Fecal microbiota diversity in survivors of adolescent/young adult Hodgkin lymphoma: a study of twins.

BACKGROUND: Adolescent/young adult Hodgkin lymphoma (AYAHL) survivors report fewer exposures to infections during childhood compared with controls, and they have functional lymphocyte aberrations. The gut microbiota plays a central role in immunity. METHODS: We investigated whether fecal microbial diversity differed between 13 AYAHL survivors and their unaffected co-twin controls. Pyrosequencing of fecal bacterial 16S rRNA amplicons yielded 252 943 edited reads that were assigned to species-level operational taxonomic units (OTUs) and standardised for sequencing depth by random sampling. Microbial diversity was compared within vs between twin pairs and by case-control status. RESULTS: The number of unique OTUs was more similar within twin pairs compared with randomly paired participants (P=0.0004). The AYAHL cases had fewer unique OTUs compared with their co-twin controls (338 vs 369, P=0.015); this difference was not significant (169 vs 183, P=0.10) when restricted to abundant OTUs. CONCLUSION: In this small study, AYAHL survivors appear to have a deficit of rare gut microbes. Further work is needed to determine if reduced microbial diversity is a consequence of the disease, its treatment, or a particularly hygienic environment.

Operon prediction without a training set.

MOTIVATION: Annotation of operons in a bacterial genome is an important step in determining an organism's transcriptional regulatory program. While extensive studies of operon structure have been carried out in a few species such as Escherichia coli, fewer resources exist to inform operon prediction in newly sequenced genomes. In particular, many extant operon finders require a large body of training examples to learn the properties of operons in the target organism. For newly sequenced genomes, such examples are generally not available; moreover, a model of operons trained on one species may not reflect the properties of other, distantly related organisms. We encountered these issues in the course of predicting operons in the genome of Bacteroides thetaiotaomicron (B.theta), a common anaerobe that is a prominent component of the normal adult human intestinal microbial community. RESULTS: We describe an operon predictor designed to work without extensive training data. We rely on a small set of a priori assumptions about the properties of the genome being annotated that permit estimation of the probability that two adjacent genes lie in a common operon. Predictions integrate several sources of information, including intergenic distance, common functional annotation and a novel formulation of conserved gene order. We validate our predictor both on the known operons of E.coli and on the genome of B.theta, using expression data to evaluate our predictions in the latter.

A molecular profile of the mouse gastric parietal cell with and without exposure to Helicobacter pylori.

The parietal cell (PC) plays an important role in normal gastric physiology and in common diseases of the stomach. Although the genes involved in acid secretion are well known, there is limited molecular information about other aspects of PC function. We have generated a comprehensive database of genes expressed preferentially in PCs relative to other gastric mucosal cell lineages. PCs were purified from FVB/N mouse stomachs by lectin panning. cRNA generated from PC-enriched (PC(+)) and PC-depleted (PC(-)) populations were used to query oligonucleotide-based microarrays. False-positive signals were filtered by using a new algorithm for noise reduction and selected results independently audited by real-time quantitative reverse transcription (RT)-PCR. The annotated database of 240 genes reveals previously unappreciated aspects of cellular function, including factors that may mediate PC regulation of gastric stem cell proliferation. PC(+) and PC(-) expression profiles were also prepared from germ-free mice 2 and 8 weeks after colonization with a clinical isolate of Helicobacter pylori (Hp)--the pathogen that produces acid-peptic disease (gastritis, ulcers) in humans. Whereas PC(+) gene expression was remarkably constant, the PC(-) fractions demonstrated a robust, evolving host response, with increased expression of genes involved in cell motility/migration, extracellular matrix interactions, and IFN responses. The consistency of PC(+) gene expression allowed identification of a cohort of 92 genes enriched in PCs under all conditions studied. These genes provide a molecular profile that can be used to define this epithelial lineage under a variety of physiologic, pharmacologic, and pathologic stimuli.

Comparison of genetic divergence and fitness between two subclones of Helicobacter pylori.

