimal immunization dose. Indeed it is possible for a combination of three individually efficient stocks to result in a mixture with which optimal immunization response might be difficult to achieve, because of averaging effects. The corresponding interpretation provides insights into why standard immunization trials for T. parva have not yielded the results that might be expected of them. The results of this work may also have implications for the use of antigen cocktails in cancer, HIV and malaria vaccine trials.     

Characterization of tRNA-dependent peptide bond formation by MurM in the synthesis of Streptococcus pneumoniae peptidoglycan

MurM is an aminoacyl ligase that adds l-serine or l-alanine as the first amino acid of a dipeptide branch to the stem peptide lysine of the pneumococcal peptidoglycan. MurM activity is essential for clinical pneumococcal penicillin resistance. Analysis of peptidoglycan from the highly penicillin-resistant Streptococcus pneumoniae strain 159 revealed that in vivo and in vitro, in the presence of the appropriate acyl-tRNA, MurM(159) alanylated the peptidoglycan epsilon-amino group of the stem peptide lysine in preference to its serylation. However, in contrast, identical analyses of the penicillin-susceptible strain Pn16 revealed that MurM(Pn16) activity supported serylation more than alanylation both in vivo and in vitro. Interestingly, both MurM(Pn16) acylation activities were far lower than the alanylation activity of MurM(159). The resulting differing stem peptide structures of 159 and Pn16 were caused by the profoundly greater catalytic efficiency of MurM(159) compared with MurM(Pn16) bought about by sequence variation between these enzymes and, to a lesser extent, differences in the in vivo tRNA(Ala):tRNA(Ser) ratio in 159 and Pn16. Kinetic analysis revealed that MurM(159) acted during the lipid-linked stages of peptidoglycan synthesis, that the d-alanyl-d-alanine of the stem peptide and the lipid II N-acetylglucosaminyl group were not essential for substrate recognition, that epsilon-carboxylation of the lysine of the stem peptide was not tolerated, and that lipid II-alanine was a substrate, suggesting an evolutionary link to staphylococcal homologues of MurM such as FemA. Kinetic analysis also revealed that MurM recognized the acceptor stem and/or the TPsiC loop stem of the tRNA(Ala). It is anticipated that definition of the minimal structural features of MurM substrates will allow development of novel resistance inhibitors that will restore the efficacy of beta-lactams for treatment of pneumococcal infection.     

Expression in promoter variant of the ERα gene in bos taurus liver

Due to the variant functions that estrogens play in the regulation of reproduction, development of the mammary gland, growth and differentiation of cells, estrogen receptors and their genes are considered as a candidates for the markers of production and functional traits in farm animals, including cattle. In the earliest study, a 2853-bp bovine ER gene 5′-region was PCR amplified and sequenced. Moreover, for the first time, a polymorphism was described within 5′ region of the bovine ERα gene—A/G transition lying upstream at position 2591 from acceptor splice site +85, possibly within its promoter—which could be recognized with RFLP-BglI. In other study we are found second polymorphism—A/G transition at position 1213 from acceptor splice site +85, located in promoter for exon B. We have examined the specific mRNA expression of ERα in various genotypes using real-time RT-PCR. We used four animals from each genotype group—AG, GG for BglI and AA, AG for SnaBI—to analyse liver ERα expression at the level of Real-time PCR. Liver samples were taken from the 16 young Friesian bulls of the different ERα genotypes, slaughtered at the local abattoir. As shown by Real-Time PCR, on the livers of animals with different genotype ERα mRNA for BglI polymorphism we didn’t found variability, but for SnaBI we have found variability between AG and AA genotypes.     

Bio-oxidation of tripropylene glycol under aerobic conditions

Aerobic biodegradation of tripropylene glycol (PG3) was investigated under the conditions of the OECD screening test 301E and the Continuous Flow Activated Sludge Simulation test (CFAS). A modified two-chamber facility with a denitrification stage was used for the CFAS test. Primary PG3 biodegradation was measured by the HPLC with fluorimetric detection and analyte derivatisation. Metabolites were identified by LC-MS with electrospray ionisation and GC-MS with electron impact ionisation, as well as semiquantitatively determined by the LC-MS technique. PG3 was found to be inherently biodegradable and it exhibits a strong poisonous effect on activated sludge after exceeding the threshold concentration (10 mg l⁻¹). Metabolite accumulation onto the activated sludge is probably responsible for this poisonous effect. Probable biotransformation products of tripropylene glycol under the aerobic conditions include metabolites with a single terminal aldehyde or a ketone group and metabolites with two terminal aldehyde or ketone groups. Their concentration rises at the end of the OECD screening test.     

