Integrative quantitative-phase and airy light-sheet imaging.

Light-sheet microscopy enables considerable speed and phototoxicity gains, while quantitative-phase imaging confers label-free recognition of cells and organelles, and quantifies their number-density that, thermodynamically, is more representative of metabolism than size. Here, we report the fusion of these two imaging modalities onto a standard inverted microscope that retains compatibility with microfluidics and open-source software for image acquisition and processing. An accelerating Airy-beam light-sheet critically enabled imaging areas that were greater by more than one order of magnitude than a Gaussian beam illumination and matched exactly those of quantitative-phase imaging. Using this integrative imaging system, we performed a demonstrative multivariate investigation of live-cells in microfluidics that unmasked that cellular noise can affect the compartmental localization of metabolic reactions. We detail the design, assembly, and performance of the integrative imaging system, and discuss potential applications in biotechnology and evolutionary biology.

101 Dothideomycetes genomes: A test case for predicting lifestyles and emergence of pathogens.

Dothideomycetes is the largest class of kingdom Fungi and comprises an incredible diversity of lifestyles, many of which have evolved multiple times. Plant pathogens represent a major ecological niche of the class Dothideomycetes and they are known to infect most major food crops and feedstocks for biomass and biofuel production. Studying the ecology and evolution of Dothideomycetes has significant implications for our fundamental understanding of fungal evolution, their adaptation to stress and host specificity, and practical implications with regard to the effects of climate change and on the food, feed, and livestock elements of the agro-economy. In this study, we present the first large-scale, whole-genome comparison of 101 Dothideomycetes introducing 55 newly sequenced species. The availability of whole-genome data produced a high-confidence phylogeny leading to reclassification of 25 organisms, provided a clearer picture of the relationships among the various families, and indicated that pathogenicity evolved multiple times within this class. We also identified gene family expansions and contractions across the Dothideomycetes phylogeny linked to ecological niches providing insights into genome evolution and adaptation across this group. Using machine-learning methods we classified fungi into lifestyle classes with >95 % accuracy and identified a small number of gene families that positively correlated with these distinctions. This can become a valuable tool for genome-based prediction of species lifestyle, especially for rarely seen and poorly studied species.

Duplications and losses of genes encoding known elements of the stress defence system of the Aspergilli contribute to the evolution of these filamentous fungi but do not directly influence their environmental stress tolerance.

The contribution of stress protein duplication and deletion events to the evolution of the Aspergilli was studied. We performed a large-scale homology analysis of stress proteins and generated and analysed three stress defence system models based on Saccharomyces cerevisiae, Schizosaccharomyces pombe and Aspergillus nidulans. Although both yeast-based and A. nidulans-based models were suitable to trace evolutionary changes, the A. nidulans-based model performed better in mapping stress protein radiations. The strong Mantel correlation found between the positions of species in the phylogenetic tree on the one hand and either in the A. nidulans-based or S. cerevisiae-based models on the other hand demonstrated that stress protein expansions and reductions contributed significantly to the evolution of the Aspergilli. Interestingly, stress tolerance attributes correlated well with the number of orthologs only for a few stress proteins. Notable examples are Ftr1 iron permease and Fet3 ferro-O2-oxidoreductase, elements of the reductive iron assimilation pathway, in the S. cerevisiae-based model, as well as MpkC, a HogA-like mitogen activated protein kinase in the A. nidulans-based model. In the case of the iron assimilation proteins, the number of orthologs showed a positive correlation with H2O2-induced stress tolerance while the number of MpkC orthologs correlated positively with Congo Red induced cell wall stress, sorbitol induced osmotic stress and H2O2 induced oxidative stress tolerances. For most stress proteins, changes in the number of orthologs did not correlate well with any stress tolerance attributes. As a consequence, stress tolerance patterns of the studied Aspergilli did not correlate with either the sets of stress response proteins in general or with the phylogeny of the species studied. These observations suggest that stress protein duplication and deletion events significantly contributed to the evolution of stress tolerance attributes of Aspergilli. In contrast, there are other processes, which may counterbalance the effects of stress gene duplications or deletions including (i) alterations in the structures of stress proteins leading to changes in their biological activities, (ii) varying biosynthesis of stress proteins, (iii) rewiring stress response regulatory networks or even (iv) acquiring new stress response genes by horizontal gene transfer. All these multilevel changes are indispensable for the successful adaptation of filamentous fungi to altering environmental conditions, especially when these organisms are entering new ecological niches.

Pregnancy rates with intrauterine insemination: comparing 1999 and 2010 World Health Organization semen analysis norms.

