The redox state of cytochrome C modulates resistance to methotrexate in human MCF7 breast cancer cells

Background: Methotrexate is a chemotherapeutic agent used to treat a variety of cancers. However, the occurrence of resistance limits its effectiveness. Cytochrome c in its reduced state is less capable of triggering the apoptotic cascade. Thus, we set up to study the relationship among redox state of cytochrome c, apoptosis and the development of resistance to methotrexate in MCF7 human breast cancer cells. Results: Cell incubation with cytochrome c-reducing agents, such as tetramethylphenylenediamine, ascorbate or reduced glutathione, decreased the mortality and apoptosis triggered by methotrexate. Conversely, depletion of glutathione increased the apoptotic action of methotrexate, showing an involvement of cytochrome c redox state in methotrexateinduced apoptosis. Methotrexate-resistant MCF7 cells showed increased levels of endogenous reduced glutathione and a higher capability to reduce exogenous cytochrome c. Using functional genomics we detected the overexpression of GSTM1 and GSTM4 in methotrexate-resistant MCF7 breast cancer cells, and determined that methotrexate was susceptible of glutathionylation by GSTs. The inhibition of these GSTM isoforms caused an increase in methotrexate cytotoxicity in sensitive and resistant cells. Conclusions: We conclude that overexpression of specific GSTMs, GSTM1 and GSTM4, together with increased endogenous reduced glutathione levels help to maintain a more reduced state of cytochrome c which, in turn, would decrease apoptosis, thus contributing to methotrexate resistance in human MCF7 breast cancer cells.

Genomics of host-pathogen interactions.

The study of immunity against infection can be framed in the context of genomics. First, long-term association with pathogens results in genomic signatures that result from positive selection. Evolutionary pressures tailor species or individual responses to pathogens, that may be associated with skewed patterns of immunity. Second, recent human population expansion carries an increasing burden of genetic mutation that can result in sporadic immunodeficiencies, and more generally, in diversity in susceptibility to infection. This review highlights current concepts and tools for the analysis of genomes and stresses the interest of these approaches in immunity.

Comparative genomics of emerging pathogens in the Candida glabrata clade

BACKGROUND: Candida glabrata follows C. albicans as the second or third most prevalent cause of candidemia worldwide. These two pathogenic yeasts are distantly related, C. glabrata being part of the Nakaseomyces, a group more closely related to Saccharomyces cerevisiae. Although C. glabrata was thought to be the only pathogenic Nakaseomyces, two new pathogens have recently been described within this group: C. nivariensis and C. bracarensis. To gain insight into the genomic changes underlying the emergence of virulence, we sequenced the genomes of these two, and three other non-pathogenic Nakaseomyces, and compared them to other sequenced yeasts. RESULTS: Our results indicate that the two new pathogens are more closely related to the non-pathogenic N. delphensis than to C. glabrata. We uncover duplications and accelerated evolution that specifically affected genes in the lineage preceding the group containing N. delphensis and the three pathogens, which may provide clues to the higher propensity of this group to infect humans. Finally, the number of Epa-like adhesins is specifically enriched in the pathogens, particularly in C. glabrata. CONCLUSIONS: Remarkably, some features thought to be the result of adaptation of C. glabrata to a pathogenic lifestyle, are present throughout the Nakaseomyces, indicating these are rather ancient adaptations to other environments. Phylogeny suggests that human pathogenesis evolved several times, independently within the clade. The expansion of the EPA gene family in pathogens establishes an evolutionary link between adhesion and virulence phenotypes. Our analyses thus shed light onto the relationships between virulence and the recent genomic changes that occurred within the Nakaseomyces.

Tissue-Specific Evolution of Protein Coding Genes in Human and Mouse.

Protein-coding genes evolve at different rates, and the influence of different parameters, from gene size to expression level, has been extensively studied. While in yeast gene expression level is the major causal factor of gene evolutionary rate, the situation is more complex in animals. Here we investigate these relations further, especially taking in account gene expression in different organs as well as indirect correlations between parameters. We used RNA-seq data from two large datasets, covering 22 mouse tissues and 27 human tissues. Over all tissues, evolutionary rate only correlates weakly with levels and breadth of expression. The strongest explanatory factors of purifying selection are GC content, expression in many developmental stages, and expression in brain tissues. While the main component of evolutionary rate is purifying selection, we also find tissue-specific patterns for sites under neutral evolution and for positive selection. We observe fast evolution of genes expressed in testis, but also in other tissues, notably liver, which are explained by weak purifying selection rather than by positive selection.