Helicobacter pylori has a very plastic genome, reflecting its high rate of recombination and point mutation. This plasticity promotes divergence of the population by the development of subclones and presumably enhances adaptation to host niches. We have investigated the genotypic and phenotypic characteristics of two such subclones isolated from one patient as well as the genetic evolution of these isolates during experimental infection. Whole-genome genotyping of the isolates using DNA microarrays revealed that they were more similar to each other than to a panel of other genotyped strains recovered from different hosts. Nonetheless, they still showed significant differences. For example, one isolate (67:21) contained the entire Cag pathogenicity island (PAI), whereas the other (67:20) had excised the PAI. Phenotypic studies disclosed that both isolates expressed adhesins that recognized human histo-blood group Lewis(b) glycan receptors produced by gastric pit and surface mucus cells. In addition, both isolates were able to colonize, to equivalent density and with similar efficiency, germ-free transgenic mice genetically engineered to synthesize Lewis(b) glycans in their pit cells (12 to 14 mice/isolate). Remarkably, the Cag PAI-negative isolate was unable to colonize conventionally raised Lewis(b) transgenic mice harboring a normal gastric microflora, whereas the Cag PAI-positive isolate colonized 74% of the animals (39 to 40 mice/isolate). The genomic evolution of both isolates during the infection of conventionally raised and germ-free mice was monitored over the course of 3 months. The Cag PAI-positive isolate was also surveyed after a 10 month colonization of conventionally raised transgenic animals (n = 9 mice). Microarray analysis of the Cag PAI and sequence analysis of the cagA, recA, and 16S rRNA genes disclosed no changes in recovered isolates. Together, these results reveal that the H. pylori population infecting one individual can undergo significant divergence, creating stable subclones with substantial genotypic and phenotypic differences.

DNA microarrays and beyond: completing the journey from tissue to cell.

For the cell biologist, identifying changes in gene expression using DNA microarrays is just the start of a long journey from tissue to cell. We discuss how chip users can first filter noise (false-positives) from daunting microarray datasets. Combining laser capture microdissection with real-time polymerase chain reaction and reverse transcription is a helpful follow-up step that allows expression of selected genes to be quantified using sensitive new in situ hybridization and immunohistochemical methods based on tyramide signal amplification.

Bringing the human genome and the revolution in bioinformatics to the medical school classroom: a case report from Washington University School of Medicine.

The human genome project is revolutionizing medical research and the practice of clinical medicine. To understand and participate in this revolution, physicians must be fluent in human genomics and bioinformatics. At Washington University School of Medicine (WUSM), the authors designed a module for teaching these skills to first-year students. The module uses clinical cases as a platform for accessing information stored in GenBank, Online Mendelian Inheritance in Man (OMIM), and PubMed databases at the National Center for Biotechnology Information (NCBI). This module, which is also designed to reinforce problem-solving skills, has been integrated into WUSM's first-year medical genetics course.

Extranuclear sequestration of phospho-Jun N-terminal kinase and distorted villi produced by activated Rac1 in the intestinal epithelium of chimeric mice.

Previously, we used a genetic mosaic system to conduct an in vivo analysis of the effects of Rac1 activation on the developing intestinal epithelium ( Stappenbeck, T. S. and Gordon, J. I. (2000) Development 127, 2629-2642). Expression of a constitutively active human Rac1 (Rac1Leu61) in the 129/Sv-derived small intestinal epithelium of C57Bl/6-ROSA26<-->129/Sv chimeric mice led to precocious differentiation of some lineages with accompanying alterations in their apical actin. We have now explored the underlying mechanisms. Rac1Leu61 leads to accumulation of the 46 kDa form of phosphorylated Jun N-terminal kinase (p-Jnk) in the apical cytoplasm, but not in the nucleus of E18.5 proliferating and differentiating intestinal epithelial cells. The effect is cell-autonomous, selective for this mitogen-activated protein kinase family member, and accompanied by apical cytoplasmic accumulation of p21-activated kinase. c-Jun, a downstream nuclear target of p-Jnk, does not show evidence of enhanced phosphorylation, providing functional evidence for cytoplasmic sequestration of p-Jnk in Rac1Leu61-expressing epithelium. In adult chimeras, Rac1 activation augments cell proliferation in crypts of Lieberkühn, without a compensatory change in basal apoptosis and produces a dramatic, very unusual widening of villi. These results reveal a novel in vivo paradigm for Rac1 activation involving p-Jnk-mediated signaling at a distinctive extra-nuclear site, with associated alterations in the actin cytoskeleton. They also provide a new perspective about the determinants of small intestinal villus morphogenesis.

Pre-steady-state kinetic studies of Saccharomyces cerevisiae myristoylCoA:protein N-myristoyltransferase mutants identify residues involved in catalysis.

