ALBERT

Description: Background: While efforts to improve cancer outcomes have typically focused on improving quality of care, recently, a growing emphasis has been placed on timely access to quality cancer care. This retrospective cohort study examines, at a population level, the relationship between quality and timeliness of colorectal cancer (CRC) care in a single Canadian province (Nova Scotia). Through the provincial cancer registry, we identified all residents diagnosed with invasive CRC between 2001 and 2005 that underwent a non-emergent resection. Using anonymized administrative databases that are individually linked at the patient level, we obtained clinicodemographic, diagnostic, and treatment event data. Selected charts were reviewed to ensure completeness of chemotherapy data.Performance on six quality indicators and the percentage of patients achieving wait time benchmarks for diagnosis, surgery, and adjuvant therapy were calculated. The relationship between quality indicators and wait time intervals was examined using logistic regression. Results: Where an association was identified, patients who received 'higher quality care' had longer wait times. Individuals who received a complete preoperative colonoscopy were less likely to meet benchmarks for time from presentation to diagnosis and from diagnosis to surgery. Those who received an appropriate radiation oncology consultation were less likely to meet benchmarks for time from diagnosis to surgery and from surgery to adjuvant therapy. Conclusions: As governments and other organizations move forward with strategies to reduce wait times, they must also focus on how to define and monitor quality care, and consider the relationship between these two dimensions of health care. Similarly, when developing quality improvement initiatives, the impact on resource utilization and potential to create longer waits for care must be considered.

Description: Background: Most PCR-based diagnostics are still considered time- and labor-intensive due to disparate purification, amplification, and detection steps. Advancements in PCR enzymes and buffer chemistry have increased inhibitor tolerance, facilitating PCR directly from crude samples. Obviating the need for DNA purification, while lacking a concentration step, these direct sample methods are particularly apt for human genetic testing. However, direct PCR protocols have traditionally employed thermal cyclers with slow ramp rates and conservative hold times that significantly increase an assay?s time-to-result. For this proof-of-principle study, our objective was to significantly reduce sample preparation and assay time for a PCR-based genetic test, for myotonic dystrophy type 1 (DM1), by pairing an inhibitor-resistant enzyme mix with a rapid thermal cycler to analyze samples directly in whole blood. Methods: DM1 genetic screening was done with an adapted conventional PCR approach that employed the Streck Philisa? Thermal Cycler, the inhibitor-resistant NEBNext? High-Fidelity 2X PCR Master Mix, and agarose gel electrophoresis or an Agilent 2100 Bioanalyzer for detection. The Gene Link? Myotonic Dystrophy Genemer? Kit was used as a reference assay kit to evaluate the rapid assay. Results: In this work, a rapid and direct PCR assay testing 10% whole blood as template has been developed as an exclusionary screening assay for DM1, a triple-repeat genetic disorder. PCR amplification was completed in 15 minutes using 30 cycles, including in situ hot-start/cell lysis. Out of the 40 donors screened, this assay identified 23 (57.5%) as DM1 negative suggesting no need for further testing. These data are 100% concordant with data collected using the commercially available Gene Link Genemer? Kit per the kit-specific PCR protocol. Conclusions: The PCR assay described in this study amplified DM1 short tandem repeats in 15 minutes. By eliminating sample purification and slower conventional PCR protocols, we demonstrated how adaptation of current PCR technology and chemistries can produce a simple-to-use exclusionary screening assay that is independent of up-front sample prep, improving a clinical lab technician?s time-to-result. We envision this direct and rapid methodology could be applied to other conventional PCR-based genetic tests and sample matrices where genomic DNA is targeted for analysis within a given molecular diagnostic platform.