Genomics and X-ray microanalysis indicate that Ca2+ and thiols mediate the aggregation and adhesion of Xylella fastidiosa

The availability of the genome sequence of the bacterial plant pathogen Xylella fastidiosa, the causal agent of citrus variegated chlorosis, is accelerating important investigations concerning its pathogenicity. Plant vessel occlusion is critical for symptom development. The objective of the present study was to search for information that would help to explain the adhesion of X. fastidiosa cells to the xylem. Scanning electron microscopy revealed that adhesion may occur without the fastidium gum, an exopolysaccharide produced by X. fastidiosa, and X-ray microanalysis demonstrated the presence of elemental sulfur both in cells grown in vitro and in cells found inside plant vessels, indicating that the sulfur signal is generated by the pathogen surface. Calcium and magnesium peaks were detected in association with sulfur in occluded vessels. We propose an explanation for the adhesion and aggregation process. Thiol groups, maintained by the enzyme peptide methionine sulfoxide reductase, could be active on the surface of the bacteria and appear to promote cell-cell aggregation by forming disulfide bonds with thiol groups on the surface of adjacent cells. The enzyme methionine sulfoxide reductase has been shown to be an auxiliary component in the adhesiveness of some human pathogens. The negative charge conferred by the ionized thiol group could of itself constitute a mechanism of adhesion by allowing the formation of divalent cation bridges between the negatively charged bacteria and predominantly negatively charged xylem walls.

Functional genomics of O-glucosyltransferases from Concord grape (Vitis labrusca)

Grape (Vitis spp.) is a culturally and economically important crop plant that has been cultivated for thousands of years, primarily for the production of wine. Grape berries accumulate a myriad of phenylpropanoid secondary metabolites, many of which are glucosylated in plantae More than 90 O-glucosyltransferases have been cloned and biochemically characterized from plants, only two of which have been isolated from Vitis spp. The world-wide economic importance of grapes as a crop plant, the human health benefits associated with increased consumption of grape-derived metabolites, the biological relevance of glucosylation, and the lack of information about Vitis glucosyltransferases has inspired the identification, cloning and biochemical characterization of five novel "family 1" O-glucosyltransferases from Concord grape (Vitis labrusca cv. Concord). Protein purification and associated protein sequencIng led to the molecular cloning of UDP-glucose: resveratrollhydroxycinnamic acid O-glucosyltransferase (VLRSGT) from Vitis labrusca berry mesocarp tissue. In addition to being the first glucosyltransferase which accepts trans-resveratrol as a substrate to be characterized in vitro, the recombinant VLRSGT preferentially produces the glucose esters of hydroxycinnamic acids at pH 6.0, and the glucosides of trans-resveratrol and flavonols at 'pH 9.0; the first demonstration of pH-dependent bifunctional glucosylation for this class of enzymes. Gene expression and metabolite profiling support a role for this enzyme in the bifuncitonal glucosylation ofstilbenes and hydroxycinnamic acids in plantae A homology-based approach to cloning was used to identify three enzymes from the Vitis vinifera TIGR grape gene index which had high levels of protein sequence iii identity to previously characterized UDP-glucose: anthocyanin 5-0-glucosyltransferases. Molecular cloning and biochemical characterization demonstrated that these enzymes (rVLOGTl, rVLOGT2, rVLOGT3) glucosylate the 7-0-position of flavonols and the xenobiotic 2,4,5-trichlorophenol (TCP), but not anthocyanins. Variable gene expression throughout grape berry development and enzyme assays with native grape berry protein are consistent with a role for these enzymes in the glucosylation of flavonols; while the broad substrate specificity, the ability of these enzymes to glucosylate TCP and expression of these genes in tissues which are subject to pathogen attack (berry, flower, bud) is consistent with a role for these genes in the plant defense response. Additionally, the Vitis labrusca UDP-glucose: flavonoid 3-0-glucosyltransferase (VL3GT) was identified, cloned and characterized. VL3GT has 96 % protein sequence identity to the previously characterized Vitis vinifera flavonoid 3-0-glucosyltransferase (VV3GT); and glucosylates the 3-0-position of anthocyanidins and flavonols in vitro. Despite high levels of protein sequence identity, VL3GT has distinct biochemical characteristics (as compared to VV3GT), including a preference for B-ring methylated flavonoids and the inability to use UDP-galactose as a donor substrate. RT-PCR analysis of VL3GT gene expression and enzyme assays with native grape protein is consistent with an in planta role for this enzyme in the glucosylation of anthocyanidins,but not flavonols. These studies reveal the power of combining several biochemistry- and molecular biology-based tools to identify, clone, biochemically characterize and elucidate the in planta function of several biologically relevant O-glucosyltransferases from Vitis spp.