Over the past 30 years, The World Health Organization has serially measured norms for human sperm. In this study, 1999 and 2010 semen analysis norms as predictors of pregnancy were compared during intrauterine insemination (IUI). A retrospective cohort study was conducted using data collected from the Stanford Fertility Center, between 2005 and 2007, with 981 couples undergoing 2231 IUI cycles. Collected semen was categorized according to total motile sperm counts (TMSC): 'normal (N.) 1999 TMSC', 'abnormal (AbN.) 1999/N. 2010 TMSC', or 'AbN. 2010 TMSC'. Sample comparison was also based on individual semen parameters: 'N. 1999 WHO', 'AbN. 1999/N. 2010 WHO', or 'AbN. 2010 WHO'. Pregnancy (defined by beta-HCG concentration) rates were calculated. Data were compared using correlation coefficients, t-tests and chi-squared tests, with and without adjusting for confounders. Pregnancy rate comparison based on TMSC ('N. 1999 TMSC', 'AbN. 1999/N. 2010 TMSC' and 'AbN. 2010 TMSC') showed a negative correlation (r = -0.41, P = 0.05). Pregnancy rate did not differ when comparisons were based on the presence of abnormal parameters, even when controlling for confounders. Therefore, TMSC based on the 1999 parameters shows best correlation with pregnancy rate for IUI; updating these norms in 2010 has little clinical implication in infertile populations.

Mixed-effects statistical model for comparative LC-MS proteomics studies.

Comparing a protein's concentrations across two or more treatments is the focus of many proteomics studies. A frequent source of measurements for these comparisons is a mass spectrometry (MS) analysis of a protein's peptide ions separated by liquid chromatography (LC) following its enzymatic digestion. Alas, LC-MS identification and quantification of equimolar peptides can vary significantly due to their unequal digestion, separation, and ionization. This unequal measurability of peptides, the largest source of LC-MS nuisance variation, stymies confident comparison of a protein's concentration across treatments. Our objective is to introduce a mixed-effects statistical model for comparative LC-MS proteomics studies. We describe LC-MS peptide abundance with a linear model featuring pivotal terms that account for unequal peptide LC-MS measurability. We advance fitting this model to an often incomplete LC-MS data set with REstricted Maximum Likelihood (REML) estimation, producing estimates of model goodness-of-fit, treatment effects, standard errors, confidence intervals, and protein relative concentrations. We illustrate the model with an experiment featuring a known dilution series of a filamentous ascomycete fungus Trichoderma reesei protein mixture. For 781 of the 1546 T. reesei proteins with sufficient data coverage, the fitted mixed-effects models capably described the LC-MS measurements. The LC-MS measurability terms effectively accounted for this major source of uncertainty. Ninety percent of the relative concentration estimates were within 0.5-fold of the true relative concentrations. Akin to the common ratio method, this model also produced biased estimates, albeit less biased. Bias decreased significantly, both absolutely and relative to the ratio method, as the number of observed peptides per protein increased. Mixed-effects statistical modeling offers a flexible, well-established methodology for comparative proteomics studies integrating common experimental designs with LC-MS sample processing plans. It favorably accounts for the unequal LC-MS measurability of peptides and produces informative quantitative comparisons of a protein's concentration across treatments with objective measures of uncertainties.

What can comparative genomics tell us about species concepts in the genus Aspergillus?

Understanding the nature of species" boundaries is a fundamental question in evolutionary biology. The availability of genomes from several species of the genus Aspergillus allows us for the first time to examine the demarcation of fungal species at the whole-genome level. Here, we examine four case studies, two of which involve intraspecific comparisons, whereas the other two deal with interspecific genomic comparisons between closely related species. These four comparisons reveal significant variation in the nature of species boundaries across Aspergillus. For example, comparisons between A. fumigatus and Neosartorya fischeri (the teleomorph of A. fischerianus) and between A. oryzae and A. flavus suggest that measures of sequence similarity and species-specific genes are significantly higher for the A. fumigatus - N. fischeri pair. Importantly, the values obtained from the comparison between A. oryzae and A. flavus are remarkably similar to those obtained from an intra-specific comparison of A. fumigatus strains, giving support to the proposal that A. oryzae represents a distinct ecotype of A. flavus and not a distinct species. We argue that genomic data can aid Aspergillus taxonomy by serving as a source of novel and unprecedented amounts of comparative data, as a resource for the development of additional diagnostic tools, and finally as a knowledge database about the biological differences between strains and species.

Effect of ozone oxidation on single-walled carbon nanotubes.

