Resource Modelling: The Missing Piece of the HTA Jigsaw?

Within health technology assessment (HTA), cost-effectiveness analysis and budget impact analyses have been broadly accepted as important components of decision making. However, whilst they address efficiency and affordability, the issue of implementation and feasibility has been largely ignored. HTA commonly takes place within a deliberative framework that captures issues of implementation and feasibility in a qualitative manner. We argue that only through a formal quantitative assessment of resource constraints can these issues be fully addressed. This paper argues the need for resource modelling to be considered explicitly in HTA. First, economic evaluation and budget impact models are described along with their limitations in evaluating feasibility. Next, resource modelling is defined and its usefulness is described along with examples of resource modelling from the literature. Then, the important issues that need to be considered when undertaking resource modelling are described before setting out recommendations for the use of resource modelling in HTA

Genetic and Epigenetic Factors at COL2A1 and ABCA4 Influence Clinical Outcome in Congenital Toxoplasmosis

Background: Primary Toxoplasma gondii infection during pregnancy can be transmitted to the fetus. At birth, infected infants may have intracranial calcification, hydrocephalus, and retinochoroiditis, and new ocular lesions can occur at any age after birth. Not all children who acquire infection in utero develop these clinical signs of disease. Whilst severity of disease is influenced by trimester in which infection is acquired by the mother, other factors including genetic predisposition may contribute.Methods and Findings: In 457 mother-child pairs from Europe, and 149 child/parent trios from North America, we show that ocular and brain disease in congenital toxoplasmosis associate with polymorphisms in ABCA4 encoding ATP-binding cassette transporter, subfamily A, member 4. Polymorphisms at COL2A1 encoding type II collagen associate only with ocular disease. Both loci showed unusual inheritance patterns for the disease allele when comparing outcomes in heterozygous affected children with outcomes in affected children of heterozygous mothers. Modeling suggested either an effect of mother's genotype, or parent-of-origin effects. Experimental studies showed that both ABCA4 and COL2A1 show isoform-specific epigenetic modifications consistent with imprinting.Conclusions: These associations between clinical outcomes of congenital toxoplasmosis and polymorphisms at ABCA4 and COL2A1 provide novel insight into the molecular pathways that can be affected by congenital infection with this parasite

Genes That Influence Swarming Motility and Biofilm Formation in Variovorax paradoxus EPS

Variovorax paradoxus is an aerobic soil bacterium associated with important biodegradative processes in nature. We use V. paradoxus EPS to study multicellular behaviors on surfaces.We recovered flanking sequence from 123 clones in a Tn5 mutant library, with insertions in 29 different genes, selected based on observed surface behavior phenotypes. We identified three genes, Varpa_4665, Varpa_4680, and Varpa_5900, for further examination. These genes were cloned into pBBR1MCS2 and used to complement the insertion mutants. We also analyzed expression of Varpa_4680 and Varpa_5900 under different growth conditions by qPCR.The 29 genes we identified had diverse predicted functions, many in exopolysaccharide synthesis. Varpa_4680, the most commonly recovered insertion site, encodes a putative N-acetyl-L-fucosamine transferase similar to WbuB. Expression of this gene in trans complemented the mutant fully. Several unique insertions were identified in Varpa_5900, which is one of three predicted pilY1 homologs in the EPS genome. No insertions in the two other putative pilY1 homologs present in the genome were identified. Expression of Varpa_5900 altered the structure of the wild type swarm, as did disruption of the chromosomal gene. The swarming phenotype was complemented by expression of Varpa_5900 from a plasmid, but biofilm formation was not restored. Both Varpa_4680 and Varpa_5900 transcripts were downregulated in biofilms and upregulated during swarming when compared to log phase culture. We identified a putative two component system (Varpa_4664-4665) encoding a response regulator (shkR) and a sensor histidine kinase (shkS), respectively. Biofilm formation increased and swarming was strongly delayed in the Varpa_4665 (shkS) mutant. Complementation of shkS restored the biofilm phenotype but swarming was still delayed. Expression of shkR in trans suppressed biofilm formation in either genetic background, and partially restored swarming in the mutant.The data presented here point to complex regulation of these surface behaviors