Transposable Elements Are a Significant Contributor to Tandem Repeats in the Human Genome

Sequence repeats are an important phenomenon in the human genome, playing important roles in genomic alteration often with phenotypic consequences. The two major types of repeat elements in the human genome are tandem repeats (TRs) including microsatellites, minisatellites, and satellites and transposable elements (TEs). So far, very little has been known about the relationship between these two types of repeats. In this study, we identified TRs that are derived from TEs either based on sequence similarity or overlapping genomic positions. We then analyzed the distribution of these TRs among TE families/subfamilies. Our study shows that at least 7,276 TRs or 23% of all minisatellites/satellites is derived from TEs, contributing ∼0.32% of the human genome. TRs seem to be generated more likely from younger/more active TEs, and once initiated they are expanded with time via local duplication of the repeat units. The currently postulated mechanisms for origin of TRs can explain only 6% of all TE-derived TRs, indicating the presence of one or more yet to be identified mechanisms for the initiation of such repeats. Our result suggests that TEs are contributing to genome expansion and alteration not only by transposition but also by generating tandem repeats.

HUMAN GENOME VARIATIONS AND EVOLUTION WITH A FOCUS ON THE ANALYSIS OF TRANSPOSABLE ELEMENTS

Genome sequence varies in numerous ways among individuals although the gross architecture is fixed for all humans. Retrotransposons create one of the most abundant structural variants in the human genome and are divided in many families, with certain members in some families, e.g., L1, Alu, SVA, and HERV-K, remaining active for transposition. Along with other types of genomic variants, retrotransponson-derived variants contribute to the whole spectrum of genome variants in humans. With the advancement of sequencing techniques, many human genomes are being sequenced at the individual level, fueling the comparative research on these variants among individuals. In this thesis, the evolution and functional impact of structural variations is examined primarily focusing on retrotransposons in the context of human evolution. The thesis comprises of three different studies on the topics that are presented in three data chapters. First, the recent evolution of all human specific AluYb members, representing the second most active subfamily of Alus, was tracked to identify their source/master copy using a novel approach. All human-specific AluYb elements from the reference genome were extracted, aligned with one another to construct clusters of similar copies and each cluster was analyzed to generate the evolutionary relationship between the members of the cluster. The approach resulted in identification of one major driver copy of all human specific Yb8 and the source copy of the Yb9 lineage. Three new subfamilies within the AluYb family – Yb8a1, Yb10 and Yb11 were also identified, with Yb11 being the youngest and most polymorphic. Second, an attempt to construct a relation between transposable elements (TEs) and tandem repeats (TRs) was made at a genome-wide scale for the first time. Upon sequence comparison, positional cross-checking and other relevant analyses, it was observed that over 20% of all TRs are derived from TEs. This result established the first connection between these two types of repetitive elements, and extends our appreciation for the impact of TEs on genomes. Furthermore, only 6% of these TE-derived TRs follow the already postulated initiation and expansion mechanisms, suggesting that the others are likely to follow a yet-unidentified mechanism. Third, by taking a combination of multiple computational approaches involving all types of genetic variations published so far including transposable elements, the first whole genome sequence of the most recent common ancestor of all modern human populations that diverged into different populations around 125,000-100,000 years ago was constructed. The study shows that the current reference genome sequence is 8.89 million base pairs larger than our common ancestor’s genome, contributed by a whole spectrum of genetic mechanisms. The use of this ancestral reference genome to facilitate the analysis of personal genomes was demonstrated using an example genome and more insightful recent evolutionary analyses involving the Neanderthal genome. The three data chapters presented in this thesis conclude that the tandem repeats and transposable elements are not two entirely distinctly isolated elements as over 20% TRs are actually derived from TEs. Certain subfamilies of TEs themselves are still evolving with the generation of newer subfamilies. The evolutionary analyses of all TEs along with other genomic variants helped to construct the genome sequence of the most recent common ancestor to all modern human populations which provides a better alternative to human reference genome and can be a useful resource for the study of personal genomics, population genetics, human and primate evolution.