Description: Background: The pig is a biomedical model to study human and livestock traits. Many of these traits are controlled by neuropeptides that result from the cleavage of prohormones by prohormone convertases. Only 45 prohormones have been confirmed in the pig. Sequence homology can be ineffective to annotate prohormone genes in sequenced species like the pig due to the multifactorial nature of the prohormone processing. The goal of this study is to undertake the first complete survey of prohormone and prohormone convertases genes in the pig genome. These genes were functionally annotated based on 35 gene expression microarray experiments. The cleavage sites of prohormone sequences into potentially active neuropeptides were predicted. Results: We identified 95 unique prohormone genes, 2 alternative calcitonin-related sequences, 8 prohormone convertases and 1 cleavage facilitator in the pig genome 10.2 assembly and trace archives. Of these, 11 pig prohormone genes have not been reported in the UniProt, UniGene or Gene databases. These genes are intermedin, cortistatin, insulin-like 5, orexigenic neuropeptide QRFP, prokineticin 2, prolactin-releasing peptide, parathyroid hormone 2, urocortin, urocortin 2, urocortin 3, and urotensin 2-related peptide. In addition, a novel neuropeptide S was identified in the pig genome correcting the previously reported pig sequence that is identical to the rabbit sequence. Most differentially expressed prohormone genes were under-expressed in pigs experiencing immune challenge relative to the un-challenged controls, in non-pregnant relative to pregnant sows, in old relative to young embryos, and in non-neural relative to neural tissues. The cleavage prediction based on human sequences had the best performance with a correct classification rate of cleaved and non-cleaved sites of 92% suggesting that the processing of prohormones in pigs is similar to humans. The cleavage prediction models did not find conclusive evidence supporting the production of the bioactive neuropeptides urocortin 2, urocortin 3, torsin family 2 member A, tachykinin 4, islet amyloid polypeptide, and calcitonin receptor-stimulating peptide 2 in the pig. Conclusions: The present genomic and functional characterization supports the use of the pig as an effective animal model to gain a deeper understanding of prohormones, prohormone convertases and neuropeptides in biomedical and agricultural research.

Description: Background: The current 'gold standard' for serotyping pneumococci is the Quellung test. This technique is laborious and requires a certain level of training to correctly perform. Commercial pneumococcal latex agglutination serotyping reagents are available, but these are expensive. In-house production of latex agglutination reagents can be a cost-effective alternative to using commercially available reagents. This paper describes a method for the production, and quality control (QC) of latex reagents, including problem solving recommendations, for pneumococcal serotyping. Results: Here we describe a method for the production of latex agglutination reagents based on the passive adsorption of antibodies to latex particles. Sixty-five latex agglutination reagents were made using the PneuCarriage Project (PCP) method, of which 35 passed QC. The other 30 reagents failed QC due to auto-agglutination (n=2), no reactivity with target serotypes (n=8) or cross-reactivity with non-target serotypes (n=20). Dilution of antisera resulted in a further 27 reagents passing QC. The remaining three reagents passed QC when prepared without centrifugation and wash steps. Protein estimates indicated that latex reagents that failed QC when prepared using the PCP method passed when made with antiserum containing

Description: Background: Specialized interactions help structure communities, but persistence of specialized organisms is puzzling because a generalist can occupy more environments and partake in more beneficial interactions. The "Jack-of-all-trades is a master of none" hypothesis asserts that specialists persist because the fitness of a generalist utilizing a particular habitat is lower than that of a specialist adapted to that habitat. Yet, there are many reasons to expect that mutualists will generalize on partners.Plant-soil feedbacks help to structure plant and microbial communities, but how frequently are soil-based symbiotic mutualistic interactions sufficiently specialized to influence species distributions and community composition? To address this question, we quantified realized partner richness and phylogenetic breadth of four wild-grown native legumes (Lupinus bicolor, L. arboreus, Acmispon strigosus and A. heermannii) and performed inoculation trials to test the ability of two hosts (L. bicolor and A. strigosus) to nodulate (fundamental partner richness), benefit from (response specificity), and provide benefit to (effect specificity) 31 Bradyrhizobium genotypes. Results: In the wild, each Lupinus species hosted a broader genetic range of Bradyrhizobium than did either Acmispon species, suggesting that Acmispon species are more specialized. In the greenhouse, however, L. bicolor and A. strigosus did not differ in fundamental association specificity: all inoculated genotypes nodulated both hosts. Nevertheless, A. strigosus exhibited more specificity, i.e., greater variation in its response to, and effect on, Bradyrhizobium genotypes. Lupinus bicolor benefited from a broader range of genotypes but averaged less benefit from each. Both hosts obtained more fitness benefit from symbionts isolated from conspecific hosts; those symbionts in turn gained greater fitness benefit from hosts of the same species from which they were isolated. Conclusions: This study affirmed two important tenets of evolutionary theory. First, as predicted by the Jack-of-all-trades is a master of none hypothesis, specialist A. strigosus obtained greater benefit from its beneficial symbionts than did generalist L. bicolor. Second, as predicted by coevolutionary theory, each test species performed better with partner genotypes isolated from conspecifics. Finally, positive fitness feedback between the tested hosts and symbionts suggests that positive plant-soil feedback could contribute to their patchy distributions in this system.