Fungal associates of the lodgepole pine beetle, Dendroctonus murrayanae

Bark beetles are well known vectors of ophiostomatoid fungi including species of Ophiostoma, Grosmannia and Ceratocystis. In this study, the most common ophiostomatoid fungi associated with the lodgepole pine beetle, Dendroctonus murrayanae, were characterized. Pre-emergent and post-attack adult beetles were collected from lodgepole pines at four sites in British Columbia, Canada. Fungi were isolated from these beetles and identified using a combination of morphology and DNA sequence comparisons of five gene regions. In all four populations, Grosmannia aurea was the most common associate (74–100% of all beetles) followed closely by Ophiostoma abietinum (29–75%). Other fungi isolated, in order of their relative prevalence with individual beetles were an undescribed Leptographium sp. (0–13%), Ophiostoma ips (0–15%), Ophiostoma piliferum (0–11%), a Pesotum sp. (0–11%) and Ophiostoma floccosum (0–1%). Comparisons of the DNA sequences of Leptographium strains isolated in this study, with ex-type isolates of G. aurea, Grosmannia robusta, Leptographium longiclavatum, and Leptographium terebrantis, as well as with sequences from GenBank, revealed a novel lineage within the Grosmannia clavigera complex. This lineage included some of the D. murrayane isolates as well as several isolates from previous studies referred to as L. terebrantis. However, the monophyly of this lineage is not well supported and a more comprehensive study will be needed to resolve its taxonomic status as one or more novel taxa.National Science Foundation grant OISE-0434171 awarded to DLS, Natural Resources Canada, Canadian Forest Service Mountain Pine Beetle Initiative Grant to ALC, members of the Tree Co-operative Programme, the THRIP initiative of the Department of Trade and Industry, South Africa

Unravelling the genome-wide contributions of specific 2-alkyl-4-quinolones and PqsE to quorum sensing in Pseudomonas aeruginosa

The pqs quorum sensing (QS) system is crucial for Pseudomonas aeruginosa virulence both in vitro and in animal models of infection and is considered an ideal target for the development of anti-virulence agents. However, the precise role played by each individual component of this complex QS circuit in the control of virulence remains to be elucidated. Key components of the pqs QS system are 2-heptyl-4-hydroxyquinoline (HHQ), 2-heptyl-3-hydroxy-4-quinolone (PQS), 2-heptyl-4-hydroxyquinoline N-oxide (HQNO), the transcriptional regulator PqsR and the PQS-effector element PqsE. To define the individual contribution of each of these components to QS-mediated regulation, transcriptomic analyses were performed and validated on engineered P. aeruginosa strains in which the biosynthesis of 2-alkyl 4-quinolones (AQs) and expression of pqsE and pqsR have been uncoupled, facilitating the identification of the genes controlled by individual pqs system components. The results obtained demonstrate that i) the PQS biosynthetic precursor HHQ triggers a PqsR-dependent positive feedback loop that leads to the increased expression of only the pqsABCDE operon, ii) PqsE is involved in the regulation of diverse genes coding for key virulence determinants and biofilm development, iii) PQS promotes AQ biosynthesis, the expression of genes involved in the iron-starvation response and virulence factor production via PqsR-dependent and PqsR-independent pathways, and iv) HQNO does not influence transcription and hence does not function as a QS signal molecule. Overall this work has facilitated identification of the specific regulons controlled by individual pqs system components and uncovered the ability of PQS to contribute to gene regulation independent of both its ability to activate PqsR and to induce the iron-starvation response

Large-Scale Pathway-Based Analysis of Bladder Cancer Genome-Wide Association Data from Five Studies of European Background

