Photo-induction of electrical current with the cyanobacteriumAgmenellum quadruplicatum PR-6

We investigated the possibility of eliciting a measurable photoinduced electrical current from the cyanobacteriumAgmenellum quadruplicatum PR-6 (Synechococcus PCC 7002). This proved virtually impossible for intact cells. However, treated PR-6 cells fixed in an alginate matrix on tin oxide as the active electrode in a three electrode electrochemical cell gave rise to a significant light response. Cell treatments involving toluene, chloroform or detergents were effective and gave current responses up to 250 nA. Drying the cyanobacterial matrix increased the current yield at least fifty-fold. These effects were observed for light wavelengths > 650 nm and were not influenced by inhibitors or enhancers of photosynthesis nor by sustained argon bubbling of the electrolyte.French pressure cell lysates facilitated distinction between two light induced current components. Lysates prepared without CaCl₂ gave current induction kinetics that were indistinguishable from those on chemically treated cells i.e. slowly rising to a stable maximum in 10–15 min. When CaCl₂ was present during lysis, a rapidly induced (<1 s) unstable component was observed. Toluenization of PR-6 either prior to or post lysing abolished the CaCl₂ related effect. CaCl₂ had no effect on current induction in strain PR-6008, which lacked the and subunits of phycocyanin and exhibited slow current induction kinetics.The observed effects are interpreted as responses of components of the photosystems of PR-6 rather than in terms of an integrated photosynthetic process.     

NAD+ binding to the Escherichia coli K+-uptake protein TrkA and sequence similarity between TrkA and domains of a family of dehydrogenases suggest a role for NAD+ in bacterial transport

The nucleotide sequence of trkA, a gene encoding a surface component of the constitutive K⁺-uptake systems TrkG and TrkH from Escherichia coli, was determined. The structure of the TrkA protein deduced from the nucleotide sequence accords with the view that TrkA is peripherally bound to the inner side of the cytoplasmic membrane. Analysis by a dot matrix revealed that TrkA is composed of similar halves. The M-terminal part of each TrkA half (residues 1–130 and 234–355, respectively) is similar to the complete NAD⁺-binding domain of NAD⁺-dependent dehydrogenases. The C-terminal part of each TrkA half (residues 131–233 and 357–458, respectively) aligns with the first 100 residues of the catalytic domain of glyceraldehyde-3-phosphate dehydrogenase. Strong u.v. illumination at 252 nm led to cross-linking of NAD⁺ or NADH, but not of ATP to the isolated TrkA protein.     

Distribution of Insect Attacks in Biological Control of Weeds: Infestation of Centaurea virgata Flowerheads by a Gall Fly

The success of biological control efforts to reduce weed density through release of insects may depend as much on the distribution of insect attacks among individual plants or plant parts as on the mean level of infestation. We used an index of dispersion to describe the distribution of Urophora quadrifasciata (Diptera: Tephritidae) galls among squarrose knapweed (Centaurea virgata) flowerheads at 18 west central Utah sites in the first 5 years following introduction of the biological control agent. Two thirds of the samples showed a significantly aggregated distribution of galls among flowerheads. Statistical analysis showed that site and year accounted for relatively small proportions of the variance in the index of dispersion. The degree of gall aggregation among flowerheads was positively correlated with the mean flowerhead quality (mean number of seeds per flowerhead; P = 0.013) and tended to be negatively correlated with the mean fly density per flowerhead at a site in a given year (P = 0.097). Our data suggest that higher quality flowerheads, and possibly higher quality plants, are preferentially attacked by U. quadrifasciata and therefore are more heavily subject to reduced reproductive potential through biological control. However, an aggregated distribution of fly attacks may undercut the potential of the fly to reduce seed production by the weed population as a whole. Understanding both the distribution of insect attacks among individual plants and the behavioral mechanisms producing such distribution patterns is important to the biological control of weeds.     

