Genomic diversity of Lactobacillus salivarius

Lactobacillus salivarius is unusual among the lactobacilli due to its multireplicon genome architecture. The circular megaplasmids harboured by L. salivarius strains encode strain-specific traits for intestinal survival and probiotic activity. L. salivarius strains are increasingly being exploited for their probiotic properties in humans and animals. In terms of probiotic strain selection, it is important to have an understanding of the level of genomic diversity present in this species. Comparative genomic hybridization (CGH) and multilocus sequence typing (MLST) were employed to assess the level of genomic diversity in L. salivarius. The wellcharacterised probiotic strains L. salivarius UCC118 was employed as a genetic reference strain. The group of test strains were chosen to reflect the range of habitats from which L. salivarius strains are frequently recovered, including human, animal, and environmental sources. Strains of L. salivarius were found to be genetically diverse when compared to the UCC118 genome. The most conserved strains were human GIT isolates, while the greatest level of divergence were identified in animal associated isolates. MLST produced a better separation of the test strains according to their isolation origins, than that produced by CGHbased strain clustering. The exopolysaccharide (EPS) associated genes of L. salivarius strains were found to be highly divergent. The EPS-producing phenotype was found to be carbonsource dependent and inversely related to a strain's ability to produce a biofilm. The genome of the porcine isolate L. salivarius JCM1046 was shown by sequencing to harbour four extrachromosomal replicons, a circular megaplasmid (pMP1046A), a putative chromid (pMP1046B), a linear megaplasmid (pLMP1046) and a smaller circular plasmid (pCTN1046) which contains an integrated Tn916-like element (Tn6224), which carries the tetracycline resistance gene tetM. pLMP1046 represents the first sequence of a linear plasmid in a Lactobacillus species. Dissemination of antibiotic resistance genes among species with food or probiotic-association is undesirable, and the identification of Tn6224-like elements in this species has implications for strain selection for probiotic applications. In summary, this thesis used a comparative genomics approach to examine the level of genotypic diversity in L. salivarius, a species which contains probiotic strains. The genome sequence of strain JCM1046 provides additional insight into the spectrum of extrachromosomal replicons present in this species.

Hypoxia-induced DNA hypermethylation in human pulmonary fibroblasts is associated with Thy-1 promoter methylation and the development of a pro-fibrotic phenotype

BACKGROUND: Pulmonary fibrosis is a debilitating and lethal disease with no effective treatment options. Understanding the pathological processes at play will direct the application of novel therapeutic avenues. Hypoxia has been implicated in the pathogenesis of pulmonary fibrosis yet the precise mechanism by which it contributes to disease progression remains to be fully elucidated. It has been shown that chronic hypoxia can alter DNA methylation patterns in tumour-derived cell lines. This epigenetic alteration can induce changes in cellular phenotype with promoter methylation being associated with gene silencing. Of particular relevance to idiopathic pulmonary fibrosis (IPF) is the observation that Thy-1 promoter methylation is associated with a myofibroblast phenotype where loss of Thy-1 occurs alongside increased alpha smooth muscle actin (α-SMA) expression. The initial aim of this study was to determine whether hypoxia regulates DNA methylation in normal human lung fibroblasts (CCD19Lu). As it has been reported that hypoxia suppresses Thy-1 expression during lung development we also studied the effect of hypoxia on Thy-1 promoter methylation and gene expression.

METHODS: CCD19Lu were grown for up to 8 days in hypoxia and assessed for global changes in DNA methylation using flow cytometry. Real-time PCR was used to quantify expression of Thy-1, α-SMA, collagen I and III. Genomic DNA was bisulphite treated and methylation specific PCR (MSPCR) was used to examine the methylation status of the Thy-1 promoter.

RESULTS: Significant global hypermethylation was detected in hypoxic fibroblasts relative to normoxic controls and was accompanied by increased expression of myofibroblast markers. Thy-1 mRNA expression was suppressed in hypoxic cells, which was restored with the demethylating agent 5-aza-2'-deoxycytidine. MSPCR revealed that Thy-1 became methylated following fibroblast exposure to 1% O2.

CONCLUSION: These data suggest that global and gene-specific changes in DNA methylation may play an important role in fibroblast function in hypoxia.

