Rapid pathogen identification and phenotypic antimicrobial susceptibility directly from urine specimens.

Implementing effective antimicrobial therapy close to the onset of infection lowers morbidity and mortality and attenuates the spread of antimicrobial resistance. Current antimicrobial susceptibility testing (AST) methods, however, require several days to determine optimal therapies. We present technology and an automated platform that identify (ID) Urinary Tract Infection pathogens in 45 min and provide phenotypic AST results in less than 5 h from urine specimens without colony isolation. The ID and AST tests count cells fluorescently labeled with specific rRNA probes using non-magnified digital imaging. The ID test detected five pathogens at ≤ 7,000 CFU/mL and had a linear range of ~ 4 orders of magnitude. For contrived specimens, AST tests gave 93.1% categorical agreement with 1.3% Very Major Errors (VME), 0.3% Major Errors (ME), and 6.3% minor Errors (mE) compared to the broth microdilution (BMD) reference method. For clinical specimens, the ID test had 98.6% agreement and the AST test had 92.3% categorical agreement with 4.2% mE, 3.4% ME and 4.0% VME compared to BMD. Data presented demonstrates that direct-from-specimen AST tests can accurately determine antimicrobial susceptibility/resistance for each pathogen in a specimen containing two pathogens. The method is robust to urine matrix effects and off-target commensal and contaminating bacteria.

How full is the evolutionary fuel tank?

A meta-analysis quantifies the heritable genetic variance in fitness-the fuel of evolution.

A Rapid, Accurate, Single Molecule Counting Method Detects Clostridium difficile Toxin B in Stool Samples.

We describe a new rapid and accurate immunoassay-based technology capable of counting single target molecules using digital imaging without magnification. Using the technology, we developed a rapid test for Clostridium difficile toxin B, which is responsible for the pathology underlying potentially fatal C. difficile infections (CDI). There are currently no tests for CDI that are rapid, sensitive, and specific. The MultiPath C. difficile toxin B test images and counts complexes of target-specific magnetic and fluorescent particles that have been tethered together by toxin B molecules in minimally processed stool samples. The performance characteristics of the 30 minute test include a limit of detection of 45 pg/mL, dynamic range covering 4-5 orders of magnitude, and coefficient of variation of less than 10%. The MultiPath test detected all toxinotypes and ribotypes tested, including the one most commonly occurring in the US and EU; shows no cross reactivity with relevant bacterial species; and is robust to potential interferants commonly present in stool samples. On a training set of 320 clinical stool samples, the MultiPath C. difficile toxin B test showed 97.0% sensitivity (95% CI, 91.4-99.4%); 98.3% specificity (95% CI, 96.8-99.2%); and 98.2% accuracy (95% CI, 96.7-99.0%) compared to the cellular cytotoxicity neutralization assay (CCNA) reference method. Based on these compelling performance characteristics, we believe the MultiPath technology can address the lack of rapid, sensitive, specific, and easy-to-use diagnostic tests for C. difficile.

Method for the mapping of a female partial-sterile locus on a molecular marker linkage map.

The female gametophyte is an absolutely essential structure for angiosperm reproduction, and female sterility has been reported in a number of crops. In this paper, a maximum-likelihood method is presented for estimating the position and effect of a female partial-sterile locus in a backcross population using the observed data of dominant or codominant markers. The ML solutions are obtained via Bailey's method. The process for the estimating of the recombination fractions and the viabilities of female gametes are described, and the variances of the estimates of the parameters are also presented. Application of the method is demonstrated using a set of simulated data. This method circumvents the problems of the traditional mapping methods for female sterile genes which were based on data from seed set or embryo-sac morphology and anatomy.

The correlations of the function and positional distribution of the cis-elements CArG around the TSS in the genes of Mus musculus.

