A Duplex PCR-Based Assay for Measuring the Amount of Bacterial Contamination in a Nucleic Acid Extract from a Culture of Free-Living Protists

Background

Cultures of heterotrophic protists often require co-culturing with bacteria to act as a source of nutrition. Such cultures will contain varying levels of intrinsic bacterial contamination that can interfere with molecular research and cause problems with the collection of sufficient material for sequencing. Measuring the levels of bacterial contamination for the purposes of molecular biology research is non-trivial, and can be complicated by the presence of a diverse bacterial flora, or by differences in the relative nucleic acid yield per bacterial or eukaryotic cell.

Principal Findings

Here we describe a duplex PCR-based assay that can be used to measure the levels of contamination from marine bacteria in a culture of loricate choanoflagellates. By comparison to a standard culture of known target sequence content, the assay can be used to quantify the relative proportions of bacterial and choanoflagellate material in DNA or RNA samples extracted from a culture. We apply the assay to compare methods of purifying choanoflagellate cultures prior to DNA extraction, to determine their effectiveness in reducing bacterial contamination. Together with measurements of the total nucleic acid concentration, the assay can then be used as the basis for determining the absolute amounts of choanoflagellate DNA or RNA present in a sample.

Conclusions

The assay protocol we describe here is a simple and relatively inexpensive method of measuring contamination levels in nucleic acid samples. This provides a new way to establish quantification and purification protocols for molecular biology and genomics in novel heterotrophic protist species. Guidelines are provided to develop a similar protocol for use with any protistan culture. This assay method is recommended where qPCR equipment is unavailable, where qPCR is not viable because of the nature of the bacterial contamination or starting material, or where prior sequence information is insufficient to develop qPCR protocols.     

A BAC-based physical map of the apple genome

Genome-wide physical mapping is an essential step toward investigating the genetic basis of complex traits as well as pursuing genomics research of virtually all plant and animal species. We have constructed a physical map of the apple genome from a total of 74,281 BAC clones representing approximately 10.5x haploid genome equivalents. The physical map consists of 2702 contigs, and it is estimated to span approximately 927 Mb in physical length. The reliability of contig assembly was evaluated by several methods, including assembling contigs using variable stringencies, assembling contigs using fingerprints from individual libraries, checking consensus maps of contigs, and using DNA markers. Altogether, the results demonstrated that the contigs were properly assembled. The apple genome-wide BAC-based physical map represents the first draft genome sequence not only for any member of the large Rosaceae family, but also for all tree species. This map will play a critical role in advanced genomics research for apple and other tree species, including marker development in targeted chromosome regions, fine-mapping and isolation of genes/QTL, conducting comparative genomics analyses of plant chromosomes, and large-scale genomics sequencing.     

A Six-Gene Signature Predicts Survival of Patients with Localized Pancreatic Ductal Adenocarcinoma

Background

Pancreatic ductal adenocarcinoma (PDAC) remains a lethal disease. For patients with localized PDAC, surgery is the best option, but with a median survival of less than 2 years and a difficult and prolonged postoperative course for most, there is an urgent need to better identify patients who have the most aggressive disease.

Methods and Findings

We analyzed the gene expression profiles of primary tumors from patients with localized compared to metastatic disease and identified a six-gene signature associated with metastatic disease. We evaluated the prognostic potential of this signature in a training set of 34 patients with localized and resected PDAC and selected a cut-point associated with outcome using X-tile. We then applied this cut-point to an independent test set of 67 patients with localized and resected PDAC and found that our signature was independently predictive of survival and superior to established clinical prognostic factors such as grade, tumor size, and nodal status, with a hazard ratio of 4.1 (95% confidence interval [CI] 1.7–10.0). Patients defined to be high-risk patients by the six-gene signature had a 1-year survival rate of 55% compared to 91% in the low-risk group.

Conclusions

Our six-gene signature may be used to better stage PDAC patients and assist in the difficult treatment decisions of surgery and to select patients whose tumor biology may benefit most from neoadjuvant therapy. The use of this six-gene signature should be investigated in prospective patient cohorts, and if confirmed, in future PDAC clinical trials, its potential as a biomarker should be investigated. Genes in this signature, or the pathways that they fall into, may represent new therapeutic targets. Please see later in the article for the Editors'' Summary     

Genomic regions involved in productivity of two interspecific poplar families in Europe. 2. Biomass production and its relationships with tree architecture and phenology

Short-rotation coppice of hybrid poplar is a promising renewable feedstock for biofuel production. Breeding for high biomass in short-rotation coppice has started only recently. Two hybrid poplar families were grown at two sites in Europe and phenotyped for a variety of biomass-related traits (1) to examine the extent of phenotypic and genetic variation in biomass production, ramification, resprouting, and phenology, (2) to search for genomic regions involved in productivity, and (3) to determine the effect of the environment on the expression of these traits. The performance of both families differed within and among sites. A pronounced heterosis was observed in most cases. Moderate to high heritability values were found. Seventeen quantitative trait loci (QTL) for biomass production, 13 for ramification, ten for resprouting, 21 for bud burst, and ten for bud set were identified. Genetic correlations and QTL colocation showed that high wood production was associated with high allocation of wood into branches and with high production of resprouts after coppicing. Correlations and QTL colocation between biomass production and phenology traits were weak. Our study provides valuable information on genomic regions involved in biomass production, ramification, and phenology and on phenotypic and genetic relationships among these three trait categories.     