Stimulating Neoblast-Like Cell Proliferation in Juvenile Fasciola hepatica Supports Growth and Progression towards the Adult Phenotype In Vitro

Fascioliasis (or fasciolosis) is a socioeconomically important parasitic disease caused by liver flukes of the genus Fasciola. Flukicide resistance has exposed the need for new drugs and/or a vaccine for liver fluke control. A rapidly improving 'molecular toolbox' for liver fluke encompasses quality genomic/transcriptomic datasets and an RNA interference platform that facilitates functional genomics approaches to drug/vaccine target validation. The exploitation of these resources is undermined by the absence of effective culture/maintenance systems that would support in vitro studies on juvenile fluke development/biology. Here we report markedly improved in vitro maintenance methods for Fasciola hepatica that achieved 65% survival of juvenile fluke after 6 months in standard cell culture medium supplemented with 50% chicken serum. We discovered that this long-term maintenance was dependent upon fluke growth, which was supported by increased proliferation of cells resembling the "neoblast" stem cells described in other flatworms. Growth led to dramatic morphological changes in juveniles, including the development of the digestive tract, reproductive organs and the tegument, towards more adult-like forms. The inhibition of DNA synthesis prevented neoblast-like cell proliferation and inhibited growth/development. Supporting our assertion that we have triggered the development of juveniles towards adult-like fluke, mass spectrometric analyses showed that growing fluke have an excretory/secretory protein profile that is distinct from that of newly-excysted juveniles and more closely resembles that of ex vivo immature and adult fluke. Further, in vitro maintained fluke displayed a transition in their movement from the probing behaviour associated with migrating stage worms to a slower wave-like motility seen in adults. Our ability to stimulate neoblast-like cell proliferation and growth in F. hepatica underpins the first simple platform for their long-term in vitro study, complementing the recent expansion in liver fluke resources and facilitating in vitro target validation studies of the developmental biology of liver fluke.

Contrasting effects of historical contingency on phenotypic and genomic trajectories during a two-step evolution experiment with bacteria

Background The impact of historical contingency, i.e. the past evolutionary history of a population, on further adaptation is mostly unknown at both the phenotypic and genomic levels. We addressed this question using a two-step evolution experiment. First, replicate populations of Escherichia coli were propagated in four different environmental conditions for 1000 generations. Then, all replicate populations were transferred and propagated for further 1000 generations to a single new environment. Results Using this two-step experimental evolution strategy, we investigated, at both the phenotypic and genomic levels, whether and how adaptation in the initial historical environments impacted evolutionary trajectories in a new environment. We showed that both the growth rate and fitness of the evolved populations obtained after the second step of evolution were contingent upon past evolutionary history. In contrast however, the genes that were modified during the second step of evolution were independent from the previous history of the populations. Conclusions Our work suggests that historical contingency affects phenotypic adaptation to a new environment. This was however not reflected at the genomic level implying complex relationships between environmental factors and the genotype-to-phenotype map.

The Expanded Human Kallikrein (KLK) gene family : genomic organisation, tissue specific expression and potential functions

The tissue kallikreins are serine proteases encoded by highly conserved multigene families. The rodent kallikrein (KLK) families are particularly large, consisting of 13 26 genes clustered in one chromosomal locus. It has been recently recognised that the human KLK gene family is of a similar size (15 genes) with the identification of another 12 related genes (KLK4-KLK15) within and adjacent to the original human KLK locus (KLK1-3) on chromosome 19q13.4. The structural organisation and size of these new genes is similar to that of other KLK genes except for additional exons encoding 5 or 3 untranslated regions. Moreover, many of these genes have multiple mRNA transcripts, a trait not observed with rodent genes. Unlike all other kallikreins, the KLK4-KLK15 encoded proteases are less related (25–44%) and do not contain a conventional kallikrein loop. Clusters of genes exhibit high prostatic (KLK2-4, KLK15) or pancreatic (KLK6-13) expression, suggesting evolutionary conservation of elements conferring tissue specificity. These genes are also expressed, to varying degrees, in a wider range of tissues suggesting a functional involvement of these newer human kallikrein proteases in a diverse range of physiological processes.