Involving the Public in Public Health Genomics: A Review of Guidelines and Policy Statements

Public health genomics raises exciting possibilities for preventing or reducing the occurrence of both rare and common disease. However, this area of research raises challenging ethical, legal and social issues that must be addressed. One way of addressing these issues is through public involvement in the policy-making process. This GenEdit reviews how international guidelines and policy statements related to public health genomics address the issue of public involvement. Key areas of discussion are the values and goals justifying public involvement, the proposed activities to increase public involvement, who is / who represents "the public", and the projected outcomes of public involvement.

Studying the Human Gut Microbiota in the Trans-Omics Era - Focus on Metagenomics and Metabonomics

The human gut microbiota comprises a diverse microbial consortium closely co-evolved with the human genome and diet. The importance of the gut microbiota in regulating human health and disease has however been largely overlooked due to the inaccessibility of the intestinal habitat, the complexity of the gut microbiota itself and the fact that many of its members resist cultivation and are in fact new to science. However, with the emergence of 16S rRNA molecular tools and "post-genomics" high resolution technologies for examining microorganisms as they occur in nature without the need for prior laboratory culture, this limited view of the gut microbiota is rapidly changing. This review will discuss the application of molecular microbiological tools to study the human gut microbiota in a culture independent manner. Genomics or metagenomics approaches have a tremendous capability to generate compositional data and to measure the metabolic potential encoded by the combined genomes of the gut microbiota. Another post-genomics approach, metabonomics, has the capacity to measure the metabolic kinetic or flux of metabolites through an ecosystem at a particular point in time or over a time course. Metabonomics thus derives data on the function of the gut microbiota in situ and how it responds to different environmental stimuli e. g. substrates like prebiotics, antibiotics and other drugs and in response to disease. Recently these two culture independent, high resolution approaches have been combined into a single "transgenomic" approach which allows correlation of changes in metabolite profiles within human biofluids with microbiota compositional metagenomic data. Such approaches are providing novel insight into the composition, function and evolution of our gut microbiota.

British Nutrition Foundation annual lecture: chips with everything? Nutritional genomics and the application of diet in disease prevention

The development of high throughput techniques ('chip' technology) for measurement of gene expression and gene polymorphisms (genomics), and techniques for measuring global protein expression (proteomics) and metabolite profile (metabolomics) are revolutionising life science research, including research in human nutrition. In particular, the ability to undertake large-scale genotyping and to identify gene polymorphisms that determine risk of chronic disease (candidate genes) could enable definition of an individual's risk at an early age. However, the search for candidate genes has proven to be more complex, and their identification more elusive, than previously thought. This is largely due to the fact that much of the variability in risk results from interactions between the genome and environmental exposures. Whilst the former is now very well defined via the Human Genome Project, the latter (e.g. diet, toxins, physical activity) are poorly characterised, resulting in inability to account for their confounding effects in most large-scale candidate gene studies. The polygenic nature of most chronic diseases offers further complexity, requiring very large studies to disentangle relatively weak impacts of large numbers of potential 'risk' genes. The efficacy of diet as a preventative strategy could also be considerably increased by better information concerning gene polymorphisms that determine variability in responsiveness to specific diet and nutrient changes. Much of the limited available data are based on retrospective genotyping using stored samples from previously conducted intervention trials. Prospective studies are now needed to provide data that can be used as the basis for provision of individualised dietary advice and development of food products that optimise disease prevention. Application of the new technologies in nutrition research offers considerable potential for development of new knowledge and could greatly advance the role of diet as a preventative disease strategy in the 21st century. Given the potential economic and social benefits offered, funding for research in this area needs greater recognition, and a stronger strategic focus, than is presently the case. Application of genomics in human health offers considerable ethical and societal as well as scientific challenges. Economic determinants of health care provision are more likely to resolve such issues than scientific developments or altruistic concerns for human health.

