A pipeline for targeted metagenomics of environmental bacteria.

BackgroundMetagenomics and single cell genomics provide a window into the genetic repertoire of yet uncultivated microorganisms, but both methods are usually taxonomically untargeted. The combination of fluorescence in situ hybridization (FISH) and fluorescence activated cell sorting (FACS) has the potential to enrich taxonomically well-defined clades for genomic analyses.MethodsCells hybridized with a taxon-specific FISH probe are enriched based on their fluorescence signal via flow cytometric cell sorting. A recently developed FISH procedure, the hybridization chain reaction (HCR)-FISH, provides the high signal intensities required for flow cytometric sorting while maintaining the integrity of the cellular DNA for subsequent genome sequencing. Sorted cells are subjected to shotgun sequencing, resulting in targeted metagenomes of low diversity.ResultsPure cultures of different taxonomic groups were used to (1) adapt and optimize the HCR-FISH protocol and (2) assess the effects of various cell fixation methods on both the signal intensity for cell sorting and the quality of subsequent genome amplification and sequencing. Best results were obtained for ethanol-fixed cells in terms of both HCR-FISH signal intensity and genome assembly quality. Our newly developed pipeline was successfully applied to a marine plankton sample from the North Sea yielding good quality metagenome assembled genomes from a yet uncultivated flavobacterial clade.ConclusionsWith the developed pipeline, targeted metagenomes at various taxonomic levels can be efficiently retrieved from environmental samples. The resulting metagenome assembled genomes allow for the description of yet uncharacterized microbial clades. Video abstract

Manifestations of Twinship in Toddler Language

Proceedings of the Sixth Annual Meeting of the Berkeley Linguistics Society (1980), pp. 361-37

Cytotoxicity of Selected Medicinal and Nonmedicinal Plant Extracts to Microbial and Cervical Cancer Cells

This study investigated the cytotoxicity of 55 species of plants. Each plant was rated as medicinal, or nonmedicinal based on the existing literature. About 79% of the medicinal plants showed some cytotoxicity, while 75% of the nonmedicinal plants showed bioactivity. It appears that Asteraceae, Labiatae, Pinaceae, and Chenopodiaceae were particularly active against human cervical cancer cells. Based on the literature, only three of the 55 plants have been significantly investigated for cytotoxicity. It is clear that there is much toxicological work yet to be done with both medicinal and nonmedicinal plants

Addressing Research Needs in the Field of Plant Virus Ecology by Defining Knowledge Gaps and Developing Wild Dicot Study Systems

Viruses are ubiquitous within all habitats that support cellular life and represent the most important emerging infectious diseases of plants. Despite this, it is only recently that we have begun to describe the ecological roles of plant viruses in unmanaged systems and the influence of ecosystem properties on virus evolution. We now know that wild plants frequently harbor infections by diverse virus species, but much remains to be learned about how viruses influence host traits and how hosts influence virus evolution and vector interactions. To identify knowledge gaps and suggest avenues for alleviating research deficits, we performed a quantitative synthesis of a representative sample of virus ecology literature, developed criteria for expanding the suite of pathosystems serving as models, and applied these criteria through a case study. We found significant gaps in the types of ecological systems studied, which merit more attention. In particular, there is a strong need for a greater diversity of logistically tractable, wild dicot perennial study systems suitable for experimental manipulations of infection status. Based on criteria developed from our quantitative synthesis, we evaluated three California native dicot perennials typically found in Mediterranean-climate plant communities as candidate models: Cucurbita foetidissima (buffalo gourd), Cucurbita palmata (coyote gourd), and Datura wrightii (sacred thorn-apple). We used Illumina sequencing and network analyses to characterize viromes and viral links among species, using samples taken from multiple individuals at two different reserves. We also compared our Illumina workflow with targeted RT-PCR detection assays of varying costs. To make this process accessible to ecologists looking to incorporate virology into existing studies, we describe our approach in detail and discuss advantages and challenges of different protocols. We also provide a bioinformatics workflow based on open-access tools with graphical user interfaces. Our study provides evidence that dicot perennials in xeric habitats support multiple, asymptomatic infections by viruses known to be pathogenic in related crop hosts. Quantifying the impacts of these interactions on plant performance and virus epidemiology in our logistically tractable host systems will provide fundamental information about plant virus ecology outside of crop environments