Description: Background: The community-associated methicillin-resistant S. aureus (CA-MRSA) ST93 clone is becoming dominant in Australia and is clinically highly virulent. In addition, sepsis and skin infection models demonstrate that ST93 CA-MRSA is the most virulent global clone of S. aureus tested to date. While the determinants of virulence have been studied in other clones of CA-MRSA, the basis for hypervirulence in ST93 CA-MRSA has not been defined. Results: Here, using a geographically and temporally dispersed collection of ST93 isolates we demonstrate that the ST93 population hyperexpresses key CA-MRSA exotoxins, in particular alpha-hemolysin, in comparison to other global clones. Gene deletion and complementation studies, and virulence comparisons in a murine skin infection model, showed unequivocally that increased expression of alpha-hemolysin is the key staphylococcal virulence determinant for this clone. Genome sequencing and comparative genomics of strains with divergent exotoxin profiles demonstrated that, like other S. aureus clones, the quorum sensing agr system is the master regulator of toxin expression and virulence in ST93 CA-MRSA. However, we also identified a previously uncharacterized AraC/XylS family regulator (AryK) that potentiates toxin expression and virulence in S. aureus. Conclusions: These data demonstrate that hyperexpression of alpha-hemolysin mediates enhanced virulence in ST93 CA-MRSA, and additional control of exotoxin production, in particular alpha-hemolysin, mediated by regulatory systems other than agr have the potential to fine-tune virulence in CA-MRSA.

Description: Background: As our world becomes warmer, agriculture is increasingly impacted by rising soil salinity and understanding plant adaptation to salt stress can help enable effective crop breeding. Salt tolerance is a complex plant phenotype and we know little about the pathways utilized by naturally tolerant plants. Legumes are important species in agricultural and natural ecosystems, since they engage in symbiotic nitrogen-fixation, but are especially vulnerable to salinity stress. Results: Our studies of the model legume Medicago truncatula in field and greenhouse settings demonstrate that Tunisian populations are locally adapted to saline soils at the metapopulation level and that saline origin genotypes are less impacted by salt than non-saline origin genotypes; these populations thus likely contain adaptively diverged alleles. Whole genome resequencing of 39 wild accessions reveals ongoing migration and candidate genomic regions that assort non-randomly with soil salinity. Consistent with natural selection acting at these sites, saline alleles are typically rare in the range-wide species' gene pool and are also typically derived relative to the sister species M. littoralis. Candidate regions for adaptation contain genes that regulate physiological acclimation to salt stress, such as abscisic acid and jasmonic acid signaling, including a novel salt-tolerance candidate orthologous to the uncharacterized gene AtCIPK21. Unexpectedly, these regions also contain biotic stress genes and flowering time pathway genes. We show that flowering time is differentiated between saline and non-saline populations and may allow salt stress escape. Conclusions: This work nominates multiple potential pathways of adaptation to naturally stressful environments in a model legume. These candidates point to the importance of both tolerance and avoidance in natural legume populations. We have uncovered several promising targets that could be used to breed for enhanced salt tolerance in crop legumes to enhance food security in an era of increasing soil salinization.

Description: Background: The Indonesia Maternal Mortality Rate (MMR) of 420/100.00 live births remains among the highest in East Asia while coverage of births assisted by skilled providers is still low. Traditional beliefs have been a key factor associated with the choice between midwives or traditional birth attendants (TBA) and the low number of antenatal care visits in rural West Sumatra. Methods: We conducted three focus groups with 16 women from rural West Java to describe their perception regarding issues related to traditional beliefs. Focus group discussions provided data for the content analysis. Results: The majority of the 16 women interviewed was from Village Dago, West Java and had only an elementary school education. Their ages ranged from 19 to 40 years. Most were multiparous housewives with an income of IDR 918.750 per month, which was lower than the monthly income in West Java (IDR. 1.172.060). Emerging from the focus group discussion were four main themes regarding their pregnancy and traditional beliefs: 1) pregnancy was a normal cycle in women's life (pregnancy is a natural phenomena, not a sickness; no recognition of danger signs during pregnancy and death of baby or mother during pregnancy was brought about by God's will); 2) women followed the traditional beliefs (positive motivation to follow the traditional beliefs and fear of not following the traditional beliefs); 3) relying on TBA called paraji rather than midwife (parajis are kind, tolerant and patient and have more experience than midwives; more accessibility than midwives and encouragement of natural birth) and 4) midwives are more secure than paraji; (they use a medical standard of care). Conclusions: Women's beliefs grounded in religion and tradition permeated the village culture making it difficult to counter their long held health practices with practices based on recent advances in health care. Use of TBA in this village was still dominant and women believed that following traditional beliefs led to a healthy pregnancy therefore, they also followed all relatives' suggestions. Understanding the complexities of local culture is the first step to improving women's awareness of how to preserve their pregnancy and prevent complications.