Pathway analysis of genome-wide association studies (GWAS) offer a unique opportunity to collectively evaluate genetic variants with effects that are too small to be detected individually. We applied a pathway analysis to a bladder cancer GWAS containing data from 3,532 cases and 5,120 controls of European background (n = 5 studies). Thirteen hundred and ninety-nine pathways were drawn from five publicly available resources (Biocarta, Kegg, NCI-PID, HumanCyc, and Reactome), and we constructed 22 additional candidate pathways previously hypothesized to be related to bladder cancer. In total, 1421 pathways, 5647 genes and ∼90,000 SNPs were included in our study. Logistic regression model adjusting for age, sex, study, DNA source, and smoking status was used to assess the marginal trend effect of SNPs on bladder cancer risk. Two complementary pathway-based methods (gene-set enrichment analysis [GSEA], and adapted rank-truncated product [ARTP]) were used to assess the enrichment of association signals within each pathway. Eighteen pathways were detected by either GSEA or ARTP at P≤0.01. To minimize false positives, we used the I2 statistic to identify SNPs displaying heterogeneous effects across the five studies. After removing these SNPs, seven pathways (‘Aromatic amine metabolism’ [PGSEA = 0.0100, PARTP = 0.0020], ‘NAD biosynthesis’ [PGSEA = 0.0018, PARTP = 0.0086], ‘NAD salvage’ [PARTP = 0.0068], ‘Clathrin derived vesicle budding’ [PARTP = 0.0018], ‘Lysosome vesicle biogenesis’ [PGSEA = 0.0023, PARTP<0.00012], ’Retrograde neurotrophin signaling’ [PGSEA = 0.00840], and ‘Mitotic metaphase/anaphase transition’ [PGSEA = 0.0040]) remained. These pathways seem to belong to three fundamental cellular processes (metabolic detoxification, mitosis, and clathrin-mediated vesicles). Identification of the aromatic amine metabolism pathway provides support for the ability of this approach to identify pathways with established relevance to bladder carcinogenesis

Phenotypic and Genome-Wide Analysis of an Antibiotic-Resistant Small Colony Variant (SCV) of Pseudomonas aeruginosa

Small colony variants (SCVs) are slow-growing bacteria, which often show increased resistance to antibiotics and cause latent or recurrent infections. It is therefore important to understand the mechanisms at the basis of this phenotypic switch.One SCV (termed PAO-SCV) was isolated, showing high resistance to gentamicin and to the cephalosporine cefotaxime. PAO-SCV was prone to reversion as evidenced by emergence of large colonies with a frequency of 10(-5) on media without antibiotics while it was stably maintained in presence of gentamicin. PAO-SCV showed a delayed growth, defective motility, and strongly reduced levels of the quorum sensing Pseudomonas quinolone signal (PQS). Whole genome expression analysis further suggested a multi-layered antibiotic resistance mechanism, including simultaneous over-expression of two drug efflux pumps (MexAB-OprM, MexXY-OprM), the LPS modification operon arnBCADTEF, and the PhoP-PhoQ two-component system. Conversely, the genes for the synthesis of PQS were strongly down-regulated in PAO-SCV. Finally, genomic analysis revealed the presence of mutations in phoP and phoQ genes as well as in the mexZ gene encoding a repressor of the mexXY and mexAB-oprM genes. Only one mutation occurred only in REV, at nucleotide 1020 of the tufA gene, a paralog of tufB, both encoding the elongation factor Tu, causing a change of the rarely used aspartic acid codon GAU to the more common GAC, possibly causing an increase of tufA mRNA translation. High expression of phoP and phoQ was confirmed for the SCV variant while the revertant showed expression levels reduced to wild-type levels.By combining data coming from phenotypic, gene expression and proteome analysis, we could demonstrate that resistance to aminoglycosides in one SCV mutant is multifactorial including overexpression of efflux mechanisms, LPS modification and is accompanied by a drastic down-regulation of the Pseudomonas quinolone signal quorum sensing system

Volatile Organic Compounds Emitted by Fungal Associates of Conifer Bark Beetles and their Potential in Bark Beetle Control

Conifer bark beetles attack and kill mature spruce and pine trees, especially during hot and dry conditions. These beetles are closely associated with ophiostomatoid fungi of the Ascomycetes, including the genera Ophiostoma, Grosmannia, and Endoconidiophora, which enhance beetle success by improving nutrition and modifying their substrate, but also have negative impacts on beetles by attracting predators and parasites. A survey of the literature and our own data revealed that ophiostomatoid fungi emit a variety of volatile organic compounds under laboratory conditions including fusel alcohols, terpenoids, aromatic compounds, and aliphatic alcohols. Many of these compounds already have been shown to elicit behavioral responses from bark beetles, functioning as attractants or repellents, often as synergists to compounds currently used in bark beetle control. Thus, these compounds could serve as valuable new agents for bark beetle management. However, bark beetle associations with fungi are very complex. Beetle behavior varies with the species of fungus, the stage of the beetle life cycle, the host tree quality, and probably with changes in the emission rate of fungal volatiles. Additional research on bark beetles and their symbiotic associates is necessary before the basic significance of ophiostomatoid fungal volatiles can be understood and their applied potential realized