BioPatML : pattern sharing for the genomic sciences

Computational biology increasingly demands the sharing of sophisticated data and annotations between research groups. Web 2.0 style sharing and publication requires that biological systems be described in well-defined, yet flexible and extensible formats which enhance exchange and re-use. In contrast to many of the standards for exchange in the genomic sciences, descriptions of biological sequences show a great diversity in format and function, impeding the definition and exchange of sequence patterns. In this presentation, we introduce BioPatML, an XML-based pattern description language that supports a wide range of patterns and allows the construction of complex, hierarchically structured patterns and pattern libraries. BioPatML unifies the diversity of current pattern description languages and fills a gap in the set of XML-based description languages for biological systems. We discuss the structure and elements of the language, and demonstrate its advantages on a series of applications, showing lightweight integration between the BioPatML parser and search engine, and the SilverGene genome browser. We conclude by describing our site to enable large scale pattern sharing, and our efforts to seed this repository.

Mutation frequency of non-ESBL phenotype SENTRY (Asia-Pacific) isolates of Klebsiella pneumoniae conversion to an ESBL positive phenotype

Extended spectrum β-lactamases or ESBLs, which are derived from non-ESBL precursors by point mutation of β-lactamase genes (bla), are spreading rapidly all over the world and have caused considerable problems in the treatment of infections caused by bacteria which harbour them. The mechanism of this resistance is not fully understood and a better understanding of these mechanisms might significantly impact on choosing proper diagnostic and treatment strategies. Previous work on SHV β-lactamase gene, blaSHV, has shown that only Klebsiella pneumoniae strains which contain plasmid-borne blaSHV are able to mutate to phenotypically ESBL-positive strains and there was also evidence of an increase in blaSHV copy number. Therefore, it was hypothesised that although specific point mutation is essential for acquisition of ESBL activity, it is not yet enough, and blaSHV copy number amplification is also essential for an ESBL-positive phenotype, with homologous recombination being the likely mechanism of blaSHV copy number expansion. In this study, we investigated the mutation rate of non-ESBL expressing K. pneumoniae isolates to an ESBL-positive status by using the MSS-maximum likelihood method. Our data showed that blaSHV mutation rate of a non-ESBL expressing isolate is lower than the mutation rate of the other single base changes on the chromosome, even with a plasmid-borne blaSHV gene. On the other hand, mutation rate from a low MIC ESBL-positive (≤ 8 µg/mL for cefotaxime) to high MIC ESBL-positive (≥16 µg/mL for cefotaxime) is very high. This is because only gene copy number increase is needed which is probably mediated by homologous recombination that typically takes place at a much higher frequencies than point mutations. Using a subinhibitory concentration of novobiocin, as a homologous recombination inhibitor, revealed that this is the case.

The Queensland study of melanoma : Environmental and genetic associations (Q-MEGA); Study design, baseline characteristics, and repeatability of phenotype and sun exposure measures

Cutaneous malignant melanoma (CMM) is a major health issue in Queensland, Australia, which has the world’s highest incidence. Recent molecular and epidemiologic studies suggest that CMM arises through multiple etiological pathways involving gene-environment interactions. Understanding the potential mechanisms leading to CMM requires larger studies than those previously conducted. This article describes the design and baseline characteristics of Q-MEGA, the Queensland Study of Melanoma: Environmental and Genetic Associations, which followed up 4 population-based samples of CMM patients in Queensland, including children, adolescents, men aged over 50, and a large sample of adult cases and their families, including twins. Q-MEGA aims to investigate the roles of genetic and environmental factors, and their interaction, in the etiology of melanoma. Three thousand, four hundred and seventy-one participants took part in the follow-up study and were administered a computer-assisted telephone interview in 2002-2005. Updated data on environmental and phenotypic risk factors, and 2777 blood samples were collected from interviewed participants as well as a subset of relatives. This study provides a large and well-described population-based sample of CMM cases with follow-up data. Characteristics of the cases and repeatability of sun exposure and phenotype measures between the baseline and the follow-up surveys, from 6 to 17 years later, are also described.