Exposing single-walled carbon nanotubes to room-temperature UV-generated ozone leads to an irreversible increase in their electrical resistance. We demonstrate that the increased resistance is due to ozone oxidation on the sidewalls of the nanotubes rather than at the end caps. Raman and X-ray photoelectron spectroscopies show an increase in the defect density due to the oxidation of the nanotubes. Using ultraviolet photoelectron spectroscopy, we show that these defects represent the removal of pi-conjugated electron states near the Fermi level, leading to the observed increase in electrical resistance. Oxidation of carbon nanotubes is an important first step in many chemical functionalization processes. Because the oxidation rate can be controlled with short exposures, UV-generated ozone offers the potential for use as a low-thermal-budget processing tool.

Nuclear import of activated D-ERK by DIM-7, an importin family member encoded by the gene moleskin.

The initiation of gene expression in response to Drosophila receptor tyrosine kinase signaling requires the nuclear import of the MAP kinase, D-ERK. However, the molecular details of D-ERK translocation are largely unknown. In this regard, we have identified D-Importin-7 (DIM-7), the Drosophila homolog of vertebrate importin 7, and its gene moleskin. DIM-7 exhibits a dynamic nuclear localization pattern that overlaps the spatial and temporal profile of nuclear, activated D-ERK. Co-immunoprecipitation experiments show that DIM-7 associates with phosphorylated D-ERK in Drosophila S2 cells. Furthermore, moleskin mutations enhance hypomorphic and suppress hypermorphic D-ERK mutant phenotypes. Deletion or mutation of moleskin dramatically reduces the nuclear localization of activated D-ERK. Directly linking DIM-7 to its nuclear import, this defect can be rescued by the expression of wild-type DIM-7. Mutations in the Drosophila Importin beta homolog Ketel, also reduce the nuclear localization of activated D-ERK. Together, these data indicate that DIM-7 and Ketel are components of the nuclear import machinery for activated D-ERK.

Simplified urinary immunoassay for 2,4-D: validation and exposure assessment.

Urinary monitoring of exposed workers by either analytic chemical methods or radioimmunoassay suggests that urinary levels of 2,4-dichlorophenoxyacetic acid (2,4-D) exceeding 30 ppb are indicative of occupational exposure. However, the current methods do not lend themselves to clinical laboratory use in the rural medical setting. The major goal of this project was to provide medical practitioners who care for members of the agricultural community with a cost-efficient way to conduct exposure assessment. This project used a direct 2,4-D enzyme immunoassay (EIA) and measurement of the ratio between 2,4-D-spiked and non-spiked samples of the same urine to quantify 2,4-D levels. This simplified approach minimizes the effects of non-specific interfering substances in urine and eliminates the need for sample extraction and clean-up. Possible urine co-contaminants (2,4-dichlorophenol and 2,5-dichlorophenol) do not significantly interfere with this immunoassay. Twenty-two forest pesticide applicators who apply and use chlorophenoxy herbicides in their work and 14 comparable control subjects were studied to validate the assay in the occupational setting. Coded urine specimens were examined for levels of 2,4-D by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and compared with immunoassay results from the same specimens. A correlation coefficient of r = 0.982 with a P value of .0001 for a plot of HPLC-MS/MS versus immunoassay demonstrated that the results from these methods were comparable over urinary dose levels ranging from not detectable (<19 ppb) to 1700 ppb 2,4-D, as determined by immunoassay.

Comparison of in vitro activity of danofloxacin, florfenicol, oxytetracycline, spectinomycin and tilmicosin against recent field isolates of Mycoplasma bovis.

The minimum inhibitory concentrations (MICS) and minimum mycoplasmacidal concentrations (MMCs) of danofloxacin, florfenicol, oxytetracycline, spectinomycin and tilmicosin against 62 recent British field isolates of Mycoplasma bovis were determined in vitro by a broth microdilution method. The isolates were most susceptible todanofloxacin with MIC90 and MMC90 values of 0.5 microg/ml and 1.0 microg/ml, respectively. They were less susceptible to florfenicol with a MIC90 of 16 microg/ml and MMC90 of 32 microg/ml. Oxytetracycline and spectinomycin had only a limited effect against the majority of isolates tested with MIC50s of 32 microg/ml and 4 microg/ml, respectively and MIC90s of 64 microg/ml and more than 128 microg/ml, respectively. Nearly 20 per cent of the isolates were highly resistant to spectinomycin, and tilmicosin was ineffective, with 92 per cent of the isolates having MIC values of 128 microg/ml or greater. There was no evidence of resistance by M bovis to danofloxacin.

Application of an indirect ELISA to milk samples to identify cows with Mycoplasma bovis mastitis.

An indirect ELISA was used to detect antibodies to Mycoplasma bovis in milk samples collected from a herd with M bovis mastitis. Antibodies were detected in samples from nine cows which had developed clinical M bovis mastitis. Milk from only three consistently antigen-negative cows tested positive for M bovis antibodies. These results indicate the potential value of the indirect ELISA for the detection of cows which have recently developed M bovis mastitis during the early stages of an outbreak.

