Bacterial charity work leads to population-wide resistance.

Bacteria show remarkable adaptability in the face of antibiotic therapeutics. Resistance alleles in drug target-specific sites and general stress responses have been identified in individual end-point isolates. Less is known, however, about the population dynamics during the development of antibiotic-resistant strains. Here we follow a continuous culture of Escherichia coli facing increasing levels of antibiotic and show that the vast majority of isolates are less resistant than the population as a whole. We find that the few highly resistant mutants improve the survival of the population's less resistant constituents, in part by producing indole, a signalling molecule generated by actively growing, unstressed cells. We show, through transcriptional profiling, that indole serves to turn on drug efflux pumps and oxidative-stress protective mechanisms. The indole production comes at a fitness cost to the highly resistant isolates, and whole-genome sequencing reveals that this bacterial altruism is made possible by drug-resistance mutations unrelated to indole production. This work establishes a population-based resistance mechanism constituting a form of kin selection whereby a small number of resistant mutants can, at some cost to themselves, provide protection to other, more vulnerable, cells, enhancing the survival capacity of the overall population in stressful environments.

Differences in healthy life expectancy for the US population by sex, race/ethnicity and geographic region: 2008.

BACKGROUND: Healthy life expectancy (HLE) varies among demographic segments of the US population and by geography. To quantify that variation, we estimated the national and regional HLE for the US population by sex, race/ethnicity and geographic region in 2008. METHODS: National HLEs were calculated using the published 2008 life table and the self-reported health status data from the National Health Interview Survey (NHIS). Regional HLEs were calculated using the combined 2007-09 mortality, population and NHIS health status data. RESULTS: In 2008, HLE in the USA varied significantly by sex, race/ethnicity and geographical regions. At 25 years of age, HLE for females was 47.3 years and ∼2.9 years greater than that for males at 44.4 years. HLE for non-Hispanic white adults was 2.6 years greater than that for Hispanic adults and 7.8 years greater than that for non-Hispanic black adults. By region, the Northeast had the longest HLE and the South had the shortest. CONCLUSIONS: The HLE estimates in this report can be used to monitor trends in the health of populations, compare estimates across populations and identify health inequalities that require attention.

An unbiased assessment of the role of imprinted genes in an intergenerational model of developmental programming.

Environmental factors during early life are critical for the later metabolic health of the individual and of future progeny. In our obesogenic environment, it is of great socioeconomic importance to investigate the mechanisms that contribute to the risk of metabolic ill health. Imprinted genes, a class of functionally mono-allelic genes critical for early growth and metabolic axis development, have been proposed to be uniquely susceptible to environmental change. Furthermore, it has also been suggested that perturbation of the epigenetic reprogramming of imprinting control regions (ICRs) may play a role in phenotypic heritability following early life insults. Alternatively, the presence of multiple layers of epigenetic regulation may in fact protect imprinted genes from such perturbation. Unbiased investigation of these alternative hypotheses requires assessment of imprinted gene expression in the context of the response of the whole transcriptome to environmental assault. We therefore analyse the role of imprinted genes in multiple tissues in two affected generations of an established murine model of the developmental origins of health and disease using microarrays and quantitative RT-PCR. We demonstrate that, despite the functional mono-allelicism of imprinted genes and their unique mechanisms of epigenetic dosage control, imprinted genes as a class are neither more susceptible nor protected from expression perturbation induced by maternal undernutrition in either the F1 or the F2 generation compared to other genes. Nor do we find any evidence that the epigenetic reprogramming of ICRs in the germline is susceptible to nutritional restriction. However, we propose that those imprinted genes that are affected may play important roles in the foetal response to undernutrition and potentially its long-term sequelae. We suggest that recently described instances of dosage regulation by relaxation of imprinting are rare and likely to be highly regulated.

Triplet repeat length bias and variation in the human transcriptome.

Length variation in short tandem repeats (STRs) is an important family of DNA polymorphisms with numerous applications in genetics, medicine, forensics, and evolutionary analysis. Several major diseases have been associated with length variation of trinucleotide (triplet) repeats including Huntington's disease, hereditary ataxias and spinobulbar muscular atrophy. Using the reference human genome, we have catalogued all triplet repeats in genic regions. This data revealed a bias in noncoding DNA repeat lengths. It also enabled a survey of repeat-length polymorphisms (RLPs) in human genomes and a comparison of the rate of polymorphism in humans versus divergence from chimpanzee. For short repeats, this analysis of three human genomes reveals a relatively low RLP rate in exons and, somewhat surprisingly, in introns. All short RLPs observed in multiple genomes are biallelic (at least in this small sample). In contrast, long repeats are highly polymorphic and some long RLPs are multiallelic. For long repeats, the chimpanzee sequence frequently differs from all observed human alleles. This suggests a high expansion/contraction rate in all long repeats. Expansions and contractions are not, however, affected by natural selection discernable from our comparison of human-chimpanzee divergence with human RLPs. Our catalog of human triplet repeats and their surrounding flanking regions can be used to produce a cost-effective whole-genome assay to test individuals. This repeat assay could someday complement SNP arrays for producing tests that assess the risk of an individual to develop a disease, or become part of personalized genomic strategy that provides therapeutic guidance with respect to drug response.