Fitting conditional survival models to meta-analytic data by using a transformation toward mixed-effects models

Summary .   Frailty models are widely used to model clustered survival data. Classical ways to fit frailty models are likelihood-based. We propose an alternative approach in which the original problem of "fitting a frailty model" is reformulated into the problem of "fitting a linear mixed model" using model transformation. We show that the transformation idea also works for multivariate proportional odds models and for multivariate additive risks models. It therefore bridges segregated methodologies as it provides a general way to fit conditional models for multivariate survival data by using mixed models methodology. To study the specific features of the proposed method we focus on frailty models. Based on a simulation study, we show that the proposed method provides a good and simple alternative for fitting frailty models for data sets with a sufficiently large number of clusters and moderate to large sample sizes within covariate-level subgroups in the clusters. The proposed method is applied to data from 27 randomized trials in advanced colorectal cancer, which are available through the Meta-Analysis Group in Cancer.     

Quantitative assessment of upper limb muscle fatigue depending on the conditions of repetitive task load

The aim of this study was to discriminate fatigue of upper limb muscles depending on the external load, through the development and analysis of a muscle fatigue index. Muscle fatigue is expressed by a fatigue index based on an amplitude parameter (calculated in the time domain) and a fatigue index based on a frequency parameter (a parameter calculated in the frequency domain). The fatigue index involves a regression function that describes changes in the EMG signal parameter, time elapsing before muscle fatigue and the probability of specific trends in changes in EMG parameters for the population under study.

The experimental study covered a group of 10 young men. During the study, they exerted force at a specific level and for a specific time in 12 load variants. During the study, EMG signals from four muscles of the upper limb were recorded (trapezius pars descendents, biceps brachii caput breve, extensor carpi radialis brevis, flexor carpi ulnaris). For each variant and for each examined muscles, the value of the fatigue index was calculated. Values of that index quantitatively expressed fatigue of a specific muscle in a specific load variant.

A statistical analysis indicated variation in the fatigue of the biceps brachii caput breve, extensor carpi radialis brevis, and flexor carpi ulnaris muscles depending on the external load (load variant) according to the task performed with the upper limb.

The study demonstrated usefulness of the fatigue index in expressing quantitatively muscle fatigue and in discriminating muscle fatigue depending on the external load.     

The herb and dwarf shrubs colonization of decaying logs in subalpine forest in the Polish Tatra Mountains

This is a study of the colonization pattern of herbs and dwarf shrubs on rotten logs in subalpine spruce forests (Plagiothecio Piceetum) in the Tatra Mountains. On four study plots (total area 1.43 ha.) all dead logs were measured and the decomposition stage was estimated using the 8-degree scale. For each log the cover of all vascular species, bryophytes and lichens was determined according to the methods of classical phytosociology. Constancy and an index of coverage were calculated for all vascular species growing on logs. The total volume of logs was relatively high (93 m³ ha^–1) and constituted 22% of the volume of living trees. Logs and stumps covered 411 m² ha^–1. These values are similar to those known from natural spruce forest from Carpathians and Scandinavia. The 8 stages of decomposition were equally represented, which indicates a constant supply of dead wood to the forest floor over time. The colonization of dead wood starts with lichens, followed by bryophytes and finally herbs and tree saplings. The first vascular plant colonists of dead logs appear at decay stage nr. 3 at least 20 years after tree death. The most suitable condition for most of the herb species corresponds to decay stage nr. 6 ca. 50 years after tree death. The herb cover is distinctively dominated by Vaccinium myrtillus. Simultaneously with herb species, tree seedlings colonize the logs. Constancy and abundance of Norway spruce saplings increases with advanced decomposition. It seems that the herb cover of logs does not hinder the regeneration of spruce.     

Microhabitat selection by Eurasian lynx and its implications for species conservation

We studied microhabitat selection of the Eurasian lynxLynx lynx (Linnaeus, 1758) at 116 hunting and 88 resting sites in Białowieża Primeval Forest (Poland) to describe its characteristics and determine the importance of habitat structure for stalking prey and for security during resting. We identified lynx-used sites by radio-tracking 3 male and 3 female lynx. When hunting, the lynx did not select for any type or age class of forest. During both summer and winter, the lynx selected sites characterized by high complexity (number of structures useful for stalking: fallen logs and branches, root plates, patches of dense bushes) and low visibility. In summer, hunting sites were often located in the vicinity of small forest glades that provided good stalking opportunities for lynx and rich foraging resources for roe deer — the main prey of lynx. The habitat at kill sites was more open than at sites where the prey was cached, with higher visibility, lower density of trees and poorer undergrowth. The most important characteristic of resting sites was very low visibility that resulted mainly from using young pine or spruce thickets in the winter and dense undergrowth of oak-lime-hornbeam and ash-alder forests in the summer. The information provided by this study could have direct implications for Eurasian lynx conservation by guiding forest restructuring to better suit the species’ biological requirements.