Comparison of in vitro activity of danofloxacin, florfenicol, oxytetracycline, spectinomycin and tilmicosin against Mycoplasma mycoides subspecies mycoides small colony type.

Minimum inhibitory concentrations (MIC) and minimum mycoplasmacidal concentrations (MMC) of the antimicrobials danofloxacin, florfenicol, oxytetracycline, spectinomycin and tilmicosin were determined in vitro for 20 isolates of Mycoplasma mycoides subspecies mycoides small colony type (MmmSC), the causative agent of contagious bovine pleuropneumonia (CBPP). The majority of strains were most susceptible to tilmicosin, followed by danofloxacin, oxytetracycline, florfenicol and spectinomycin with MIC50 values of 0.015, 0.25, 0.5, 1 and 8 microg/ml, and MMC50 values of 0.06, 0.5, 8, 8 and 16 microg/ml, respectively. However, tilmicosin had poor mycoplasmacidal activity against two recent strains from Portugal. There was no evidence of resistance to danofloxacin in any of the strains.

Quantification of selected pesticide metabolites in human urine using isotope dilution high-performance liquid chromatography/tandem mass spectrometry.

The annual domestic use of pesticides is continually increasing, virtually ensuring that everyone is exposed to some level of pesticides on a regular basis through diet or environment. The potential developmental and physical adverse effects these chronic pesticide exposures have on children are of increasing concern. To adequately evaluate the potential adverse effects resulting from these exposures, accurate methods to measure the amount of the pesticide absorbed by the body must be developed. We have developed a sensitive method to measure the urinary metabolites of atrazine, diazinon, malathion, 2,4-dichlorophenoxyacetic acid (2,4-D), and certain synthetic pyrethroids in human urine. In our method, stable isotopically labeled analogues of the metabolites were spiked into the urine, which was subsequently extracted at both a neutral and acidic pH using organic solvents. The extracts were analyzed by high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) using atmospheric pressure chemical ionization. Our method has limits of detection ranging from 20 to 500 ng/l (parts per trillion) and relative standard deviations of less than 11%. This method has been used to measure the internal doses of these pesticides in both adults and children (n = 130) with no documented exposure to the pesticides. We detected atrazine and synthetic pyrethroid metabolites in less than 12% of the samples analyzed. The metabolites of 2,4-D, malathion, and diazinon were detected in 22%, 32%, and 57% of the samples, respectively.

A cell signal pathway involving laminin-5, alpha3beta1 integrin, and mitogen-activated protein kinase can regulate epithelial cell proliferation.

Laminin-5 (LN5) is a matrix component of epithelial tissue basement membranes and plays an important role in the initiation and maintenance of epithelial cell anchorage to the underlying connective tissue. Here we show that two distinct LN5 function-inhibitory antibodies, both of which bind the globular domain of the alpha3 subunit, inhibit proliferation of epithelial cells. These same antibodies also induce a decrease in mitogen-activated protein kinase activity. Inhibition of proliferation by the function-perturbing LN5 antibodies is reversed upon removal of the antibodies and can be overcome by providing the antibody-treated cells with exogenous LN5 and rat tail collagen. Because epithelial cells use the integrin receptor alpha3beta1 to interact with both LN5 and rat tail collagen, we next investigated the possibility that integrin alpha3beta1 is involved in mediating the proliferative impact of LN5. Proliferation of human epithelial cells is significantly inhibited by a function-perturbing alpha3 integrin antibody. In addition, antibody activation of beta1 integrin restores the proliferation of epithelial cells treated with LN5 function-perturbing antibodies. These data indicate that a complex comprising LN5 and alpha3beta1 integrin is multifunctional and contributes not only to epithelial cell adhesion but also to the regulation of cell growth via a signaling pathway involving mitogen-activated protein kinase. We discuss our study in light of recent evidence that LN5 expression is up-regulated at the leading tips of tumors, where it may play a role in tumor cell proliferation.

The PS integrins are required for a regulatory event during Drosophila wing morphogenesis.

The PS1 and PS2 integrins are required for morphogenesis of the adult Drosophila wing. Clonal analysis experiments have shown that both integrins are necessary to maintain adhesion between the dorsal and ventral wing epithelia. We have found that early in wing morphogenesis, the integrins are also required for a regulatory event, and this may explain why PS1 and PS2 must be expressed on opposite surfaces of the wing at the onset of pupariation. Overexpression of integrin subunits during this early phase can lead to separation of dorsal and ventral surfaces, and we present evidence here that this dominant phenotype (the Blistermaker phenotype) results from a gain of integrin function, as opposed to negative interference from free integrin subunits. A possible model for an integrin signaling requirement in the wing is discussed.