MyristoylCoA:protein N-myristoyltransferase (Nmt, EC 2.3.1.97), a member of the GCN5 acetyltransferase (GNAT) superfamily, is an essential eukaryotic enzyme that catalyzes covalent attachment of myristate (C14:0) to the N-terminal Gly of proteins involved in myriad cellular functions. The 2.5 A resolution structure of a ternary complex of Saccharomyces cerevisiae Nmt1p with a bound substrate peptide (GLYASKLA) and nonhydrolyzable myristoylCoA analogue [Farazi, T. A., et al. (2001) Biochemistry 40, 6335] was used as the basis for a series of mutagenesis experiments designed to define the enzyme's catalytic mechanism. The kinetic properties of an F170A/L171A Nmt mutant are consistent with the proposal that their main chain amides, located in a beta-bulge structure conserved among GNATs, function as an oxyanion hole to polarize the thioester carbonyl of bound myristoylCoA prior to subsequent nucleophilic attack. Removal of the two C-terminal residues (M454 and L455) produces a 300--400-fold reduction in the chemical transformation rate and converts the rate-limiting step from a step after the transformation to the transformation event itself. This finding is consistent with the main chain C-terminal carboxylate of L455 functioning as a catalytic base that abstracts a proton from the N-terminal Gly ammonium of the bound peptide to generate the nucleophilic amine. Mutating N169 and T205 in concert reduces the rate of the chemical transformation, supporting their role as components of an H-bonding network that facilitates attack of the Gly1 amine and stabilizes the tetrahedral intermediate.

Enhanced gluconeogenesis and increased energy storage as hallmarks of aging in Saccharomyces cerevisiae.

A relationship between life span and cellular glucose metabolism has been inferred from genetic manipulations and caloric restriction of model organisms. In this report, we have used the Snf1p glucose-sensing pathway of Saccharomyces cerevisiae to explore the genetic and biochemical linkages between glucose metabolism and aging. Snf1p is a serine/threonine kinase that regulates cellular responses to glucose deprivation. Loss of Snf4p, an activator of Snf1p, extends generational life span whereas loss of Sip2p, a presumed repressor of the kinase, causes an accelerated aging phenotype. An annotated data base of global age-associated changes in gene expression in isogenic wild-type, sip2Delta, and snf4Delta strains was generated from DNA microarray studies. The transcriptional responses suggested that gluconeogenesis and glucose storage increase as wild-type cells age, that this metabolic evolution is exaggerated in rapidly aging sip2Delta cells, and that it is attenuated in longer-lived snf4Delta cells. To test this hypothesis directly, we applied microanalytic biochemical methods to generation-matched cells from each strain and measured the activities of enzymes and concentrations of metabolites in the gluconeogenic, glycolytic, and glyoxylate pathways, as well as glycogen, ATP, and NAD(+). The sensitivity of the assays allowed comprehensive biochemical profiling to be performed using aliquots of the same cell populations employed for the transcriptional profiling. The results provided additional evidence that aging in S. cerevisiae is associated with a shift away from glycolysis and toward gluconeogenesis and energy storage. They also disclosed that this shift is forestalled by two manipulations that extend life span, caloric restriction and genetic attenuation of the normal age-associated increase in Snf1p activity. Together, these findings indicate that Snf1p activation is not only a marker of aging but also a candidate mediator, because a shift toward energy storage over expenditure could impact myriad aspects of cellular maintenance and repair.

A new approach for filtering noise from high-density oligonucleotide microarray datasets.

Although DNA microarrays are powerful tools for profiling gene expression, the dynamic range and the sheer number of signals produced require efficient procedures for distinguishing false positive results (noise) from changes in expression that are 'real' (independently reproducible). We have developed an approach to filter noise from datasets generated when high density oligonucleotide-based microarrays are used to compare two distinct RNA populations. First, we performed comparisons between chips hybridized with cRNAs prepared from an identical starting RNA population; an 'Increase' or 'Decrease' call in such a comparison was defined as a false positive. Plotting the average distribution of these false positive signal intensities across 18 such comparisons of nine independent RNA preparations allowed us to develop a series of noise-filtering look-up tables (LUTs). Using a database of 70 separate chip-to-chip comparisons between distinct RNA preparations prepared by different workers at different sites and at different times, we show that the LUTs can be used to predict the likelihood that a given transcript called Increased or Decreased in one comparison will again be called Increased or Decreased in a replicate comparison. Evidence is presented that this LUT-based scoring system provides greater predictive value for reproducible microarray results than imposition of arbitrary fold-change thresholds and accurately predicts which microarray-identified changes will be validated by independent assays such as quantitative real-time PCR.

