The biological and structural characterization of Mycobacterium tuberculosis UvrA provides novel insights into its mechanism of action

Mycobacterium tuberculosis is an extremely well adapted intracellular human pathogen that is exposed to multiple DNA damaging chemical assaults originating from the host defence mechanisms. As a consequence, this bacterium is thought to possess highly efficient DNA repair machineries, the nucleotide excision repair (NER) system amongst these. Although NER is of central importance to DNA repair in M. tuberculosis, our understanding of the processes in this species is limited. The conserved UvrABC endonuclease represents the multi-enzymatic core in bacterial NER, where the UvrA ATPase provides the DNA lesion-sensing function. The herein reported genetic analysis demonstrates that M. tuberculosis UvrA is important for the repair of nitrosative and oxidative DNA damage. Moreover, our biochemical and structural characterization of recombinant M. tuberculosis UvrA contributes new insights into its mechanism of action. In particular, the structural investigation reveals an unprecedented conformation of the UvrB-binding domain that we propose to be of functional relevance. Taken together, our data suggest UvrA as a potential target for the development of novel anti-tubercular agents and provide a biochemical framework for the identification of small-molecule inhibitors interfering with the NER activity in M. tuberculosi

Mycobacterium tuberculosis RuvA Induces Two Distinct Types of Structural Distortions between the Homologous and Heterologous Holliday Junctions

A central step in the process of homologous genetic recombination is the strand exchange between two homologous DNA molecules, leading to the formation of the Holliday junction intermediate. Several lines of evidence, both in vitro and in vivo, suggest a concerted role for the Escherichia coli RuvABC protein complex in the process of branch migration and the resolution of the Holliday junctions. A number of investigations have examined the role of RuvA protein in branch migration of the Holliday junction in conjunction with its natural cellular partner, RuvB. However, it remains unclear whether the RuvABC protein complex or its individual subunits function differently in the context of DNA repair and homologous recombination. In this study, we have specifically investigated the function of RuvA protein using Holliday junctions containing either homologous or heterologous arms. Our data show that Mycobocterium tuberculosis ruvA complements E. coli Delta ruvA mutants for survival to genotoxic stress caused by different DNA-damaging agents, and the purified RuvA protein binds HJ in preference to any other substrates. Strikingly, our analysis revealed two distinct types of structural distortions caused by M. tuberculosis RuvA between the homologous and heterologous Holliday junctions. We interpret these data as evidence that local distortion of base pairing in the arms of homologous Holliday junctions by RuvA might augment branch migration catalyzed by RuvB. The biological significance of two modes of structural distortion caused by M. tuberculosis RuvA and the implications for its role in DNA repair and homologous recombination are discussed

Urinary excretion of glycosaminoglycans, hydroxyproline and hydroxylysine in rabbits after excessive ingestion of fluoride

1. Urinary levels of glycosaminoglycans (GAG) and hydroxyproline from normal and fluoride treated rabbits were estimated. The hydroxylysine content of serum and urine of rabbits after excessive ingestion of fluoride was also investigated. 2. There was a progressive decrease in GAG content, reduction in hydroxylysine, whereas the hydroxyproline content was increased after fluoride ingestion. 3. Enhanced hydroxyproline in urinary excretion is due to collagen breakdown after fluoride ingestion. 4. The reduction in hydroxylysine content is due to reduced collagen cross-link formation. 5. The report suggests the possibility of using the urinary levels of GAG or hydroxyproline or hydroxylysine as an index of fluoride intoxication

Application of DNA and Immunoassay Analytical Methods for GMO Testing 1

Abstract: The introduction of new agricultural commodities and products derived from modern biotechnology may have an impact on human and animal health, the environment and economies of countries. As more Genetically Modified Organisms (GMO) enter markets worldwide, the monitoring of GMOs is being preferred for obvious reasons such as determination of seed purity, verification of non-GMO status of agricultural crops and fulfilling GMO labeling provisions, to mention a few. Numerous GMO analytical methods which include screening, identification and quantification have been developed to reliably determine the presence and/or amount of GMO in agricultural commodities, in raw agricultural materials and in processed and refined ingredients. The detection of GMOs relies on the detection of transgenic DNA or protein material. For routine analysis, a good sample preparation technique should reproducibly generate DNA/protein of sufficient quality, purity and yield while minimizing the effects of inhibition and contamination. The key sample preparation steps include homogenization, pretreatment, extraction and purification. Due to the fact that analytical laboratories receive samples that are often processed and refined, the quality and quantity of transgenic target analyte (e.g. protein and DNA) frequently challenge the sensitivity of any detection method. With the development of GMO analysis techniques, the Polymerase Chain Reaction (PCR) technique has been the mainstay for GMO detection, and the real-time PCR is the most effective and important method for GMO quantification. The choice of target sequence; for example a promoter, a terminator, a gene, or a junction between two of these elements, is the single most important factor controlling the specificity of the PCR method. Recent developments include event-specific methods, particularly useful for identification and quantification of GM content. Although PCR technology has obvious limitations, the potentially high degree of sensitivity and specificity explains why PCR in its various formats, is currently the leading analytical technology employed in GMO analysis. Comparatively, immunoassays are becoming attractive tools for rapid field monitoring for the integrity of agricultural commodities in identity preservation systems, whereby non-specialised personnel can employ them in cost-effective manner. This review discusses various popular extraction methodologies and summarises the current status of the most widely used and easily applicable GMO analysis technologies in laboratories, namely the PCR and immunoassay technologies