Microevolution of Anthrax from a Young Ancestor (M.A.Y.A.) Suggests a Soil-Borne Life Cycle of Bacillus anthracis

During an anthrax outbreak at the Pollino National Park (Basilicata, Italy) in 2004, diseased cattle were buried and from these anthrax-foci Bacillus anthracis endospores still diffuse to the surface resulting in local accumulations. Recent data suggest that B. anthracis multiplies in soil outside the animal-host body. This notion is supported by the frequent isolation of B. anthracis from soil lacking one or both virulence plasmids. Such strains represent an evolutionary dead end, as they are likely no longer able to successfully infect new hosts. This loss of virulence plasmids is explained most simply by postulating a soil-borne life cycle of the pathogen. To test this hypothesis we investigated possible microevolution at two natural anthrax foci from the 2004 outbreak. If valid, then genotypes of strains isolated from near the surface at these foci should be on a different evolutionary trajectory from those below residing in deeper-laying horizons close to the carcass. Thus, the genetic diversity of B. anthracis isolates was compared conducting Progressive Hierarchical Resolving Assays using Nucleic Acids (PHRANA) and next generation Whole Genome Sequencing (WGS). PHRANA was not discriminatory enough to resolve the fine genetic relationships between the isolates. Conversely, WGS of nine isolates from near-surface and nine from near-carcass revealed five isolate specific SNPs, four of which were found only in different near-surface isolates. In support of our hypothesis, one surface-isolate lacked plasmid pXO1 and also harbored one of the unique SNPs. Taken together, our results suggest a limited soil-borne life cycle of B. anthracis.     

Methodological reflections on using pilot data from fracture patients to develop a qualitative study

Background

Qualitative studies are particularly valued for their exploratory nature but, like other research methods, they do require careful planning to ensure rigorous study design. Our objective was to undertake a pilot study to inform the development of a larger qualitative study.

Results

We conducted a series of brief interviews with out-patients in a hospital setting. The interviews were designed to elicit superficial information about whether (and how) post-fracture osteoporosis investigation and/or treatment were being initiated among patients receiving treatment or follow-up for a current or recent fracture. We used thematic analysis to identify key themes in the data that related to the broader research questions.We analysed data obtained from 11 out of a total of 12 interviews conducted. Participants were male and female, aged 19-83 years of age (median age 57 years). Participants attended 2-8 medical appointments to seek treatment and follow up for a current or recent fracture. The following four overarching themes emerged from thematic analysis of the data: fracture event, referral pathway, osteoporosis investigation and/or treatment, and communication by health practitioners and staff.

Conclusions

This pilot study was necessarily tentative and exploratory in nature, but provided a helpful snapshot of some typical experiences in the public health system following fracture. Several themes emerged for consideration in the design of the main study.Despite its critics, theoretical sampling and saturation continue to provide sustainable methods for ensuring that relevant themes and categories are covered in sufficient depth and breadth, appropriate to the needs of the study.

     

Extreme challenges and advances in archaeal proteomics

Archaea display amazing physiological properties that are of interest to understand at the molecular level including the ability to thrive at extreme environmental conditions, the presence of novel metabolic pathways (e.g. methanogenesis, methylaspartate cycle) and the use of eukaryotic-like protein machineries for basic cellular functions. Coupling traditional genetic and biochemical approaches with advanced technologies, such as genomics and proteomics, provides an avenue for scientists to discover new aspects related to the molecular physiology of archaea. This review emphasizes the unusual properties of archaeal proteomes and how high-throughput and specialized mass spectrometry-based proteomic studies have provided insight into the molecular properties of archaeal cells.     

Functional Profiling Reveals Critical Role for miRNA in Differentiation of Human Mesenchymal Stem Cells

Background

Mesenchymal stem (MS) cells are excellent candidates for cell-based therapeutic strategies to regenerate injured tissue. Although human MS cells can be isolated from bone marrow and directed to differentiate by means of an osteogenic pathway, the regulation of cell-fate determination is not well understood. Recent reports identify critical roles for microRNAs (miRNAs), regulators of gene expression either by inhibiting the translation or by stimulating the degradation of target mRNAs.

Methodology/Principal Findings

In this study, we employed a library of miRNA inhibitors to evaluate the role of miRNAs in early osteogenic differentiation of human MS cells. We discovered that miR-148b, -27a and -489 are essential for the regulation of osteogenesis: miR-27a and miR-489 down-regulate while miR-148b up-regulates differentiation. Modulation of these miRNAs induced osteogenesis in the absence of other external differentiation cues and restored osteogenic potential in high passage number human MS cells.

Conclusions/Significance

Overall, we have demonstrated the utility of the functional profiling strategy for unraveling complex miRNA pathways. Our findings indicate that miRNAs regulate early osteogenic differentiation in human MS cells: miR-148b, -27a, and -489 were found to play a critical role in osteogenesis.     