Studying the Human Gut Microbiota in the Trans-Omics Era - Focus on Metagenomics and Metabonomics

The human gut microbiota comprises a diverse microbial consortium closely co-evolved with the human genome and diet. The importance of the gut microbiota in regulating human health and disease has however been largely overlooked due to the inaccessibility of the intestinal habitat, the complexity of the gut microbiota itself and the fact that many of its members resist cultivation and are in fact new to science. However, with the emergence of 16S rRNA molecular tools and "post-genomics" high resolution technologies for examining microorganisms as they occur in nature without the need for prior laboratory culture, this limited view of the gut microbiota is rapidly changing. This review will discuss the application of molecular microbiological tools to study the human gut microbiota in a culture independent manner. Genomics or metagenomics approaches have a tremendous capability to generate compositional data and to measure the metabolic potential encoded by the combined genomes of the gut microbiota. Another post-genomics approach, metabonomics, has the capacity to measure the metabolic kinetic or flux of metabolites through an ecosystem at a particular point in time or over a time course. Metabonomics thus derives data on the function of the gut microbiota in situ and how it responds to different environmental stimuli e.g. substrates like prebiotics, antibiotics and other drugs and in response to disease. Recently these two culture independent, high resolution approaches have been combined into a single "transgenomic" approach which allows correlation of changes in metabolite profiles within human biofluids with microbiota compositional metagenomic data. Such approaches are providing novel insight into the composition, function and evolution of our gut microbiota.

Post-genomics approaches towards monitoring changes within the microbial ecology of the gut

The human gut microbiota, comprising many hundreds of different microbial species, has closely co-evolved with its human host over the millennia. Diet has been a major driver of this co-evolution, in particular dietary non-digestible carbohydrates. This dietary fraction reaches the colon and becomes available for microbial fermentation, and it is in the colon that the great diversity of gut microorganisms resides. For the vast majority of our evolutionary history humans followed hunter-gatherer life-styles and consumed diets with many times more non-digestible carbohydrates, fiber and whole plant polyphenol rich foods than typical Western style diets today.

Virulotyping and antimicrobial resistance typing of salmonella enterica serovars relevant to human health in Europe

The combination of virulence gene and antimicrobial resistance gene typing using DNA arrays is a recently developed genomics-based approach to bacterial molecular epidemiology. We have now applied this technology to 523 Salmonella enterica subsp. enterica strains collected from various host sources and public health and veterinary institutes across nine European countries. The strain set included the five predominant Salmonella serovars isolated in Europe (Enteritidis, Typhimurium, Infantis, Virchow, and Hadar). Initially, these strains were screened for 10 potential virulence factors (avrA, ssaQ, mgtC, siiD, sopB, gipA, sodC1, sopE1, spvC, and bcfC) by polymerase chain reaction. The results indicated that only 14 profiles comprising these genes (virulotypes) were observed throughout Europe. Moreover, most of these virulotypes were restricted to only one (n = 9) or two (n = 4) serovars. The data also indicated that the virulotype did not vary significantly with host source or geographical location. Subsequently, a representative subset of 77 strains was investigated using a microarray designed to detect 102 virulence and 49 resistance determinants. The results confirmed and extended the previous observations using the virulo-polymerase chain reaction screen. Strains belonging to the same serovar grouped together, indicating that the broader virulence-associated gene complement corresponded with the serovar. There were, however, some differences in the virulence gene profiles between strains belonging to an individual serovar. This variation occurred primarily within those virulence genes that were prophage encoded, in fimbrial clusters or in the virulence plasmid. It seems likely that such changes enable Salmonella to adapt to different environmental conditions, which might be reflected in serovar-specific ecology. In this strain subset a number of resistance genes were detected and were serovar restricted to a varying degree. Once again the profiles of those genes encoding resistance were similar or the same for each serovar in all hosts and countries investigated.