Estimating healthy life expectancies using longitudinal survey data: methods and techniques in population health measures.

Objective-Summary measures of population health are statistics that combine mortality and morbidity to represent overall population health in a single index. Such measures include healthy life expectancy, also called disability-free life expectancy and active life expectancy. Healthy life expectancy can be calculated using cross-sectional or longitudinal survey data. This report presents a comprehensive discussion of a method for calculating healthy life expectancy using data from longitudinal surveys. Methods-Healthy life expectancies are calculated using the multistate life table model. Expected life in various states of health is estimated using data from the Second Longitudinal Study of Aging and the Medicare Current Beneficiary Survey to illustrate the calculation of the statistics and the discussion of data and methodology related issues. Results-The study shows that estimating summary measures of population health using longitudinal survey data provides the opportunity of using incidence rather than prevalence rates. Health measures estimated based on incidence reflect the most recent health status of the population. Models that use longitudinal survey data measure transitions from good to poor health as well as poor to good health. That is, the models account for recovery from morbidity or illness. Longitudinal survey data canalsobeusedtocalculate healthy or active life expectancies by initial health states.

Complete genome sequence of Treponema pallidum ssp. pallidum strain SS14 determined with oligonucleotide arrays.

BACKGROUND: Syphilis spirochete Treponema pallidum ssp. pallidum remains the enigmatic pathogen, since no virulence factors have been identified and the pathogenesis of the disease is poorly understood. Increasing rates of new syphilis cases per year have been observed recently. RESULTS: The genome of the SS14 strain was sequenced to high accuracy by an oligonucleotide array strategy requiring hybridization to only three arrays (Comparative Genome Sequencing, CGS). Gaps in the resulting sequence were filled with targeted dideoxy-terminators (DDT) sequencing and the sequence was confirmed by whole genome fingerprinting (WGF). When compared to the Nichols strain, 327 single nucleotide substitutions (224 transitions, 103 transversions), 14 deletions, and 18 insertions were found. On the proteome level, the highest frequency of amino acid-altering substitution polymorphisms was in novel genes, while the lowest was in housekeeping genes, as expected by their evolutionary conservation. Evidence was also found for hypervariable regions and multiple regions showing intrastrain heterogeneity in the T. pallidum chromosome. CONCLUSION: The observed genetic changes do not have influence on the ability of Treponema pallidum to cause syphilitic infection, since both SS14 and Nichols are virulent in rabbit. However, this is the first assessment of the degree of variation between the two syphilis pathogens and paves the way for phylogenetic studies of this fascinating organism.

Compensatory evolution of interacting gene products through multifunctional intermediates.

When two mutations are singly deleterious but neutral or beneficial together, compensatory evolution can occur. The accumulation of derived, compensated genotypes contributes to the evolution of genetic incompatibilities between diverging populations or species. Previous two locus/two allele models have shown that compensatory evolution is appreciable only with tight linkage, the possibility of nearly simultaneous mutations, and/or a way to overcome negative selection against the singly mutated genotype. These conditions are often not met. Even when they are met, compensatory evolution is still predicted to be extremely slow, and in many scenarios selective advantage of the compensated genotype does little to accelerate it. Despite these obstacles, empirical studies suggest that it occurs readily. We describe here a set of related two locus/three allele models that invoke plausible neutral intermediates capable of productive interaction with both ancestral and compensated products of the interacting locus. These models are explored with analytical and computer simulation methods. The effect of these stepping-stone alleles on the evolution of ancestor-descendant incompatibilities is often profound, making the difference between evolution and stasis in several situations, including in small populations, when codominance or haploidy prevents shielding of mismatched genotypes, and in the absence of positive selection on the derived genotype. However, in large populations these intermediates can either speed or slow the evolution of incompatible genotypes relative to the two-allele case, depending on the specific fitness model. These results suggest that population size, the source of adaptive benefit, and the structural details of heteromeric gene product complexes interact to influence the path by which intergenic incompatibility evolves.

Genetic insulin resistance is a potent regulator of gene expression and proliferation in human iPS cells.