Description: Background: Tools for high throughput sequencing and de novo assembly make the analysis of transcriptomes (i.e. the suite of genes expressed in a tissue) feasible for almost any organism. Yet a challenge for biologists is that it can be difficult to assign identities to gene sequences, especially from non-model organisms. Phylogenetic analyses are one useful method for assigning identities to these sequences, but such methods tend to be time-consuming because of the need to re-calculate trees for every gene of interest and each time a new data set is analyzed. In response, we employed existing tools for phylogenetic analysis to produce a computationally efficient, tree-based approach for annotating transcriptomes or new genomes that we term Phylogenetically-Informed Annotation (PIA), which places uncharacterized genes into pre-calculated phylogenies of gene families. Results: We generated maximum likelihood trees for 109 genes from a Light Interaction Toolkit (LIT), a collection of genes that underlie the function or development of light-interacting structures in metazoans. To do so, we searched protein sequences predicted from 30 fully-sequenced genomes and built trees using tools for phylogenetic analysis in the Osiris package of Galaxy (an open-source workflow management system). Next, to rapidly annotate transcriptomes from organisms that lack sequenced genomes, we repurposed a maximum likelihood-based Evolutionary Placement Algorithm (implemented in RAxML) to place sequences of potential LIT genes on to our pre-calculated gene trees. Finally, we implemented PIA in Galaxy and used it to search for LIT genes in 28 newly-sequenced transcriptomes from the light-interacting tissues of a range of cephalopod mollusks, arthropods, and cubozoan cnidarians. Our new trees for LIT genes are available on the Bitbucket public repository (http://bitbucket.org/osiris_phylogenetics/pia/) and we demonstrate PIA on a publicly-accessible web server (http://galaxy-dev.cnsi.ucsb.edu/pia/). Conclusions: Our new trees for LIT genes will be a valuable resource for researchers studying the evolution of eyes or other light-interacting structures. We also introduce PIA, a high throughput method for using phylogenetic relationships to identify LIT genes in transcriptomes from non-model organisms. With simple modifications, our methods may be used to search for different sets of genes or to annotate data sets from taxa outside of Metazoa.

Description: Background: Multiple tick saliva proteins, the majority of which are unknown, confer tick resistance in repeatedly infested animals. The objective of this study was to identify the 24-48 h fed Amblyomma americanum tick saliva immuno-proteome. The 24-48 h tick-feeding phase is critical to tick parasitism as it precedes important events in tick biology, blood meal feeding and disease agent transmission. Fed male, 24 and 96 h fed female phage display cDNA expression libraries were biopanned using rabbit antibodies to 24 and 48 h fed A. americanum female tick saliva proteins. Biopanned immuno-cDNA libraries were subjected to next generation sequencing, de novo assembly, and bioinformatic analysis. Results: More than 800 transcripts that code for 24-48 h fed A. americanum immuno-proteins are described. Of the 895 immuno-proteins, 52% (464/895) were provisionally identified based on matches in GenBank. Of these, ~19% (86/464) show high level of identity to other tick hypothetical proteins, and the rest include putative proteases (serine, cysteine, leukotriene A-4 hydrolase, carboxypeptidases, and metalloproteases), protease inhibitors (serine and cysteine protease inhibitors, tick carboxypeptidase inhibitor), and transporters and/or ligand binding proteins (histamine binding/lipocalin, fatty acid binding, calreticulin, hemelipoprotein, IgG binding protein, ferritin, insulin-like growth factor binding proteins, and evasin). Others include enzymes (glutathione transferase, cytochrome oxidase, protein disulfide isomerase), ribosomal proteins, and those of miscellaneous functions (histamine release factor, selenoproteins, tetraspanin, defensin, heat shock proteins). Conclusions: Data here demonstrate that A. americanum secretes a complex cocktail of immunogenic tick saliva proteins during the first 24-48 h of feeding. Of significance, previously validated immunogenic tick saliva proteins including AV422 protein, calreticulin, histamine release factor, histamine binding/lipocalins, selenoproteins, and paramyosin were identified in this screen, supporting the specificity of the approach in this study. While descriptive, this study opens opportunities for in-depth tick feeding physiology studies.