Multicenter evaluation of reverse line blot assay for detection of drug resistance in Mycobacterium tuberculosis clinical isolates

A multicenter study was conducted with the objective to evaluate a reverse line blot (RLB) assay to detect resistance to rifampin (RIF), isoniazid (INH), streptomycin (STR), and ethambutol (EMB) in clinical isolates of Mycobacterium tuberculosis. Oligonucleotides specific for wild type and mutant (drug resistance linked) alleles of the selected codons in the genes rpoB, inhA, ahpC, rpsL, rrs, embB, were immobilized on a nylon membrane. The RLB assay conditions were optimized following analysis of DNA samples with known sequences of the targeted genes. For validation of the method at different geographical locations, the membranes were sent to seven laboratories in six countries representing the regions with high burdens of multudrug-resistant tuberculosis. The reproducibility of the assay for detection of rpoB genotypes was initially evaluated on a blinded set of twenty reference DNA samples with known allele types and overall concordant results were obtained. Further mutation analysis was performed by each laboratory on the local strains. Upon RLB analysis of 315 clinical isolates from different countries, 132 (85.2%) of 155 RIF-resistant and 28 (51.0%) of 55 EMB-resistant isolates were correctly identified, showing applicability of the assay when targeting the rpoB hot-spot region and embB306. Mutations in the inhA and ahpC promoter regions, conferring resistance to INH, were successfully identified in respectively 16.9% and 13.2% of INH-resistant strains. Likewise, mutations in rrs513 and rpsL88 that confer resistance to STR were identified in respectively 15.1% and 10.7% of STR-resistant strains. It should be mentioned that mutation analysis of the above targets usually requires rather costly DNA sequencing to which the proposed RLB assay presents rapid and inexpensive alternative. Furthermore, the proposed method requires the same simple equipment as that used for spoligotyping and permits simultaneous analysis of up to 40 samples. This technique is a first attempt to combine different targets in a single assay for prediction of antituberculosis drugs resistance. It is open to further development as it allows easy incorporation of new probes for detection of mutations in other genes associated with resistance to second-line (e.g., fluoroquinolones) and new antituberculosis compounds.     

Basic amino acid residues in the beta-structure region contribute, but not critically, to presynaptic neurotoxicity of ammodytoxin A

The molecular mechanism of action of presynaptically toxic secreted phospholipases A2 (sPLA2s) isolated from snake venoms is not completely understood. It has been proposed that the positive charge in the beta-structure region is important for their toxic activity. To test this hypothesis, we characterised several mutants of ammodytoxin A (AtxA) possessing substitution of all five basic residues in this region. The mutations had relatively little influence on the catalytic activity of AtxA, either on charge-neutral or anionic phospholipid vesicles. An exception was R72 when replaced by a hydrophobic (higher activity) or an acidic (lower activity) residue. Lethal potencies of the eight single site mutants were up to four times lower than that of the wild-type, whereas the triple mutant (K74S/H76S/R77L) was 13-fold less toxic. The substitutions also lowered the affinity of the toxin, slightly to moderately, for the neuronal receptors R25 and R180. Interaction with calmodulin was only slightly affected by substitutions of K86, more by those of the K74/H76/R77 cluster and most by those of R72 (up to 11-fold lower binding affinity). The results clearly indicate that the basic amino acid residues in the beta-region of AtxA contribute to, but are not necessary for, its neurotoxic effect.     

A multi-criteria decision-making model for classifying wood products with respect to their impact on environment

Background, aim and scope  

Although life cycle assessment is frequently used in scientific studies of product comparison, many practitioners are looking for improvements in the normalisation, grouping and weighting of life cycle inventory results. Local conditions, which are well known to local experts, are very important to these steps. The goal of this work was to develop a computer-based decision support system for classifying wood products according to their influence on the environment in their whole life cycle. The model specifically addresses local conditions in the Republic of Slovenia and was developed by Slovenian experts.     