While many studies of cis-elements CArG bound by serum response factor (SRF) are in progress, little is known about the positional distribution of the functional CArG elements around the transcription start site (TSS) of genes that they influence. We use a validated CArG data set to calculate the distance distribution of functional CArG elements around the TSS. Distances between adjacent CArGs were also analyzed. We compare these distributions with those derived using a control set of randomly selected CArGs (that were not experimentally validated for function). Our results show that most functional CArG elements (108 of 152, 71%) exist upstream of the annotated TSS, with copy number increasing as one moves closer to the TSS. Moreover, the average number of the CArG elements in the CArG-containing genes is significantly more than that in the control genes. Our study extends earlier bioinformatic analyses of functional CArG elements and provides an application of comparative sequence data to the identification of transcription factor binding sites.

Quantitative genetics, version 3.0: where have we gone since 1987 and where are we headed?

The last 20 years since the previous World Congress have seen tremendous advancements in quantitative genetics, in large part due to the advancements in genomics, computation, and statistics. One central theme of this last 20 years has been the exploitation of the vast harvest of molecular markers--examples include QTL and association mapping, marker-assisted selection and introgression, scans for loci under selection, and methods to infer degree of coancestry, population membership, and past demographic history. One consequence of this harvest is that phenotyping, rather than genotyping, is now the bottleneck in molecular quantitative genetics studies. Equally important have been advances in statistics, many developed to effectively use this treasure trove of markers. Computational improvements in statistics, and in particular Markov Chain Monte Carlo (MCMC) methods, have facilitated many of these methods, as have significantly improved computational abilities for mixed models. Indeed, one could argue that mixed models have had at least as great an impact in quantitative genetics as have molecular markers. A final important theme over the past 20 years has been the fusion of population and quantitative genetics, in particular the importance of coalescence theory with its applications for association mapping, scans for loci under selection, and estimation of the demography history of a population. What are the future directions of the field? While obviously important surprises await us, the general trend seems to be moving into higher and higher dimensional traits and, in general, dimensional considerations. We have methods to deal with infinite-dimensional traits indexed by a single variable (such as a trait varying over time), but the future will require us to treat much more complex objects, such as infinite-dimensional traits indexed over several variables and with graphs and dynamical networks. A second important direction is the interfacing of quantitative genetics with physiological and developmental models as a step towards both the gene-phenotype map as well as predicting the effects of environmental changes. The high-dimensional objects we will need to consider almost certainly have most of their variation residing on a lower (likely much lower) dimensional subspace, and how to treat these constraints will be an important area of future research. Conversely, the univariate traits we currently deal with are themselves projections of more complex structures onto a lower dimensional space, and simply treating these as univariate traits can result in serious errors in understanding their selection and biology. As a field, our future is quite bright. We have new tools and techniques, and (most importantly) new talent with an exciting international group of vibrant young investigators who have received their degrees since the last Congress. One cloud for concern, however, has been the replacement at many universities of plant and animal breeders with plant and animal molecular biologists. Molecular tools are now an integral part of breeding, but breeding is not an integral part of molecular biology.

Joint match probabilities for Y chromosomal and autosomal markers.

Empirical tests of association between Y chromosome and autosomal markers are presented and a theoretical framework for determining a joint match probability is recommended. Statistical analyses of association were performed in 16 US populations between the autosomal genotypes from loci CSF1PO, FGA, THO1, TPOX, vWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S512, D21S11 and Y chromosome haplotypes from loci DYS19, DYS385ab, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS438, and DYS439. The sample populations include individuals of European-, African-, Hispanic-, Native-, and Asian-American ancestry. The results are consistent with independence of Y and autosomal markers, although small amounts of dependence would likely have escaped our tests. Given the data in hand, we suggest it is appropriate to compute joint match probabilities by multiplying the Y haplotype frequency with the appropriately corrected autosomal frequency. In addition to correcting for autosomal frequency differences between groups, a further correction may be required. Since two individuals sharing the same Y haplotype are likely to be more recently related than two randomly chosen individuals, the autosomal frequencies have to be adjusted to account for this, akin to the theta correction used to account for population substructure. The structure imposed on the autosomal frequencies conditioned in a Y match is a function of the number of markers scored and their mutation rate. However, in most settings theta<0.01. When population structure is already present in the autosomes, the additional effect due to conditioning on the Y is small. For example, if the amount of structure in the population is theta=0.01 or 0.03 (the NRCII range), then the effect of conditioning on the Y results in only a trivial increase in theta to 0.02-0.04, respectively.