In Vitro Evaluation of Non-Protein Adsorbing Breast Cancer Theranostics Based on 19F-Polymer Containing Nanoparticles

Eight fluorinated nanoparticles (NPs) are synthesized, loaded with doxorubicin (DOX), and evaluated as theranostic delivery platforms to breast cancer cells. The multifunctional NPs are formed by self-assembly of either linear or star-shaped amphiphilic block copolymers, with fluorinated segments incorporated in the hydrophilic corona of the carrier. The sizes of the NPs confirm that small circular NPs are formed. The release kinetics data of the particles reveals clear hydrophobic core dependence, with longer sustained release from particles with larger hydrophobic cores, suggesting that the DOX release from these carriers can be tailored. Viability assays and flow cytometry evaluation of the ratios of apoptosis/necrosis indicate that the materials are non-toxic to breast cancer cells before DOX loading; however, they are very efficient, similar to free DOX, at killing cancer cells after drug encapsulation. Both flow cytometry and confocal microscopy confirm the cellular uptake of NPs and DOX-NPs into breast cancer cells, and in vitro 19F-MRI measurement shows that the fluorinated NPs have strong imaging signals, qualifying them as a potential in vivo contrast agent for 19F-MRI

Substrate limitations for heterotrophs: Implications for models that estimate the seasonal cycle of atmospheric CO_2

We examine the sensitivity of the seasonal cycle of heterotrophic respiration to model estimates of litterfall seasonality, herbivory, plant allocation, tissue chemistry, and land use. As a part of this analysis, we compare heterotrophic respiration models based solely on temperature and soil moisture controls (zero‐order models) with models that depend on available substrate as well (first‐order models). As indicators of regional and global CO_2 exchange, we use maps of monthly global net ecosystem production, growing season net flux (GSNF), and simulated atmospheric CO_2 concentrations from an atmospheric tracer transport model. In one first‐order model, CASA, variations on the representation of the seasonal flow of organic matter from plants to heterotrophs can increase global GSNF as much as 60% (5.7 Pg C yr^(−1)) above estimates obtained from a zero‐order model. Under a new first‐order scheme that includes separate seasonal dynamics for leaf litterfall, fine root mortality, coarse woody debris, and herbivory, we observe an increase in GSNF of 8% (0.7 Pg C yr^(−1)) over that predicted by the zero‐order model. The increase in seasonality of CO2 exchange in first‐order models reflects the dynamics of labile litter fractions; specifically, the rapid decomposition of a pulse of labile leaf and fine root litter that enters the heterotrophic community primarily from the middle to the end of the growing season shifts respiration outside the growing season. From the perspective of a first‐order model, we then explore the consequences of land use change and winter temperature anomalies on the amplitude of the seasonal cycle of atmospheric CO_2. Agricultural practices that accelerate decomposition may drive a long‐term increase in the amplitude, independent of human impacts on plant production. Consideration of first‐order litter decomposition dynamics may also help explain year‐to‐year variation in the amplitude