If Cell Mechanics Can Be Described by Elastic Modulus: Study of Different Models and Probes Used in Indentation Experiments

Here we investigated the question whether cells, being highly heterogeneous objects, could be described with the elastic modulus (effective Young’s modulus) in a self-consistent way. We performed a comparative analysis of the elastic modulus derived from the indentation data obtained with atomic force microscopy (AFM) on human cervical epithelial cells (both normal and cancerous). Both sharp (cone) and dull (2500-nm radius sphere) AFM probes were used. The indentation data were processed through different elastic models. The cell was approximated as a homogeneous elastic medium that had either 1), smooth hemispherical boundary (Hertz/Sneddon models) or 2), the boundary covered with a layer of glycocalyx and membrane protrusions (“brush” models). Consistency of these approximations was investigated. Specifically, we tested the independence of the elastic modulus of the indentation depth, which is assumed in these models. We demonstrated that only one model showed consistency in treating cells as a homogeneous elastic medium, namely, the brush model, when processing the indentation data collected with the dull AFM probe. The elastic modulus demonstrated strong depth dependence in all models: Hertz/Sneddon models (no brush taken into account), and when the brush model was applied to the data collected with sharp conical probes. We conclude that it is possible to describe the elastic properties of the cell body by means of an effective elastic modulus, used in a self-consistent way, when using the brush model to analyze data collected with a dull AFM probe. The nature of these results is discussed.     

Molecular enzymology of lipoxygenases

Lipoxygenases (LOXs) are lipid peroxidizing enzymes, implicated in the pathogenesis of inflammatory and hyperproliferative diseases, which represent potential targets for pharmacological intervention. Although soybean LOX1 was discovered more than 60 years ago, the structural biology of these enzymes was not studied until the mid 1990s. In 1993 the first crystal structure for a plant LOX was solved and following this protein biochemistry and molecular enzymology became major fields in LOX research. This review focuses on recent developments in molecular enzymology of LOXs and summarizes our current understanding of the structural basis of LOX catalysis. Various hypotheses explaining the reaction specificity of different isoforms are critically reviewed and their pros and cons briefly discussed. Moreover, we summarize the current knowledge of LOX evolution by profiling the existence of LOX-related genomic sequences in the three kingdoms of life. Such sequences are found in eukaryotes and bacteria but not in archaea. Although the biological role of LOXs in lower organisms is far from clear, sequence data suggests that this enzyme family might have evolved shortly after the appearance of atmospheric oxygen on earth.     

Description of Idiomarina insulisalsae sp. nov., isolated from the soil of a sea salt evaporation pond,proposal to transfer the species of the genus Pseudidiomarina to the genus Idiomarina and emended description of the genus Idiomarina

A halophilic, aerobic Gram-negative bacterium, designated strain CVS-6^T, was isolated from a sea salt evaporation pond on the Island of Sal in the Cape Verde Archipelago. Phylogenetic analysis of the 16S rRNA gene sequence revealed a clear affiliation of the organism with members of the family Idiomarinaceae. Sequence similarities between CVS-6^T and the type strains of the species of the genera Pseudidiomarina and Idiomarina ranged from 93.7% to 96.9%. The major isoprenoid quinone was ubiquinone 8 (Q-8). The major cellular fatty acids were 15:0 iso (21.8%), 17:0 iso (12.5%), 17:1 iso ω9c (10.7%), and 16:1 ω7c (10.6%). The DNA G+C content was 51.6 mol%. The species represented by strain CVS-6^T could be distinguished from the species of the genera Pseudidiomarina and Idiomarina; however, it was not possible to distinguish both genera from each other using the phenotypic or chemotaxonomic characteristics examined. Consequently, we propose that the species classified in the genus Pseudidiomarina should be transferred to the genus Idiomarina. We also propose that, on the basis of physiological and biochemical characteristics, strain CVS-6^T (=LMG 23123=CIP 108836) represents a new species which we name Idiomarina insulisalsae.     

Three-dimensional chromatin ensemble reconstruction via stochastic embedding

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Lamin A as a Determinant of Mechanical Properties of the Cell Nucleus in Health and Disease

Biochemistry (Moscow) - One of the main factors associated with worse prognosis in oncology is metastasis, which is based on the ability of tumor cells to migrate from the primary source and to...