Morphologic and genomic characterisation of the koala Chlamydia pneumoniae strain

Chlamydia pneumoniae is a common human and animal pathogen associated with a wide range of upper and lower respiratory tract infections. In more recent years there has been increasing evidence to suggest a link between C. pneumoniae and chronic diseases in humans, including atherosclerosis, stroke and Alzheimer’s disease. C. pneumoniae human strains show little genetic variation, indicating that the human-derived strain originated from a common ancestor in the recent past. Despite extensive information on the genetics and morphology processes of the human strain, knowledge concerning many other hosts (including marsupials, amphibians, reptiles and equines) remains virtually unexplored. The koala (Phascolarctos cinereus) is a native Australian marsupial under threat due to habitat loss, predation and disease. Koalas are very susceptible to chlamydial infections, most commonly affecting the conjunctiva, urogenital tract and/or respiratory tract. To address this gap in the literature, the present study (i) provides a detailed description of the morphologic and genomic architecture of the C. pneumoniae koala (and human) strain, and shows that the koala strain is microscopically, developmentally and genetically distinct from the C. pneumoniae human strain, and (ii) examines the genetic relationship of geographically diverse C. pneumoniae isolates from human, marsupial, amphibian, reptilian and equine hosts, and identifies two distinct lineages that have arisen from animal-to-human cross species transmissions. Chapter One of this thesis explores the scientific problem and aims of this study, while Chapter Two provides a detailed literature review of the background in this field of work. Chapter Three, the first results chapter, describes the morphology and developmental stages of C. pneumoniae koala isolate LPCoLN, as revealed by fluorescence and transmission electron microscopy. The profile of this isolate, when cultured in HEp-2 human epithelial cells, was quite different to the human AR39 isolate. Koala LPCoLN inclusions were larger; the elementary bodies did not have the characteristic pear-shaped appearance, and the developmental cycle was completed within a shorter period of time (as confirmed by quantitative real-time PCR). These in vitro findings might reflect biological differences between koala LPCoLN and human AR39 in vivo. Chapter Four describes the complete genome sequence of the koala respiratory pathogen, C. pneumoniae LPCoLN. This is the first animal isolate of C. pneumoniae to be fully-sequenced. The genome sequence provides new insights into genomic ‘plasticity’ (organisation), evolution and biology of koala LPCoLN, relative to four complete C. pneumoniae human genomes (AR39, CWL029, J138 and TW183). Koala LPCoLN contains a plasmid that is not shared with any of the human isolates, there is evidence of gene loss in nucleotide salvage pathways, and there are 10 hot spot genomic regions of variation that were previously not identified in the C. pneumoniae human genomes. Sequence (partial-length) from a second, independent, wild koala isolate (EBB) at several gene loci confirmed that the koala LPCoLN isolate was representative of a koala C. pneumoniae strain. The combined sequence data provides evidence that the C. pneumoniae animal (koala LPCoLN) genome is ancestral to the C. pneumoniae human genomes and that human infections may have originated from zoonotic infections. Chapter Five examines key genome components of the five C. pneumoniae genomes in more detail. This analysis reveals genomic features that are shared by and/or contribute to the broad ecological adaptability and evolution of C. pneumoniae. This analysis resulted in the identification of 65 gene sequences for further analysis of intraspecific variation, and revealed some interesting differences, including fragmentation, truncation and gene decay (loss of redundant ancestral traits). This study provides valuable insights into metabolic diversity, adaptation and evolution of C. pneumoniae. Chapter Six utilises a subset of 23 target genes identified from the previous genomic comparisons and makes a significant contribution to our understanding of genetic variability among C. pneumoniae human (11) and animal (6 amphibian, 5 reptilian, 1 equine and 7 marsupial hosts) isolates. It has been shown that the animal isolates are genetically diverse, unlike the human isolates that are virtually clonal. More convincing evidence that C. pneumoniae originated in animals and recently (in the last few hundred thousand years) crossed host species to infect humans is provided in this study. It is proposed that two animal-to-human cross species events have occurred in the context of the results, one evident by the nearly clonal human genotype circulating in the world today, and the other by a more animal-like genotype apparent in Indigenous Australians. Taken together, these data indicate that the C. pneumoniae koala LPCoLN isolate has morphologic and genomic characteristics that are distinct from the human isolates. These differences may affect the survival and activity of the C. pneumoniae koala pathogen in its natural host, in vivo. This study, by utilising the genetic diversity of C. pneumoniae, identified new genetic markers for distinguishing human and animal isolates. However, not all C. pneumoniae isolates were genetically diverse; in fact, several isolates were highly conserved, if not identical in sequence (i.e. Australian marsupials) emphasising that at some stage in the evolution of this pathogen, there has been an adaptation/s to a particular host, providing some stability in the genome. The outcomes of this study by experimental and bioinformatic approaches have significantly enhanced our knowledge of the biology of this pathogen and will advance opportunities for the investigation of novel vaccine targets, antimicrobial therapy, or blocking of pathogenic pathways.

