ALBERT

Description: Background: The bacterium Caulobacter crescentus is a popular model for the study of cell cycle regulation and senescence. The large prolate siphophage phiCbK has been an important tool in C. crescentus biology, and has been studied in its own right as a model for viral morphogenesis. Although a system of some interest, to date little genomic information is available on phiCbK or its relatives. Results: Five novel phiCbK-like C. crescentus bacteriophages, CcrMagneto, CcrSwift, CcrKarma, CcrRogue and CcrColossus, were isolated from the environment. The genomes of phage phiCbK and these five environmental phage isolates were obtained by 454 pyrosequencing. The phiCbK-like phage genomes range in size from 205 kb encoding 318 proteins (phiCbK) to 280 kb encoding 448 proteins (CcrColossus), and were found to contain nonpermuted terminal redundancies of 10 to 17 kb. A novel method of terminal ligation was developed to map genomic termini, which confirmed termini predicted by coverage analysis. This suggests that sequence coverage discontinuities may be useable as predictors of genomic termini in phage genomes. Genomic modules encoding virion morphogenesis, lysis and DNA replication proteins were identified. The phiCbK-like phages were also found to encode a number of intriguing proteins; all contain a clearly T7-like DNA polymerase, and five of the six encode a possible homolog of the C. crescentus cell cycle regulator GcrA, which may allow the phage to alter the host cell's replicative state. The structural proteome of phage phiCbK was determined, identifying the portal, major and minor capsid proteins, the tail tape measure and possible tail fiber proteins. All six phage genomes are clearly related; phiCbK, CcrMagneto, CcrSwift, CcrKarma and CcrRogue form a group related at the DNA level, while CcrColossus is more diverged but retains significant similarity at the protein level. Conclusions: Due to their lack of any apparent relationship to other described phages, this group is proposed as the founding cohort of a new phage type, the phiCbK-like phages. This work will serve as a foundation for future studies on morphogenesis, infection and phage-host interactions in C. crescentus.

Description: Background The bacterium Caulobacter crescentus is a popular model for the study of cell cycle regulation and senescence. The large prolate siphophage phiCbK has been an important tool in C. crescentus biology, and has been studied in its own right as a model for viral morphogenesis. Although a system of some interest, to date little genomic information is available on phiCbK or its relatives. Results Five novel phiCbK-like C. crescentus bacteriophages, CcrMagneto, CcrSwift, CcrKarma, CcrRogue and CcrColossus, were isolated from the environment. The genomes of phage phiCbK and these five environmental phage isolates were obtained by 454 pyrosequencing. The phiCbK-like phage genomes range in size from 205 kb encoding 318 proteins (phiCbK) to 280 kb encoding 448 proteins (CcrColossus), and were found to contain nonpermuted terminal redundancies of 10 to 17 kb. A novel method of terminal ligation was developed to map genomic termini, which confirmed termini predicted by coverage analysis. This suggests that sequence coverage discontinuities may be useable as predictors of genomic termini in phage genomes. Genomic modules encoding virion morphogenesis, lysis and DNA replication proteins were identified. The phiCbK-like phages were also found to encode a number of intriguing proteins; all contain a clearly T7-like DNA polymerase, and five of the six encode a possible homolog of the C. crescentus cell cycle regulator GcrA, which may allow the phage to alter the host cell’s replicative state. The structural proteome of phage phiCbK was determined, identifying the portal, major and minor capsid proteins, the tail tape measure and possible tail fiber proteins. All six phage genomes are clearly related; phiCbK, CcrMagneto, CcrSwift, CcrKarma and CcrRogue form a group related at the DNA level, while CcrColossus is more diverged but retains significant similarity at the protein level. Conclusions Due to their lack of any apparent relationship to other described phages, this group is proposed as the founding cohort of a new phage type, the phiCbK-like phages. This work will serve as a foundation for future studies on morphogenesis, infection and phage-host interactions in C. crescentus.