Crystallographic and modelling studies on Mycobacterium tuberculosis RuvA: Additional role of RuvB-binding domain and inter species variability

RuvA, along with RuvB, is involved in branch migration of heteroduplex DNA in homologous recombination. The structures of three new crystal forms of RuvA from Mycobacterium tuberculosis (MtRuvA) have been determined. The RuvB-binding domain is cleaved off in one of them. Detailed models of the complexes of octameric RuvA from different species with the Holliday junction have also been constructed. A thorough examination of the structures presented here and those reported earlier brings to light the hitherto unappreciated role of the RuvB-binding domain in determining inter-domain orientation and oligomerization. These structures also permit an exploration of the interspecies variability of structural features such as oligomerization and the conformation of the loop that carries the acidic pin, in terms of amino acid substitutions. These models emphasize the additional role of the RuvB-binding domain in Holliday junction binding. This role along with its role in oligomerization could have important biological implications

The biological and structural characterization of Mycobacterium tuberculosis UvrA provides novel insights into its mechanism of action

Mycobacterium tuberculosis is an extremely well adapted intracellular human pathogen that is exposed to multiple DNA damaging chemical assaults originating from the host defence mechanisms. As a consequence, this bacterium is thought to possess highly efficient DNA repair machineries, the nucleotide excision repair (NER) system amongst these. Although NER is of central importance to DNA repair in M. tuberculosis, our understanding of the processes in this species is limited. The conserved UvrABC endonuclease represents the multi-enzymatic core in bacterial NER, where the UvrA ATPase provides the DNA lesion-sensing function. The herein reported genetic analysis demonstrates that M. tuberculosis UvrA is important for the repair of nitrosative and oxidative DNA damage. Moreover, our biochemical and structural characterization of recombinant M. tuberculosis UvrA contributes new insights into its mechanism of action. In particular, the structural investigation reveals an unprecedented conformation of the UvrB-binding domain that we propose to be of functional relevance. Taken together, our data suggest UvrA as a potential target for the development of novel anti-tubercular agents and provide a biochemical framework for the identification of small-molecule inhibitors interfering with the NER activity in M. tuberculosis

Structure of Mycobacterium tuberculosis RuvA, a protein involved in recombination

RuvA, a protein from M. tuberculosis H37Rv involved in recombination, has been cloned, expressed, purified and analysed by X-ray crystallography

Early and intensive motor training to enhance neurological recovery in people with spinal cord injury:trial protocol

Study designProtocol for a multi-centre randomised controlled trial (the SCI-MT trial).ObjectivesTo determine whether 10 weeks of intensive motor training enhances neurological recovery in people with recent spinal cord injury (SCI).SettingFifteen spinal injury units in Australia, Scotland, England, Italy, Netherlands, Norway, and Belgium.MethodsA pragmatic randomised controlled trial will be undertaken. Two hundred and twenty people with recent SCI (onset in the preceding 10 weeks, American Spinal Injuries Association Impairment Scale (AIS) A lesion with motor function more than three levels below the motor level on one or both sides, or an AIS C or D lesion) will be randomised to receive either usual care plus intensive motor training (12 h of motor training per week for 10 weeks) or usual care alone. The primary outcome is neurological recovery at 10 weeks, measured with the Total Motor Score from the International Standards for Neurological Classification of SCI. Secondary outcomes include global measures of motor function, ability to walk, quality of life, participants' perceptions about ability to perform self-selected goals, length of hospital stay and participants' impressions of therapeutic benefit at 10 weeks and 6 months. A cost-effectiveness study and process evaluation will be run alongside the trial. The first participant was randomised in June 2021 and the trial is due for completion in 2025.ConclusionsThe findings of the SCI-MT Trial will guide recommendations about the type and dose of inpatient therapy that optimises neurological recovery in people with SCI