Observer ratings of neighborhoods: Comparison of two methods

BACKGROUND: Although neighborhood characteristics have important relationships with health outcomes, direct observation involves imperfect measurement. The African American Health (AAH) study included two observer neighborhood rating systems (5-item Krause and 18-item AAH Neighborhood Assessment Scale [NAS]), initially fielded at two different waves. Good measurement characteristics were previously shown for both, but there was more rater variability than desired. In 2010 both measures were re-fielded together, with enhanced training and field methods implemented to decrease rater variability while maintaining psychometric properties. METHODS: AAH included a poor inner city and more heterogeneous suburban areas. Four interviewers rated 483 blocks, with 120 randomly-selected blocks rated by two interviewers. We conducted confirmatory factor analysis of scales and tested the Krause (5-20 points), AAH 18-item NAS (0-28 points), and a previous 7-item and new 5-item versions of the NAS (0-17 points, 0-11 points). Retest reliability for items (kappa) and scales (Intraclass Correlation Coefficient [ICC]) were calculated overall and among pre-specified subgroups. Linear regression assessed interviewer effects on total scale scores and assessed concurrent validity on lung and lower body functions. Mismeasurement effects on self-rated health were also assessed. RESULTS: Scale scores were better in the suburbs than in the inner city. ICC was poor for the Krause scale (ICC=0.19), but improved if the retests occurred within 10 days (ICC=0.49). The 7- and 5-item NAS scales had better ICCs (0.56 and 0.62, respectively), and were higher (0.71 and 0.73) within 10 days. Rater variability for the Kraus and 5- and 7-item NAS scales was 1-3 points (compared to the supervising rater). Concurrent validity was modest, with residents living in worse neighborhood conditions having worse function. Unadjusted estimates were biased towards the null compared with measurement-error corrected estimates. CONCLUSIONS: Enhanced field protocols and rater training did not improve measurement quality. Specifically, retest reliability and interviewer variability remained problematic. Measurement error partially reduced, but did not eliminate concurrent validity, suggesting there are robust associations between neighborhood characteristics and health outcomes. We conclude that the 5-item AAH NAS has sufficient reliability and validity for further use. Additional research on the measurement properties of environmental rating methods is encouraged

Three-year measured weight change in the African American health study

OBJECTIVE: This study examines 3-year weight change in African Americans. METHOD: Nine hundred and ninety-eight participants 49 to 65 years old were assessed at baseline and 3 years later. Weight was measured, and weight change was defined as clinically meaningful increases or decreases (+/- 5 kg). Potential risk factors were investigated using multinomial logistic regression. RESULTS: In-home measured weights were available for 752 participants (75%): 504 (67%) had stable weights, 131 (17%) gained more than 5 kg, and 117 (16%) lost more than 5 kg. Among all participants, the risks for weight gains were cancer, chronic obstructive pulmonary disease, lower income, and Medicaid status; the risks for weight losses were angina, cancer, high measured systolic blood pressure, asthma, and physical inactivity. Sex-stratified analyses reveal differences involving age, socioeconomic status, cancer, blood pressure, and lower body function. DISCUSSION: Three-year weight changes in middle-aged African Americans were frequent and significantly associated with several risk factors

Different secretion patterns of matrix metalloproteinases and IL-8 and effect of corticotropin-releasing hormone in preterm and term cervical fibroblasts

The aims of the present study were to compare the levels of mRNA and protein expression of matrix metalloproteinase (MMP)-1, -3, -8 and -9 in human cervical tissue in preterm and term labor as well as not in labor and to determine if corticotropin-releasing hormone (CRH) has an effect on MMP-1, -3 and interleukin (IL)-8 secretion in both preterm and term cervical fibroblasts. Cervical biopsies were taken from 60 women: 18 at preterm labor, 7 at preterm not in labor, 18 at term labor and 17 at term not in labor. ELISA and Immulite were used for protein and real-time RT–PCR for mRNA analysis. Cervical fibroblast cultures were incubated for 18 h with different CRH concentrations (10−13–10−6 M). The mRNA expression of MMP-1, -3 and -9 was higher in laboring groups compared with term not in labor. Protein levels of MMP-8 and -9 were higher in term in labor group compared with non-laboring groups. There were no significant differences in mRNA and protein expression between the preterm and respective term control groups. CRH significantly increased secretion of IL-8 in preterm and term cervical fibroblasts compared with controls. The secretion of IL-8 and MMP-1 was significantly higher and MMP-3 secretion lower in preterm cervical fibroblasts. In conclusion, cervical ripening at preterm seems to be a similar inflammatory process as at term with CRH involved. However, preterm and term cervical fibroblasts might have different phenotypes based on different secretion patterns of IL-8, MMP-1 and MMP-3