Description: Background: Blood--brain barrier (BBB) disruption is an integral feature of numerous neurological disorders. However, there is a relative lack of knowledge regarding the underlying molecular mechanisms of immune-mediated BBB disruption. We have previously shown that CD8 T cells and perforin play critical roles in initiating altered permeability of the BBB in the peptide-induced fatal syndrome (PIFS) model developed by our laboratory. Additionally, despite having indistinguishable CD8 T cell responses, C57BL/6J (B6) mice are highly susceptible to PIFS, exhibiting functional motor deficits, increased astrocyte activation, and severe CNS vascular permeability, while 129S1/SvImJ (129S1) mice remain resistant. Therefore, to investigate the potential role of genetic factors, we performed a comprehensive genetic analysis of (B6 x 129S1) F2 progeny to define quantitative trait loci (QTL) linked to the phenotypic characteristics stated above that mediate CD8 T cell-initiated BBB disruption. Results: Using single nucleotide polymorphism (SNP) markers and a 95% confidence interval, we identified one QTL (PIFS1) on chromosome 12 linked to deficits in motor function (SNP markers rs6292954, rs13481303, rs3655057, and rs13481324, LOD score = 3.3). In addition we identified a second QTL (PIFS2) on chromosome 17 linked to changes in CNS vascular permeability (SNP markers rs6196216 and rs3672065, LOD score = 3.7). Conclusions: The QTL critical intervals discovered have allowed for compilation of a list of candidate genes implicated in regulating functional deficit and CNS vascular permeability. These genes encode for factors that may be potential targets for therapeutic approaches to treat disorders characterized by CD8 T cell-mediated BBB disruption.

Description: Background: Exposure to early adverse events can result in the development of later psychopathology, and is often associated with cognitive impairment. This may be due to accelerated cell aging, which can be catalogued by attritioned telomeres. Exercise enhances neurogenesis and has been proposed to buffer the effect of psychological stress on telomere length. This study aimed to investigate the impact of early developmental stress and voluntary exercise on telomere length in the ventral hippocampus (VH) and prefrontal cortex (PFC) of the rat. Forty-five male Sprague--Dawley rats were categorised into four groups: maternally separated runners (MSR), maternally separated non-runners (MSnR), non-maternally separated runners (nMSR) and non-maternally separated non-runners (nMSnR). Behavioural analyses were conducted to assess anxiety-like behaviour and memory performance in the rats, after which relative telomere length was measured using qPCR. Results: Maternally separated (MS) rats exhibited no significant differences in either anxiety levels or memory performance on the elevated-plus maze and the open field compared to non-maternally separated rats at 49 days of age. Exercised rats displayed increased levels of anxiety on the day that they were removed from the cages with attached running wheels, as well as improved spatial learning and temporal recognition memory compared to non-exercised rats. Exploratory post-hoc analyses revealed that maternally separated non-exercised rats exhibited significantly longer telomere length in the VH compared to those who were not maternally separated; however, exercise appeared to cancel this effect since there was no difference in VH telomere length between maternally separated and non-maternally separated runners. Conclusions: The increased telomere length in the VH of maternally separated non-exercised rats may be indicative of reduced cellular proliferation, which could, in turn, indicate hippocampal dysfunction. This effect on telomere length was not observed in exercised rats, indicating that voluntary exercise may buffer against the progressive changes in telomere length caused by alterations in maternal care early in life. In future, larger sample sizes will be needed to validate results obtained in the present study and obtain a more accurate representation of the effect that psychological stress and voluntary exercise have on telomere length.

Description: Background: MAP is a suspected zoonotic pathogen and the causative agent of Johne's Disease in cattle and other ruminant animals. With over $1 billion dollars in loss to the dairy industry due to Johne's Disease, efforts to eliminate or reduce MAP from cattle are of importance. The purpose of this study was to determine if daily intake of probiotics could eliminate or reduce Johne's Disease associated symptoms and pathogenesis by MAP. Post infection, animals are often asymptomatic carriers with limited shedding of the pathogen, proving early detection to be difficult. Disease and symptoms often appear 3--4 years after infection with antibiotic treatment proving ineffective. Symptoms include chronic gastrointestinal inflammation leading to severe weight-loss from poor feed and water intake cause a wasting disease. These symptoms are similar to those found in individuals with Crohn's Disease (CD); MAP has been implicated by not proven to be the causative agent of CD. Probiotics administered to livestock animals, including dairy and beef cattle have demonstrated improvements in cattle performance and health. Our objectives included determining the benefits of Lactobacillus animalis (strain name: NP-51) in MAP infected BALB/c mice by evaluating systemic and gastrointestinal response by the host and gut microbiota. Male and female animals were fed 1x106CFU/g probiotics in sterile, powdered mouse chow daily and infected with 1 x 107 CFU/ml MAP and compared to controls. Animals were evaluated for 180 days to assess acute and chronic stages of disease, with sample collection from animals every 45 days. MAP concentrations from liver and intestinal tissues were examined using real time-PCR methods and the expression of key inflammatory markers were measured during MAP infection (interferon-gamma [IFN-Upsilon], Interleukin-1alpha, IL-12, IL-10, IL-6, and Tumor necrosis factor alpha [TNF-alpha]) Results: Our results demonstrate administration of probiotics reduces production of IFN-Upsilon and IL-6 while increasing TNF-alpha and IL-17 in chronic disease; healthful immune responses that reduce chronic inflammation associated to MAP infection. Conclusions: We observed that the immune system's response in the presence of probiotics to MAP contributes towards host health by influencing the activity of the immune system and gut microbial populations.