Actin microfilament aggregation induced by withaferin A is mediated by annexin II

The actin cytoskeleton supports diverse cellular processes such as endocytosis, oriented growth, adhesion and migration. The dynamic nature of the cytoskeleton, however, has made it difficult to define the roles of the many accessory molecules that modulate actin organization, especially the multifunctional adapter protein annexin II. We now report that the compound withaferin A (1) can alter cytoskeletal architecture in a previously unknown manner by covalently binding annexin II and stimulating its basal F-actin cross-linking activity. Drug-mediated disruption of F-actin organization is dependent on annexin II expression by cells and markedly limits their migratory and invasive capabilities at subcytotoxic concentrations. Given the extensive ethnobotanical history of withaferin-containing plant preparations in the treatment of cancer and inflammatory and neurological disorders, we suggest that annexin II represents a feasible, previously unexploited target for therapeutic intervention by small-molecule drugs.     

Highly potent and selective NaV1.7 inhibitors for use as intravenous agents and chemical probes

The discovery and selection of a highly potent and selective Na_V1.7 inhibitor PF-06456384, designed specifically for intravenous infusion, is disclosed. Extensive in vitro pharmacology and ADME profiling followed by in vivo preclinical PK and efficacy model data are discussed. A proposed protein–ligand binding mode for this compound is also provided to rationalise the high levels of potency and selectivity over inhibition of related sodium channels. To further support the proposed binding mode, potent conjugates are described which illustrate the potential for development of chemical probes to enable further target evaluation.     

Morphology and electrostatics play active role in neuronal differentiation processes on flexible conducting substrates

This commentary discusses and summarizes the key highlights of our recently reported work entitled “Neuronal Differentiation of Embryonic Stem Cell Derived Neuronal Progenitors Can Be Regulated by Stretchable Conducting Polymers.” The prospect of controlling the mechanical-rigidity and the surface conductance properties offers a unique combination for tailoring the growth and differentiation of neuronal cells. We emphasize the utility of transparent elastomeric substrates with coatings of electrically conducting polymer to realize the desired substrate-characteristics for cellular development processes. Our study showed that neuronal differentiation from ES cells is highly influenced by the specific substrates on which they are growing. Thus, our results provide a better strategy for regulated neuronal differentiation by using such functional conducting surfaces.     

Effects of sinusoidal 60-Hz electric and magnetic fields on ATP and oxygen levels in the slime mold, Physarum polycephalum

We have previously reported that exposing the vegetative plasmodia stage of Physarum polycephalum to either individual or simultaneously applied electric and magnetic fields (45-75 Hz, 0.14-2.0 G, and 0.035-0.7 V/m) lengthens their mitotic cycle, depresses their rate of reversible shuttle streaming, and lowers their respiration rate. In this article we report the effects of simultaneously applied electromagnetic fields (60 Hz, 1.0 G, 1.0 V/m), electric fields only (60 Hz, 1.0 V/m), magnetic fields only (60 Hz, 1.0 G) on the haploid amoeba of Physarum exposed for 120-180 days. Statistically significant depressions (about 8-11%) in ATP levels were observed with all field conditions; however, respiration was significantly decreased only when amoebae were subjected to either combined fields or electric fields alone. Magnetic fields alone failed to induce a significant decrease in respiration.     

Molecular determination of van genes among clinical isolates of enterococci at a hospital setting

Vancomycin-resistant enterococci (VRE) poses a formidable challenge to public health due to its inherent resistance to multiple antibiotics coupled with the ability to transfer genetic determinants to dangerous pathogens like Methicillin-resistant Staphylococcus aureus (MRSA). The purpose of this study was to investigate the incidence of vancomycin resistance in enterococci among clinical isolates at a tertiary care military hospital in the eastern region of Saudi Arabia and to detect van genes using multiplex-PCR. Overall, 246 isolates of enterococci were collected from various clinical specimens. The isolates were identified, and antimicrobial susceptibility testing was done using the Vitek 2 system. Multiplex PCR was performed on the VRE isolates, thus identified to determine the van genes harbored. A total of 15 VRE were identified, of which 14 (93.3%) were Enterococcus faecium, and 1(6.7%) was Enterococcus casseliflavus with intrinsic vanC resistance. Of the 14 vancomycin-resistant Enterococcus faecium, 8 (57.1%) harbored vanB genes, while 6 (42.8%) harbored vanA genes. All the VRE were susceptible to linezolid and tigecycline. Our study detected a low prevalence (6.1%) of VRE among clinical isolates of enterococci and that the vanB gene predominates in such strains. Susceptibility profiles indicated that linezolid and tigecycline are still effective against these multidrug-resistant pathogens. Pus specimens yielded the highest percentage (53.3%) of isolates from which VRE was obtained, and this finding is novel among studies done in Saudi Arabia.