Pathogen quantitation in complex matrices: a multi-operator comparison of DNA extraction methods with a novel assessment of PCR inhibition

Background

Mycobacterium bovis is the aetiological agent of bovine tuberculosis (bTB), an important recrudescent zoonosis, significantly increasing in British herds in recent years. Wildlife reservoirs have been identified for this disease but the mode of transmission to cattle remains unclear. There is evidence that viable M. bovis cells can survive in soil and faeces for over a year.

Methodology/Principal Findings

We report a multi-operator blinded trial for a rigorous comparison of five DNA extraction methods from a variety of soil and faecal samples to assess recovery of M. bovis via real-time PCR detection. The methods included four commercial kits: the QIAamp Stool Mini kit with a pre-treatment step, the FastDNA® Spin kit, the UltraClean™ and PowerSoil™ soil kits and a published manual method based on phenol:chloroform purification, termed Griffiths. M. bovis BCG Pasteur spiked samples were extracted by four operators and evaluated using a specific real-time PCR assay. A novel inhibition control assay was used alongside spectrophotometric ratios to monitor the level of inhibitory compounds affecting PCR, DNA yield, and purity. There were statistically significant differences in M. bovis detection between methods of extraction and types of environmental samples; no significant differences were observed between operators. Processing times and costs were also evaluated. To improve M. bovis detection further, the two best performing methods, FastDNA® Spin kit and Griffiths, were optimised and the ABI TaqMan environmental PCR Master mix was adopted, leading to improved sensitivities.

Conclusions

M. bovis was successfully detected in all environmental samples; DNA extraction using FastDNA® Spin kit was the most sensitive method with highest recoveries from all soil types tested. For troublesome faecal samples, we have used and recommend an improved assay based on a reduced volume, resulting in detection limits of 4.25×10⁵ cells g⁻¹ using Griffiths and 4.25×10⁶ cells g⁻¹ using FastDNA® Spin kit.     

Heme-Oxygenases during Erythropoiesis in K562 and Human Bone Marrow Cells

In mammalian cells, heme can be degraded by heme-oxygenases (HO). Heme-oxygenase 1 (HO-1) is known to be the heme inducible isoform, whereas heme-oxygenase 2 (HO-2) is the constitutive enzyme. Here we investigated the presence of HO during erythroid differentiation in human bone marrow erythroid precursors and K562 cells. HO-1 mRNA and protein expression levels were below limits of detection in K562 cells. Moreover, heme was unable to induce HO-1, at the protein and mRNA profiles. Surprisingly, HO-2 expression was inhibited upon incubation with heme. To evaluate the physiological relevance of these findings, we analyzed HO expression during normal erythropoiesis in human bone marrow. Erythroid precursors were characterized by lack of significant expression of HO-1 and by progressive reduction of HO-2 during differentiation. FLVCR expression, a recently described heme exporter found in erythroid precursors, was also analyzed. Interestingly, the disruption in the HO detoxification system was accompanied by a transient induction of FLVCR. It will be interesting to verify if the inhibition of HO expression, that we found, is preventing a futile cycle of concomitant heme synthesis and catabolism. We believe that a significant feature of erythropoiesis could be the replacement of heme breakdown by heme exportation, as a mechanism to prevent heme toxicity.     

The intrinsic resolution limit in the atomic force microscope: implications for heights of nano-scale features

Background

Accurate mechanical characterization by the atomic force microscope at the highest spatial resolution requires that topography is deconvoluted from indentation. The measured height of nanoscale features in the atomic force microscope (AFM) is almost always smaller than the true value, which is often explained away as sample deformation, the formation of salt deposits and/or dehydration. We show that the real height of nano-objects cannot be obtained directly: a result arising as a consequence of the local probe-sample geometry.

Methods and Findings

We have modeled the tip-surface-sample interaction as the sum of the interaction between the tip and the surface and the tip and the sample. We find that the dynamics of the AFM cannot differentiate between differences in force resulting from 1) the chemical and/or mechanical characteristics of the surface or 2) a step in topography due to the size of the sample; once the size of a feature becomes smaller than the effective area of interaction between the AFM tip and sample, the measured height is compromised. This general result is a major contributor to loss of height and can amount to up to ∼90% for nanoscale features. In particular, these very large values in height loss may occur even when there is no sample deformation, and, more generally, height loss does not correlate with sample deformation. DNA and IgG antibodies have been used as model samples where experimental height measurements are shown to closely match the predicted phenomena.