Characterising the homeostatic and hedonic markers of the susceptible phenotype

Over the years, approaches to obesity prevention and treatment have gone from focusing on genetic and other biological factors to exploring a diversity of diets and individual behavior modification interventions anchored primarily in the power of the mind, to the recent shift focusing on societal interventions to design ";temptation-proof"; physical, social, and economic environments. In spite of repeated calls to action, including those of the World Health Organization (WHO), the pandemic continues to progress. WHO recently projected that if the current lifestyle trend in young and adult populations around the world persist, by 2012 in countries like the USA, health care costs may amount to as much as 17.7% of the GDP. Most importantly, in large part due to the problems of obesity, those children may be the first generation ever to have a shorter life expectancy than that of their parents. This work presents the most current research and proposals for addressing the pandemic. Past studies have focused primarly on either genetic or behavioral causes for obesity, however today's research indicates that a strongly integrated program is the best prospect for success in overcoming obesity. Furthermore, focus on the role of society in establishing an affordable, accessible and sustainable program for implementing these lifestyle changes is vital, particularly for those in economically challenged situations, who are ultimately at the highest risk for obesity. Using studies from both neuroscience and behavioral science to present a comprehensive overview of the challenges and possible solutions, The brain-to-society approach to obesity prevention focuses on what is needed in order to sustain a healthy, pleasurable and affordable lifestyle.

Candidate metastasis suppressor genes uncovered by array comparative genomic hybridization in a mouse allograft model of prostate cancer.

Background The purpose of this study was to identify candidate metastasis suppressor genes from a mouse allograft model of prostate cancer (NE-10). This allograft model originally developed metastases by twelve weeks after implantation in male athymic nude mice, but lost the ability to metastasize after a number of in vivo passages. We performed high resolution array comparative genomic hybridization on the metastasizing and non-metastasizing allografts to identify chromosome imbalances that differed between the two groups of tumors. Results This analysis uncovered a deletion on chromosome 2 that differed between the metastasizing and non-metastasizing tumors. Bioinformatics filters were employed to mine this region of the genome for candidate metastasis suppressor genes. Of the 146 known genes that reside within the region of interest on mouse chromosome 2, four candidate metastasis suppressor genes (Slc27a2, Mall, Snrpb, and Rassf2) were identified. Quantitative expression analysis confirmed decreased expression of these genes in the metastasizing compared to non-metastasizing tumors. Conclusion This study presents combined genomics and bioinformatics approaches for identifying potential metastasis suppressor genes. The genes identified here are candidates for further studies to determine their functional role in inhibiting metastases in the NE-10 allograft model and human prostate cancer.

Single-stranded DNA-binding protein hSSB1 is critical for genomic stability

Single-strand DNA (ssDNA)-binding proteins (SSBs) are ubiquitous and essential for a wide variety of DNA metabolic processes, including DNA replication, recombination, DNA damage detection and repair1. SSBs have multiple roles in binding and sequestering ssDNA, detecting DNA damage, stimulating nucleases, helicases and strand-exchange proteins, activating transcription and mediating protein–protein interactions. In eukaryotes, the major SSB, replication protein A (RPA), is a heterotrimer1. Here we describe a second human SSB (hSSB1), with a domain organization closer to the archaeal SSB than to RPA. Ataxia telangiectasia mutated (ATM) kinase phosphorylates hSSB1 in response to DNA double-strand breaks (DSBs). This phosphorylation event is required for DNA damage-induced stabilization of hSSB1. Upon induction of DNA damage, hSSB1 accumulates in the nucleus and forms distinct foci independent of cell-cycle phase. These foci co-localize with other known repair proteins. In contrast to RPA, hSSB1 does not localize to replication foci in S-phase cells and hSSB1 deficiency does not influence S-phase progression. Depletion of hSSB1 abrogates the cellular response to DSBs, including activation of ATM and phosphorylation of ATM targets after ionizing radiation. Cells deficient in hSSB1 exhibit increased radiosensitivity, defective checkpoint activation and enhanced genomic instability coupled with a diminished capacity for DNA repair. These findings establish that hSSB1 influences diverse endpoints in the cellular DNA damage response.