PubMed · 13127537
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K MAIGATTER. 1953. [Preventive measures].. https://pubmed.ncbi.nlm.nih.gov/13127537/
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UNLABELLED: Genomic analysis of mycobacteria has become increasingly crucial for understanding drug-resistance mechanisms, molecular epidemiology, and pathogenesis. However, efficient extraction of high-molecular-weight genomic DNA from these organisms remains challenging because of their thick mycolic acid-rich cell walls. In this study, we report the chloroform-bead method, a universal DNA extraction protocol that combines chemical and mechanical disruptions to overcome these challenges. Multi-laboratory evaluation (16 sites) demonstrated the chloroform-bead method's superiority over conventional methods for Mycobacterium tuberculosis (DNA yield: 17.9 vs 1.9 µg, purity A260/A230: 1.86 vs 1.22, both P < 0.001). Single-facility assessment extended these findings to >32 nontuberculous mycobacterial species (n = 1,058), showing performance comparable to M. tuberculosis (n = 1,000), with both achieving median yields of 22.2 µg DNA and consistent quality metrics. The chloroform-bead method significantly reduced the processing time from 2 to 3 days to 2 h while ensuring complete sample sterilization, eliminating the need for species-specific optimization. This streamlined and universally applicable protocol represents a practical advancement in mycobacterial DNA extraction methodology, ideal for high-throughput genomic studies and routine clinical diagnostics. IMPORTANCE: Mycobacterial genomics is crucial for understanding pathogenesis and drug resistance; however, DNA extraction remains a significant challenge because of its unique cell wall. Traditional methods rely on enzymatic treatments, resulting in complex and time-consuming protocols with variable results. The chloroform-bead method introduces a paradigm shift by chemically and mechanically disrupting the mycolic acid layer and eliminating the need for enzymatic treatment. This standardized approach ensures consistent, high-quality DNA extraction across diverse mycobacterial species, thereby enhancing research capabilities and clinical applications.
Electro-osmosis, a coupled-flow phenomenon in which an applied electrical potential gradient drives water flow, may be used to induce water flow through fine-grained sediments. Test cell measurements of electro-osmotic transport in clayey cores extracted from the 27-31 m depth range of the Lawrence Livermore National Laboratory site indicate the importance of pH control within the anode and cathode reservoirs. In our first experiment, pH was not controlled. As a result, carbonate precipitation and metals precipitation occurred near the cathode end of the core, with acidification near the anode. The combination of these acid and base reactions led to the decline of electro-osmotic flow by a factor of 2 in less than one pore volume. In a second experiment, long-term water transport (>21 pore volumes) at stable electro-osmotic conductivity (k(eo) approximately 1 x 10(-9) m2/s-V) was effected with anode reservoir pH > 8, and cathode reservoir pH < 6. Hydraulic conductivity (k(h)) of the same core was 4 x 10(-10) m/s under a 0.07 MPa hydraulic gradient without electro-osmosis. Stable electro-osmotic flow was measured at a velocity of 4 x 10(-7) m/s under a 4 V/cm voltage gradient, and no hydraulic gradient-3 orders of magnitude greater than the hydraulic flow. We also observed chloroform production in the anode reservoir, resulting from electrochemical production of chlorine gas reacting with trace organics. The chloroform was transported electro-osmotically to the cathode, without measurable loss to adsorption, volatilization, or degradation.
Methyl-coenzyme M reductase (MCR) from Methanothermobacter marburgensis (Mtm), catalyses the final step in methane synthesis in all methanogenic organisms. Methane is produced by coenzyme B-dependent two-electron reduction of methyl-coenzyme M. At the active site of MCR is the corphin cofactor F(430), which provides four-coordination through the pyrrole nitrogens to a central Ni ion in all states of the enzyme. The important MCRox1 ("ready") and MCRred1 ("active") states contain six-coordinate Ni(I) and differ in their upper axial ligands; furthermore, red1 appears to be two-electrons more reduced than in ox1 and other Ni(II) states that have been studied. On the basis of the reactivity of MCRred1 and MCRox1 with a substrate analogue and inhibitor (3-bromopropanesulfonate) and other small molecules (chloroform, dichloromethane, mercaptoethanol, and nitric oxide), we present evidence that the six-coordinate Ni(I) centers in the MCRred1 and MCRox1 states exhibit markedly different inherent reactivities. MCRred1 reacts faster with chloroform (2100-fold or 35000-fold when corrected for temperature effects), nitric oxide (90-fold), and 3-bromopropanesulfonate (10(6)-fold) than MCRox1. MCRred1 reacts with chloroform and dichloromethane and, like F(430), can catalyze dehalogenation reactions and produce lower halogenated products. We conclude that the enhanced reactivity of MCRred1 is due to the replacement of a relatively exchange-inert thiol ligand in MCRox1 with a weakly coordinating upper axial ligand in red1 that can be easily replaced by incoming ligands.