Insulin resistance is central to diabetes and metabolic syndrome. To define the consequences of genetic insulin resistance distinct from those secondary to cellular differentiation or in vivo regulation, we generated induced pluripotent stem cells (iPSCs) from individuals with insulin receptor mutations and age-appropriate control subjects and studied insulin signaling and gene expression compared with the fibroblasts from which they were derived. iPSCs from patients with genetic insulin resistance exhibited altered insulin signaling, paralleling that seen in the original fibroblasts. Insulin-stimulated expression of immediate early genes and proliferation were also potently reduced in insulin resistant iPSCs. Global gene expression analysis revealed marked differences in both insulin-resistant iPSCs and corresponding fibroblasts compared with control iPSCs and fibroblasts. Patterns of gene expression in patients with genetic insulin resistance were particularly distinct in the two cell types, indicating dependence on not only receptor activity but also the cellular context of the mutant insulin receptor. Thus, iPSCs provide a novel approach to define effects of genetically determined insulin resistance. This study demonstrates that effects of insulin resistance on gene expression are modified by cellular context and differentiation state. Moreover, altered insulin receptor signaling and insulin resistance can modify proliferation and function of pluripotent stem cell populations.

Expected years of life free of chronic condition-induced activity limitations - United States, 1999-2008.

Over the 20th century, the U.S. population has witnessed major changes in fatal and nonfatal health outcomes. Mortality has declined, and life expectancy has increased continuously; chronic conditions have replaced acute diseases as leading causes of both illness and death. During 1900-2008, average life expectancy at birth for the total U.S. population increased from 47.3 years in 1900 to 78.1 years in 2008, a gain of 30.8 years. In addition, an increasing proportion of the U.S. population is aged >65 years. According to the U.S. Census Bureau estimates, at the beginning of the 20th century, the U.S. population aged >65 years constituted only 4.1 percent of the total population; by 2008, the percentage of the total U.S. population aged >65 years was 12.8%. However, declines in mortality are not necessarily associated with declines in morbidity or the consequences of chronic conditions on life activities. The possibility that longer life might be accompanied by poor health makes it essential to develop measures that account for both mortality and morbidity at the same time. Hence, over the past 40 years, a new set of health measures (e.g., "healthy life expectancies") have been developed that account for both mortality and life spent free of the consequences of ill health. One of these newly developed set of measures (called "active life expectancy") is the average number of years expected to be lived without activity limitations.

Anatomical localization, gene expression profiling and functional characterization of adult human neck brown fat.

The imbalance between energy intake and expenditure is the underlying cause of the current obesity and diabetes pandemics. Central to these pathologies is the fat depot: white adipose tissue (WAT) stores excess calories, and brown adipose tissue (BAT) consumes fuel for thermogenesis using tissue-specific uncoupling protein 1 (UCP1). BAT was once thought to have a functional role in rodents and human infants only, but it has been recently shown that in response to mild cold exposure, adult human BAT consumes more glucose per gram than any other tissue. In addition to this nonshivering thermogenesis, human BAT may also combat weight gain by becoming more active in the setting of increased whole-body energy intake. This phenomenon of BAT-mediated diet-induced thermogenesis has been observed in rodents and suggests that activation of human BAT could be used as a safe treatment for obesity and metabolic dysregulation. In this study, we isolated anatomically defined neck fat from adult human volunteers and compared its gene expression, differentiation capacity and basal oxygen consumption to different mouse adipose depots. Although the properties of human neck fat vary substantially between individuals, some human samples share many similarities with classical, also called constitutive, rodent BAT.

Life Expectancy Free of Chronic Condition-induced Activity Limitations Among White and Black Americans, 2000-2006.

Objective-Life expectancy without activity limitations or active life expectancy is one of the health expectancy measures that is used to summarize population health. The measure differentiates the remaining years of life that are expected to be spent with activity limitations from expected years of life without activity limitations. The objective of this study was to estimate life expectancy with and without activity limitations for the white and black populations of the United States in the years 2000-2006, focusing on expected years free of chronic condition-induced activity limitations. Methods-Life expectancies for the total as well as the white and black populations for the years 2000-2006 were calculated separately using abridged single decrement life tables. Expected years of life with and without chronic condition-induced activity limitations were calculated using Sullivan's method. The statistical analysis is based on data from the U.S. Census Bureau and the National Center for Health Statistics. Results-Results of the study show that during the 7-year period, expected years free of chronic condition-induced activity limitations increased for the total population as well as the white and black populations of both sexes. For the total population, all males and all females, years free of chronic condition-induced activity limitations increased significantly at all ages except at 85 and over. Expected years free of chronic condition-induced activity limitations increased at age 75 and under for the white population and at age 65 and under for the black population.

Retrospective information on health status and its application for population health measures.