Description: Background: Population stratification is a systematic difference in allele frequencies between subpopulations. This can lead to spurious association findings in the case--control genome wide association studies (GWASs) used to identify single nucleotide polymorphisms (SNPs) associated with disease-linked phenotypes. Methods such as self-declared ancestry, ancestry informative markers, genomic control, structured association, and principal component analysis are used to assess and correct population stratification but each has limitations. We provide an alternative technique to address population stratification. Results: We propose a novel machine learning method, ETHNOPRED, which uses the genotype and ethnicity data from the HapMap project to learn ensembles of disjoint decision trees, capable of accurately predicting an individual's continental and sub-continental ancestry. To predict an individual's continental ancestry, ETHNOPRED produced an ensemble of 3 decision trees involving a total of 10 SNPs, with 10-fold cross validation accuracy of 100% using HapMap II dataset. We extended this model to involve 29 disjoint decision trees over 149 SNPs, and showed that this ensemble has an accuracy of 〉= 99.9%, even if some of those 149 SNP values were missing. On an independent dataset, predominantly of Caucasian origin, our continental classifier showed 96.8% accuracy and improved genomic control's lamda from 1.22 to 1.11. We next used the HapMap III dataset to learn classifiers to distinguish European subpopulations (North-Western vs. Southern), East Asian subpopulations (Chinese vs. Japanese), African subpopulations (Eastern vs. Western), North American subpopulations (European vs. Chinese vs. African vs. Mexican vs. Indian), and Kenyan subpopulations (Luhya vs. Maasai). In these cases, ETHNOPRED produced ensembles of 3, 39, 21, 11, and 25 disjoint decision trees, respectively involving 31, 502, 526, 242 and 271 SNPs, with 10-fold cross validation accuracy of 86.5% +/- 2.4%, 95.6% +/- 3.9%, 95.6% +/- 2.1%, 98.3% +/- 2.0%, and 95.9% +/- 1.5%. However, ETHNOPRED was unable to produce a classifier that can accurately distinguish Chinese in Beijing vs. Chinese in Denver. Conclusions: ETHNOPRED is a novel technique for producing classifiers that can identify an individual's continental and sub-continental heritage, based on a small number of SNPs. We show that its learned classifiers are simple, cost-efficient, accurate, transparent, flexible, fast, applicable to large scale GWASs, and robust to missing values.

Description: Background: Characterising genetic diversity through the analysis of massively parallel sequencing (MPS) data offers enormous potential to significantly improve our understanding of the genetic basis for observed phenotypes, including predisposition to and progression of complex human disease. Great challenges remain in resolving which genetic variants are genuinely associated with disease from the millions of 'bystanders' and artefactual signals. Results: FAVR is a suite of new methods designed to work with commonly used MPS analysis pipelines to assist in the resolution of some of these issues with a focus on relatively rare genetic variants. To the best of our knowledge, no equivalent has previously been described. The most important and novel aspect of FAVR is the use of signatures in comparator sequence alignment files during variant filtering, and annotation of variants potentially shared between individuals. The FAVR methods use these signatures to facilitate filtering of (i) platform-specific artefacts, (ii) common genetic variants, and, where relevant, (iii) artefacts derived from imbalanced paired-end sequencing, as well as annotation of genetic variants based on evidence of co-occurrence in individuals. By comparing conventional variant calling with or without downstream processing by FAVR methods applied to whole-exome sequencing datasets, we demonstrate a 3-fold smaller rare single nucleotide variant shortlist with no detected reduction in sensitivity. This analysis included Sanger sequencing of rare variant signals not evident in dbSNP131, assessment of known variant signal preservation, and comparison of observed and expected rare variant numbers across a range of first cousin pairs. The principles described herein were applied in our recent publication identifying XRCC2 as a new breast cancer risk gene and have been made publically available as a suite of software tools. Conclusions: FAVR is a platform-agnostic suite of methods that significantly enhances the analysis of large volumes of sequencing data for the study of rare genetic variants and their influence on phenotypes.