Human Flt3L Generates Dendritic Cells from Canine Peripheral Blood Precursors: Implications for a Dog Glioma Clinical Trial

Background

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults and carries a dismal prognosis. We have developed a conditional cytotoxic/immunotherapeutic approach using adenoviral vectors (Ads) encoding the immunostimulatory cytokine, human soluble fms-like tyrosine kinase 3 ligand (hsFlt3L) and the conditional cytotoxic molecule, i.e., Herpes Simplex Type 1- thymide kinase (TK). This therapy triggers an anti-tumor immune response that leads to tumor regression and anti-tumor immunological memory in intracranial rodent cancer models. We aim to test the efficacy of this immunotherapy in dogs bearing spontaneous GBM. In view of the controversy regarding the effect of human cytokines on dog immune cells, and considering that the efficacy of this treatment depends on hsFlt3L-stimulated dendritic cells (DCs), in the present work we tested the ability of Ad-encoded hsFlt3L to generate DCs from dog peripheral blood and compared its effects with canine IL-4 and GM-CSF.

Methodology/Principal Findings

Our results demonstrate that hsFlT3L expressed form an Ad vector, generated DCs from peripheral blood cultures with very similar morphological and phenotypic characteristics to canine IL-4 and GM-CSF-cultured DCs. These include phagocytic activity and expression of CD11c, MHCII, CD80 and CD14. Maturation of DCs cultured under both conditions resulted in increased secretion of IL-6, TNF-α and IFN-γ. Importantly, hsFlt3L-derived antigen presenting cells showed allostimulatory potential highlighting their ability to present antigen to T cells and elicit their proliferation.

Conclusions/Significance

These results demonstrate that hsFlt3L induces the proliferation of canine DCs and support its use in upcoming clinical trials for canine GBM. Our data further support the translation of hsFlt3L to be used for dendritic cells'' vaccination and gene therapeutic approaches from rodent models to canine patients and its future implementation in human clinical trials.     

Antibody Targeting of Cathepsin S Inhibits Angiogenesis and Synergistically Enhances Anti-VEGF

Background

Angiogenesis is a key hallmark of tumourigenesis and its inhibition is a proven strategy for the development of novel anti-cancer therapeutics. An important aspect of early angiogenesis is the co-ordinated migration and invasion of endothelial cells through the hypoxic tumour tissue. Cathepsin S has been shown to play an important role in angiogenesis as has vascular endothelial growth factor (VEGF). We sought to assess the anti-angiogenic effect of Fsn0503, a novel cathepsin S inhibitory antibody, when combined with anti-VEGF on vascular development.

Methodology/Principal Findings

Cathepsin S expression and secretion from endothelial cells was characterised using RT-PCR and western blotting. We further show that cathepsin S promotes pericellular hydrolysis of extracellular matrix components in the tumour microenvironment and facilitates endothelial invasion. The cathepsin S inhibitory antibody, Fsn0503, blocks extracellular proteolysis, inhibiting endothelial invasion and tube formation in cell-based assays. The anti-angiogenic effects of Fsn0503 were also shown in vivo where it significantly retarded the development of vasculature in human xenograft models. Furthermore, when Fsn0503 was combined with an anti-VEGF antibody, a synergistic inhibition of microvascular development was observed.

Conclusions/Significance

Taken together, this data demonstrates that the antibody-mediated targeting of cathepsin S represents a novel method of inhibiting angiogenesis. Furthermore, when used in combination with anti-VEGF therapies, Fsn0503 has the potential to significantly enhance current treatments of tumour neovascularisation and may also be of use in the treatment of other conditions associated with inappropriate angiogenesis.     

The Characterization of Twenty Sequenced Human Genomes

We present the analysis of twenty human genomes to evaluate the prospects for identifying rare functional variants that contribute to a phenotype of interest. We sequenced at high coverage ten “case” genomes from individuals with severe hemophilia A and ten “control” genomes. We summarize the number of genetic variants emerging from a study of this magnitude, and provide a proof of concept for the identification of rare and highly-penetrant functional variants by confirming that the cause of hemophilia A is easily recognizable in this data set. We also show that the number of novel single nucleotide variants (SNVs) discovered per genome seems to stabilize at about 144,000 new variants per genome, after the first 15 individuals have been sequenced. Finally, we find that, on average, each genome carries 165 homozygous protein-truncating or stop loss variants in genes representing a diverse set of pathways.