Conclusions

Being able to measure the true height of single nanoscale features is paramount in many nanotechnology applications since phenomena and properties in the nanoscale critically depend on dimensions. Our approach allows accurate predictions for the true height of nanoscale objects and will lead to reliable mechanical characterization at the highest spatial resolution.     

Measuring generalization of visuomotor perturbations in wrist movements using mobile phones

Recent studies in motor control have shown that visuomotor rotations for reaching have narrow generalization functions: what we learn during movements in one direction only affects subsequent movements into close directions. Here we wanted to measure the generalization functions for wrist movement. To do so we had 7 subjects performing an experiment holding a mobile phone in their dominant hand. The mobile phone's built in acceleration sensor provided a convenient way to measure wrist movements and to run the behavioral protocol. Subjects moved a cursor on the screen by tilting the phone. Movements on the screen toward the training target were rotated and we then measured how learning of the rotation in the training direction affected subsequent movements in other directions. We find that generalization is local and similar to generalization patterns of visuomotor rotation for reaching.     

Drosophila Ten-m and filamin affect motor neuron growth cone guidance

The Drosophila Ten-m (also called Tenascin-major, or odd Oz (odz)) gene has been associated with a pair-rule phenotype. We identified and characterized new alleles of Drosophila Ten-m to establish that this gene is not responsible for segmentation defects but rather causes defects in motor neuron axon routing. In Ten-m mutants the inter-segmental nerve (ISN) often crosses segment boundaries and fasciculates with the ISN in the adjacent segment. Ten-m is expressed in the central nervous system and epidermal stripes during the stages when the growth cones of the neurons that form the ISN navigate to their targets. Over-expression of Ten-m in epidermal cells also leads to ISN misrouting. We also found that Filamin, an actin binding protein, physically interacts with the Ten-m protein. Mutations in cheerio, which encodes Filamin, cause defects in motor neuron axon routing like those of Ten-m. During embryonic development, the expression of Filamin and Ten-m partially overlap in ectodermal cells. These results suggest that Ten-m and Filamin in epidermal cells might together influence growth cone progression.     

A specific interdomain interaction preserves the structural and binding properties of the ModA protein from the phytopathogen Xanthomonas citri domain interaction and transport in ModA

The periplasmic-binding proteins in ATP-binding cassette systems (ABC Transporters) are responsible for the capture and delivery of ligands to their specific transporters, triggering a series of ATP-driven conformational changes that leads to the transport of the ligand. Structurally consisting of two lobes, the proteins change conformation after interaction with the ligand. The structure of the molybdate-binding protein (ModA) from Xanthomonas citri, bound to molybdate, was previously solved by our group and an interdomain interaction, mediated by a salt bridge between K127 and D59, apparently supports the binding properties and keeps the domains closed. To determinate the importance of this interaction, we built two ModA mutants, K127S and D59A, and analysed their functional and structural properties. Based on a set of spectroscopic experiments, crystallisation trials, structure determination and molecular dynamics (MD) simulations, we showed that the salt bridge is essential to maintain the structure and binding properties. Additionally, the MD simulations revealed that this mutant adopted a more compact structure that packed down the ligand-binding pocket. From the closed bound to open structure, the positioning of the helices forming the dipole and the salt bridge are essential to induce an intermediate state.     

The C8ORF38 homologue Sicily is a cytosolic chaperone for a mitochondrial complex I subunit

Mitochondrial complex I (CI) is an essential component in energy production through oxidative phosphorylation. Most CI subunits are encoded by nuclear genes, translated in the cytoplasm, and imported into mitochondria. Upon entry, they are embedded into the mitochondrial inner membrane. How these membrane-associated proteins cope with the hydrophilic cytoplasmic environment before import is unknown. In a forward genetic screen to identify genes that cause neurodegeneration, we identified sicily, the Drosophila melanogaster homologue of human C8ORF38, the loss of which causes Leigh syndrome. We show that in the cytoplasm, Sicily preprotein interacts with cytosolic Hsp90 to chaperone the CI subunit, ND42, before mitochondrial import. Loss of Sicily leads to loss of CI proteins and preproteins in both mitochondria and cytoplasm, respectively, and causes a CI deficiency and neurodegeneration. Our data indicate that cytosolic chaperones are required for the subcellular transport of ND42.     