Models for navigating biological complexity in breeding improved crop plants.

Progress in breeding higher-yielding crop plants would be greatly accelerated if the phenotypic consequences of making changes to the genetic makeup of an organism could be reliably predicted. Developing a predictive capacity that scales from genotype to phenotype is impeded by biological complexities associated with genetic controls, environmental effects and interactions among plant growth and development processes. Plant modelling can help navigate a path through this complexity. Here we profile modelling approaches for complex traits at gene network, organ and whole plant levels. Each provides a means to link phenotypic consequence to changes in genomic regions via stable associations with model coefficients. A unifying feature of the models is the relatively coarse level of granularity they use to capture system dynamics. Much of the fine detail is not directly required. Robust coarse-grained models might be the tool needed to integrate phenotypic and molecular approaches to plant breeding.

Genetic structure among 38 populations from the United States based on 11 U.S. core Y chromosome STRs.

A DNA database consisting of the 11 Y chromosome short-tandem-repeat (Y-STR) recommended by the Scientific Working Group on DNA Analysis Methods is constructed for 2517 individuals from 38 populations in the United States. The population samples derive from five ethnic groups currently living in 10 states. A multidimensional scaling (MDS) plot places the populations into four discrete clusters (African Americans (AA), European Americans (EA), Hispanic Americans (HA), and Asian Americans (SA)) and one dispersed cluster of Native Americans. An analysis of molecular variance (AMOVA) indicates that a large proportion of the total genetic variance is partitioned among ethnic groups (24.8%), whereas only a small amount (1.5%) is found among-populations within ethnic groups. Separate AMOVA analyses within each ethnic group show that only the NA sample contains statistically significant among-population variation. Pair wise population differentiation tests do uncover heterogeneity among EA and among HA populations; however, this is due to only a single sample within each group. The analyses support the creation of AA, EA, HA, and Asian American databases in which samples from different geographic regions within the United States are pooled. We recommend that separate databases be constructed for different NA groups.

Population structure of Y chromosome SNP haplogroups in the United States and forensic implications for constructing Y chromosome STR databases.

A set of 61 Y chromosome single-nucleotide-polymorphisms (Y-SNPs) is typed in a sample of 2517 individuals from 38 populations to infer the geographic origins of Y chromosomes in the United States and to test for paternal admixture among African-, European-, Hispanic-, Asian-, and Native-Americans. All of the samples were previously typed with the 11 core U.S. Y chromosome short tandem repeats (Y-STRs) recommended by SWGDAM, which revealed high levels of among ethnic group variation and low levels of among-population-within-ethnic-group variation. Admixture estimates vary greatly among populations and ethnic groups. The frequencies of non-European (3.4%) and non-Asian (4.5%) Y chromosomes are generally low in European-American and Asian-American populations, respectively. The frequencies of European Y chromosomes in Native-American populations range widely (i.e., 7-89%) and follow a West to East gradient, whereas they are relatively consistent in African-American populations (26.4+/-8.9%) from different locations. The European (77.8+/-9.3%) and Native-American (13.7+/-7.4%) components of the Hispanic paternal gene pool are also relatively constant among geographic regions; however, the African contribution is much higher in the Northeast (10.5+/-6.4%) than in the Southwest (1.5+/-0.9%) or Midwest (0%). To test for the effects of inter-ethnic admixture on the structure of Y-STR diversity in the U.S., we perform subtraction analyses in which Y chromosomes inferred to be admixed by Y-SNP analysis are removed from the database and pairwise population differentiation tests are implemented on the remaining Y-STR haplotypes. Results show that low levels of heterogeneity previously observed between pairs of Hispanic-American populations disappear when African-derived chromosomes are removed from the analysis. This is not the case for an unusual sample of European-Americans from New York City when its African-derived chromosomes are removed, or for Native-American populations when European-derived chromosomes are removed. We infer that both inter-ethnic admixture and population structure in ancestral source populations may contribute to fine scale Y-STR heterogeneity within U.S. ethnic groups.