Description: Background: Professional societies recommend shared decision making (SDM) for prostate cancer screening, however, most efforts have promoted informed rather than shared decision making. The objective of this study is to 1) examine the effects of a prostate cancer screening intervention to promote SDM and 2) determine whether framing prostate information in the context of other clearly beneficial men's health services affects decisions. Methods: We conducted two separate randomized controlled trials of the same prostate cancer intervention (with or without additional information on more clearly beneficial men's health services). For each trial, we enrolled a convenience sample of 2 internal medicine practices, and their interested physicians and male patients with no prior history of prostate cancer (for a total of 4 practices, 28 physicians, and 128 men across trials). Within each practice site, we randomized men to either 1) a video-based decision aid and researcher-led coaching session or 2) a highway safety video. Physicians at each site received a 1-hour educational session on prostate cancer and SDM. To assess intervention effects, we measured key components of SDM, intent to be screened, and actual screening. After finding that results did not vary by trial, we combined data across sites, adjusting for the random effects of both practice and physician. Results: Compared to an attention control, our prostate cancer screening intervention increased men's perceptions that screening is a decision (absolute difference +41%; 95% CI 25 to 57%) and men's knowledge about prostate cancer screening (absolute difference +34%; 95% CI 19% to 50%), but had no effect on men's self-reported participation in shared decisions or their participation at their preferred level. Overall, the intervention decreased screening intent (absolute difference -34%; 95% CI -50% to -18%) and actual screening rates (absolute difference -22%; 95% CI -38 to -7%) with no difference in effect by frame. Conclusions: SDM interventions can increase men's knowledge, alter their perceptions of prostate cancer screening, and reduce actual screening. However, they may not guarantee an increase in shared decisions.Trial registration#NCT00630188

Description: Background: Previous studies have proposed that mammalian toll like receptors (TLRs) have evolvedunder diversifying selection due to their role in pathogen detection. To determine if this is thecase, we examined the extent of adaptive evolution in the TLR5 gene in both individualspecies and defined clades of the mammalia. Results: In support of previous studies, we find evidence of adaptive evolution of mammalian TLR5.However, we also show that TLR5 genes of domestic livestock have a concentration of singlenucleotide polymorphisms suggesting a specific signature of adaptation. Using codon modelsof evolution we have identified a concentration of rapidly evolving codons within the TLR5extracellular domain a site of interaction between host and the bacterial surface proteinflagellin. Conclusions: The results suggest that interactions between pathogen and host may be driving adaptivechange in TLR5 by competition between species. In support of this, we have identified singlenucleotide polymorphisms (SNP) in sheep and cattle TLR5 genes that are co-localised andco-incident with the predicted adaptive codons suggesting that adaptation in this region of theTLR5 gene is on-going in domestic species.

Description: Background: The challenge in extracting genome-wide chromatin features from limiting clinical samples poses a significant hurdle in identification of regulatory marks that impact the physiological or pathological state. Current methods that identify nuclease accessible chromatin are reliant on large amounts of purified nuclei as starting material. This complicates analysis of trace clinical tissue samples that are often stored frozen. We have developed an alternative nuclease based procedure to bypass nuclear preparation to interrogate nuclease accessible regions in frozen tissue samples. Results: Here we introduce a novel technique that specifically identifies Tissue Accessible Chromatin (TACh). The TACh method uses pulverized frozen tissue as starting material and employs one of the two robust endonucleases, Benzonase or Cyansase, which are fully active under a range of stringent conditions such as high levels of detergent and DTT. As a proof of principle we applied TACh to frozen mouse liver tissue. Combined with massive parallel sequencing TACh identifies accessible regions that are associated with euchromatic features and accessibility at transcriptional start sites correlates positively with levels of gene transcription. Accessible chromatin identified by TACh overlaps to a large extend with accessible chromatin identified by DNase I using nuclei purified from freshly isolated liver tissue as starting material. The similarities are most pronounced at highly accessible regions, whereas identification of less accessible regions tends to be more divergence between nucleases. Interestingly, we show that some of the differences between DNase I and Benzonase relate to their intrinsic sequence biases and accordingly accessibility of CpG islands is probed more efficiently using TACh. Conclusion: The TACh methodology identifies accessible chromatin derived from frozen tissue samples. We propose that this simple, robust approach can be applied across a broad range of clinically relevant samples to allow demarcation of regulatory elements of considerable prognostic significance.