Diurnal changes in leaflet gas exchange, water status and antioxidant responses in Carapa guianensis plants under water-deficit conditions

Young Carapa guianensis plants were examined under well-watered (control) and water-deficit conditions with the aim to evaluate possible relationship between diurnal changes in leaflet gas exchange with lipid peroxidation and adjustments in antioxidative responses. Treatment comparisons were assessed when leaflet water potential (Ψ_w) in water-stressed plants reached around ?2.5 ± 0.5 MPa at pre-dawn. Regardless of watering regime, the highest net CO₂ assimilation rate and stomatal conductance were recorded until 9:00 h. Control plants showed diurnal increases in transpiration, while it was strongly decreased in water-stressed plants. Diurnal decreases in intercellular to ambient CO₂ concentration ratio were just observed in stressed plants. Regardless of watering regime, non-significant changes (P > 0.05) in Ψ_w and relative water content were registered throughout the day; however, both variables were significantly lower (P < 0.05) in stressed plants. Malondialdehyde concentration did not vary throughout the day, but it was higher in stressed plants. Excepting for guaiacol-type peroxidase, the antioxidant enzyme activities varied throughout the day regardless of watering regimes. Nevertheless, increases in antioxidant enzymes were more expressive in water-stressed plants. Despite, a relationship between diurnal changes in A and g _s and lipid peroxidation or antioxidant enzymes was unclear regardless of watering regimes. Thus, we conclude that although plants from both watering regimes were able to adjust antioxidant enzymes activities throughout the day, the water-stressed plants were more susceptible to damages to net CO₂ assimilation and suffered more expressive oxidative damages to lipids than plants grown under well-watered conditions.     

Biomass-to-bio-products application of feruloyl esterase from Aspergillus clavatus

The structural polysaccharides contained in plant cell walls have been pointed to as a promising renewable alternative to petroleum and natural gas. Ferulic acid is a ubiquitous component of plant polysaccharides, which is found in either monomeric or dimeric forms and is covalently linked to arabinosyl residues. Ferulic acid has several commercial applications in food and pharmaceutical industries. The study herein introduces a novel feruloyl esterase from Aspergillus clavatus (AcFAE). Along with a comprehensive functional and biophysical characterization, the low-resolution structure of this enzyme was also determined by small-angle X-ray scattering. In addition, we described the production of phenolic compounds with antioxidant capacity from wheat arabinoxylan and sugarcane bagasse using AcFAE. The ability to specifically cleave ester linkages in hemicellulose is useful in several biotechnological applications, including improved accessibility to lignocellulosic enzymes for biofuel production.     

Genetic structure of Mexican Mestizos with type 2 diabetes mellitus based on three STR loci

Background

The aims of this population genetics study were: 1) to ascertain whether Mexicans with type 2 diabetes mellitus (DM) were genetically homogeneous and 2) to compare the genetic structure of this selected population with the previously reported data of four random populations (Nuevo León, Hispanics, Chihuahua, and Central Region of Mexico).

Methods

A sample of 103 unrelated individuals with DM and whose 4 grandparents were born in five zones of Mexico was interviewed in 32 Medical Units in the Mexican Institute of Social Security (IMSS). The non-coding STRs D16S539, D7S820, and D13S317 were analyzed.

Results

Genotype distribution was in agreement with Hardy–Weinberg expectations for all three markers. Allele frequencies were found to be similar between the selected population and the four random populations. Gene diversity analysis suggested that more than 99.57% of the total gene diversity could be attributed to variation between individuals within the population and 0.43% between the populations.

Conclusions

According to the present and previous studies using molecular and non-molecular nuclear DNA markers not associated with any disease, the Mexican Mestizo population is found to be genetically homogeneous and therefore the genetic causes of DM are less heterogeneous, thereby simplifying genetic epidemiological studies as has been found in a previous study with the same design in Mexican women with breast cancer.