Polymorphisms in the human monomethylarsonic acid (MMA V) reductase/hGSTO1 gene and changes in urinary arsenic profiles.

Large interindividual variability in urinary arsenic profiles, following chronic inorganic arsenic exposure, is well-known in humans. To understand this variability, we studied the relationship between polymorphisms in the gene for human monomethylarsonic acid (MMA(V)) reductase/hGSTO1 and the urinary arsenic profiles of individuals chronically exposed to arsenic in their drinking water. To ensure that we did not overlook rare polymorphisms, not included in the public databases, we amplified and sequenced all six exons of the gene and their flanking regions, using DNA isolated from peripheral blood samples of 75 subjects, living in the vicinity of Torreon, Mexico. Four groups, based on the levels of arsenic (9-100 microg/L) in their drinking water, were studied. We identified six novel polymorphisms and two reported previously. The novel polymorphisms were a three base pair deletion (delGGC) in the first intron; a G > C transversion, leading to a serine-to-cysteine substitution at amino acid 86; a G > T transversion and a A > T transversion in intron 5; a G > A transition resulting in glutamate-to-lysine substitution in amino acid 208; and a C > T transition producing an alanine-to-valine substitution in amino acid 236. Two subjects displayed significant differences in patterns of urinary arsenic; they had increased levels of urinary inorganic arsenic and reduced levels of methylated urinary arsenic species as compared to the rest of the study population. These two subjects had the same unique polymorphisms in hGSTO1 in that they were heterozygous for E155del and Glu208Lys. The identified SNPs may be one of the reasons for the large interindividual variability in the response of humans to chronic inorganic arsenic exposure. The findings suggest the need for further studies to identify unambiguously specific polymorphisms that may account for interindividual variability in the human response to chronic inorganic arsenic exposure.

Population-genetic models of the fates of duplicate genes.

The ultimate fate of a duplicated gene is that it either silenced through inactivating mutations or both copies are maintained by selection. This later fate can occur via neofunctionalization wherein one copy acquires a new function or by subfunctionalization wherein the original function of the gene is partitioned across both copies. The relative probabilities of these three different fates involve often very subtle iterations between of population size, mutation rate, and selection. All three of these fates are critical to the expansion and diversification of gene families.

Gene flow from the Indian subcontinent to Australia: evidence from the Y chromosome.

Phenotypic similarities between Australian Aboriginal People and some tribes of India were noted by T.H. Huxley during the voyage of the Rattlesnake (1846-1850). Anthropometric studies by Birdsell led to his suggestion that a migratory wave into Australia included populations with affinities to tribal Indians. Genetic evidence for an Indian contribution to the Australian gene pool is contradictory; most studies of autosomal markers have not supported this hypothesis (; and references therein). On the other hand, affinities between Australian Aboriginal People and southern Indians were suggested based on maternally inherited mitochondrial DNA. Here, we show additional DNA evidence in support of Huxley's hypothesis of an Indian-Australian connection using single-nucleotide polymorphisms (SNPs) and short tandem repeats (STRs) on the nonrecombining portion of the Y chromosome (NRY). Phylogenetic analyses of STR variation associated with a major Australian SNP lineage indicated tight clustering with southern Indian/Sri Lankan Y chromosomes. Estimates of the divergence time for these Indian and Australian chromosomes overlap with important changes in the archaeological and linguistic records in Australia. These results provide strong evidence for an influx of Y chromosomes from the Indian subcontinent to Australia that may have occurred during the Holocene.