Structures of Saccharomyces cerevisiae N-myristoyltransferase with bound myristoylCoA and peptide provide insights about substrate recognition and catalysis.

MyristoylCoA:protein N-myristoyltransferase (Nmt) attaches myristate to the N-terminal Gly residue of proteins involved in a variety of signal transduction cascades, and other critical cellular functions. To gain insight about the structural basis of substrate recognition and catalysis, we determined the structures of a binary complex of Saccharomyces cerevisiae Nmt1p with myristoylCoA to 2.2 A resolution and of a ternary complex of Nmt1p with a nonhydrolyzable myristoylCoA analogue [S-(2-oxo)pentadecylCoA] and an octapeptide substrate (GLYASKLA) to 2.5 A resolution. The binary complex reveals how myristoylCoA alters the conformation of the enzyme to promote binding of both myristoylCoA and peptide and identifies the backbone amides of F170 and L171 as an oxyanion hole which polarizes the reactive thioester carbonyl. The ternary complex structure reveals details of the enzyme's peptide binding specificity and illuminates its mechanism of acyl transfer. The N-terminal Gly ammonium is positioned in close proximity to the C-terminal carboxylate of the protein, where it is poised to undergo the required deprotonation to an amine. In this conformation, the nucleophile is 6.3 A away from the thioester carbonyl. A catalytic mechanism is proposed whereby, once deprotonation is initiated, the N-terminal Gly amine can approximate the thioester carbonyl by rotating along Psi. This motion is facilitated by a H-bond network and leads to reaction between the glycine nitrogen nucleophile and the carbonyl. Loss of CoA from the tetrahedral intermediate may be facilitated by intramolecular H-bonding of the sulfur to the adenylamine of CoA. This affords a compact leaving group and lends a role for the observed bends in the CoA structure. The absolute requirement for Gly at the N-terminus of substrates is explained by the requirement for flexible rotation of its amine.

Genomics and proteomics converge on Helicobacter pylori.

During the past year, a series of studies have provided new perspectives about genetic diversity in Helicobacter pylori. The results illustrate how the current revolution in genomics and proteomics is being used to understand how this organism co-evolves with its host. The approaches should have broad applications to other host-bacterium relationships.

Commensal host-bacterial relationships in the gut.

One potential outcome of the adaptive coevolution of humans and bacteria is the development of commensal relationships, where neither partner is harmed, or symbiotic relationships, where unique metabolic traits or other benefits are provided. Our gastrointestinal tract is colonized by a vast community of symbionts and commensals that have important effects on immune function, nutrient processing, and a broad range of other host activities. The current genomic revolution offers an unprecedented opportunity to identify the molecular foundations of these relationships so that we can understand how they contribute to our normal physiology and how they can be exploited to develop new therapeutic strategies.

Glycans as legislators of host-microbial interactions: spanning the spectrum from symbiosis to pathogenicity.

The number of microbes associated with our gut likely exceeds our total number of somatic and germ cells. Despite their numbers, almost nothing is known about the molecular mechanisms that determine whether the interaction between a microbial species and its host will be beneficial. Recent results obtained from in vivo models have revealed critical roles for glycoconjugates in helping define the outcome of two such host-microbial relationships. In one case, attachment of Helicobacter pylori to fucosylated or sialylated glycans produced by various gastric epithelial lineages and their progenitors skews the destiny of colonization toward pathogenicity. In the second case, a molecular dissection of how Bacteroides thetaiotaomicron, a normal inhabitant of the distal small intestine, is able to communicate with intestinal epithelial cells has revealed a novel role for host fucosylated glycans in forging a mutually beneficial relationship. These observations lend support to the hypothesis that the capacity to synthesize diverse carbohydrate structures may have arisen in part from our need to both evade pathogenic relationships and to coevolve symbiotic relationships with our nonpathogenic resident microbes.

Molecular analysis of commensal host-microbial relationships in the intestine.

Human beings contain complex societies of indigenous microbes, yet little is known about how resident bacteria shape our physiology. We colonized germ-free mice with Bacteroides thetaiotaomicron, a prominent component of the normal mouse and human intestinal microflora. Global intestinal transcriptional responses to colonization were observed with DNA microarrays, and the cellular origins of selected responses were established by laser-capture microdissection. The results reveal that this commensal bacterium modulates expression of genes involved in several important intestinal functions, including nutrient absorption, mucosal barrier fortification, xenobiotic metabolism, angiogenesis, and postnatal intestinal maturation. These findings provide perspectives about the essential nature of the interactions between resident microorganisms and their hosts.