Healthy life expectancies are almost always calculated by using health data from cross-sectional surveys. This type of calculation is done partly because data from longitudinal surveys are not always available, and when they are available, they are collected at intervals that are longer than one year. In such cases, collecting health information retrospectively for the years skipped by the survey is useful. The main purpose of this paper is to show how retrospective health information can be used to estimate life expectancies in different health states. Healthy life expectancies are estimated with and without using data on retrospective health information, and the corresponding estimates are compared. The two sets of estimates are similar. We conclude that retrospectively assessed health information based on a one-year recall period can be used to estimate years of life in various health states and that estimates based on such information will closely approximate estimates based on concurrent health information.

Genome-wide in situ exon capture for selective resequencing.

Increasingly powerful sequencing technologies are ushering in an era of personal genome sequences and raising the possibility of using such information to guide medical decisions. Genome resequencing also promises to accelerate the identification of disease-associated mutations. Roughly 98% of the human genome is composed of repeats and intergenic or non-protein-coding sequences. Thus, it is crucial to focus resequencing on high-value genomic regions. Protein-coding exons represent one such type of high-value target. We have developed a method of using flexible, high-density microarrays to capture any desired fraction of the human genome, in this case corresponding to more than 200,000 protein-coding exons. Depending on the precise protocol, up to 55-85% of the captured fragments are associated with targeted regions and up to 98% of intended exons can be recovered. This methodology provides an adaptable route toward rapid and efficient resequencing of any sizeable, non-repeat portion of the human genome.

Direct selection of human genomic loci by microarray hybridization.

We applied high-density microarrays to the enrichment of specific sequences from the human genome for high-throughput sequencing. After capture of 6,726 approximately 500-base 'exon' segments, and of 'locus-specific' regions ranging in size from 200 kb to 5 Mb, followed by sequencing on a 454 Life Sciences FLX sequencer, most sequence reads represented selection targets. These direct selection methods supersede multiplex PCR for the large-scale analysis of genomic regions.

Mutation discovery in bacterial genomes: metronidazole resistance in Helicobacter pylori.

We developed a microarray hybridization-based method, 'comparative genome sequencing' (CGS), to find mutations in bacterial genomes and used it to study metronidazole resistance in H. pylori. CGS identified mutations in several genes, most likely affecting metronidazole activation, and produced no false positives in analysis of three megabases. We conclude that CGS identifies mutations in bacterial genomes efficiently, should enrich understanding of systems biology and genome evolution, and help track pathogens during outbreaks.

Achieving national health objectives: the impact on life expectancy and on healthy life expectancy.

Our study quantifies the impact of achieving specific Healthy People 2010 targets and of eliminating racial/ethnic health disparities on summary measures of health. We used life table methods to calculate gains in life expectancy and healthy life expectancy that would result from achievement of Healthy People 2010 objectives or of current mortality rates in the Asian/Pacific Islander (API) population. Attainment of Healthy People 2010 mortality targets would increase life expectancy by 2.8 years, and reduction of population wide mortality rates to current API rates would add 4.1 years. Healthy life expectancy would increase by 5.8 years if Healthy People 2010 mortality and assumed morbidity targets were attained and by 8.1 years if API mortality and activity limitation rates were attained. Achievement of specific Healthy People 2010 targets would produce significant increases in longevity and health, and elimination of racial/ethnic health disparities could result in even larger gains.

A self-tuning method for one-chip SNP identification.

Current methods for interpreting oligonucleotide-based SNP-detection microarrays, SNP chips, are based on statistics and require extensive parameter tuning as well as extremely high-resolution images of the chip being processed. We present a method, based on a simple data-classification technique called nearest-neighbors that, on haploid organisms, produces results comparable to the published results of the leading statistical methods and requires very little in the way of parameter tuning. Furthermore, it can interpret SNP chips using lower-resolution scanners of the type more typically used in current microarray experiments. Along with our algorithm, we present the results of a SNP-detection experiment where, when independently applying this algorithm to six identical SARS SNP chips, we correctly identify all 24 SNPs in a particular strain of the SARS virus, with between 6 and 13 false positives across the six experiments.

Gene expression analysis using oligonucleotide arrays produced by maskless photolithography.

Microarrays containing 195,000 in situ synthesized oligonucleotide features have been created using a benchtop, maskless photolithographic instrument. This instrument, the Maskless Array Synthesizer (MAS), uses a digital light processor (DLP) developed by Texas Instruments. The DLP creates the patterns of UV light used in the light-directed synthesis of oligonucleotides. This digital mask eliminates the need for expensive and time-consuming chromium masks. In this report, we describe experiments in which we tested this maskless technology for DNA synthesis on glass surfaces. Parameters examined included deprotection rates, repetitive yields, and oligonucleotide length. Custom gene expression arrays were manufactured and hybridized to Drosophila melanogaster and mouse samples. Quantitative PCR was used to validate the gene expression data from the mouse arrays.