Impaired prostate tumorigenesis in Egr1-deficient mice.

The transcription factor early growth response protein 1 (EGR1) is overexpressed in a majority of human prostate cancers and is implicated in the regulation of several genes important for prostate tumor progression. Here we have assessed the effect of Egr1 deficiency on tumor development in two transgenic mouse models of prostate cancer (CR2-T-Ag and TRAMP). Using a combination of high-resolution magnetic resonance imaging and histopathological and survival analyses, we show that tumor progression was significantly impaired in Egr1-/- mice. Tumor initiation and tumor growth rate were not affected by the lack of Egr1; however, Egr1 deficiency significantly delayed the progression from prostatic intra-epithelial neoplasia to invasive carcinoma. These results indicate a unique role for Egr1 in regulating the transition from localized, carcinoma in situ to invasive carcinoma.

Transient-state kinetic analysis of Saccharomyces cerevisiae myristoylCoA:protein N-myristoyltransferase reveals that a step after chemical transformation is rate limiting.

MyristoylCoA:protein N-myristoyltransferase is a member of the superfamily of GCN5-related N-acetyltransferases and catalyzes the covalent attachment of myristate to the N-terminal Gly residue of proteins with diverse functions. Saccharomyces cerevisiae Nmt1p is a monomeric protein with an ordered bi-bi reaction mechanism: myristoylCoA is bound prior to peptide substrate; after catalysis, CoA is released followed by myristoylpeptide. Analysis of the X-ray structure of Nmt1p with bound substrate analogues indicates that the active site contains an oxyanion hole and a catalytic base and that catalysis proceeds through the nucleophilic addition-elimination mechanism. To determine the rate-limiting step in the enzyme reaction, pre-steady-state kinetic analyses were performed using a new, sensitive nonradioactive assay that detects CoA. Multiple turnover quenched flow studies disclosed that a step after the chemical transformation limits the overall rate of the reaction. Multiple and single turnover analyses revealed that the rate for the chemical transformation step is 13.8+/-0.6 s(-1) while the slower steady-state phase is 0.10+/-0.01 s(-1). Stopped flow kinetic studies of substrate acquisition indicated that binding of myristoylCoA to the apo-enzyme occurs through at least a two-step process, with a fast phase rate of 3.2 x 10(8) M(-1) s(-1) and a slow phase rate of 23+/-2 s(-1) (defined at 5 degrees C). Binding of an octapeptide substrate, representing the N-terminal sequence of a known yeast N-myristoylprotein (Cnb1p), to a binary complex composed of Nmt1p and a nonhydrolyzable myristoylCoA analogue (S-(2-oxo)pentadecylCoA) has a second-order rate constant of 2.1+/-0.3 x 10(6) M(-1) s(-1) and a dissociation rate of 26+/-15 s(-1) (defined at 10 degrees C). These results are interpreted in light of the X-ray structures of this enzyme.

Genetic mosaic analysis based on Cre recombinase and navigated laser capture microdissection.

Defining molecular interactions that occur at the interface between "normal" and "abnormal" cell populations represents an important but often underexplored aspect of the pathogenesis of diseases with focal origins. Here, we illustrate an approach for conducting such analyses based on mosaic patterns of Cre recombinase expression in the adult mouse intestinal epithelium. Transgenic mice were generated that express Cre in the stem cell niche of crypts located in specified regions of their intestine. Some of these mice were engineered to allow for doxycycline-inducible Cre expression. Recombination in all pedigrees was mosaic: Cre-expressing crypts that supported recombination in all of their active multipotent stem cells were located adjacent to "control" crypts that did not express Cre at detectable levels. Cre-mediated recombination of a floxed LacZ reporter provided direct evidence that adult small-intestinal crypts contain more than one active multipotent stem cell, and that these cells can be retained in both small-intestinal and colonic crypts for at least 80 d. A method was developed to recover epithelial cells from crypts with or without recombination for subsequent gene expression profiling. Stained sections of intestine were used to create electronic image templates to guide laser capture microdissection (LCM) of adjacent frozen sections. This navigated form of LCM overcomes problems with mRNA degradation encountered when cells are marked directly by immunohistochemical methods. Combining Cre-engineered genetic mosaic mice with navigated-LCM will allow biology and pathobiology to be explored at the junction between normal and perturbed cellular cohorts.