ThermoPhyl : a software tool for selecting phylogenetically optimized conventional and quantitative-PCR taxon-targeted assays for use with complex samples

The ability to specifically and sensitively target genotypes of interest is critical for the success of many PCR-based analyses of environmental or clinical samples that contain multiple templates.Next-generation sequence data clearly show that such samples can harbour hundreds to thousands of operational taxonomic units; a richness which precludes the manual evaluation of candidate assay specificity and sensitivity using multiple sequence alignments. To solve this problem we have developed and validated a free software tool which automates the identification of PCR assays targeting specific genotypes in complex samples. ThermoPhyl uses user-defined target and non-target sequence databases to assess the phylogenetic sensitivity and specificity of thermodynamically optimised candidate assays derived from primer design software packages. ThermoPhyl takes its name from its central premise of testing Thermodynamically optimal assays for Phylogenetic specificity and sensitivity and can be used for two primer (traditional PCR) or two primers with an internal probe (e.g. TaqMan® qPCR) applications and potentially for oligonucleotide probes.Here we describe the use of ThermoPhyl for traditional PCR and qPCR assays. PCR assays selected using ThermoPhyl were validated using 454 pyrosequencing of a traditional specific PCR assay and with a set of four genotype-specific qPCR assays applied to estuarine sediment samples

Diversity Matters: Race, Gender and Ethnicity in Legal Education.

This Article presents more evidence of the inequality that persists in legal education for students. Based on a survey of University of Florida law students conducted in 2001, this study reaffirms the existence of differential experience and an inegalitarian culture in legal education. However, it also demonstrates the importance of diversity and the recognition by a significant majority of students of the value of race and gender pluralism. These competing findings provide a clear guide to the future direction of legal education

Influence of wet distillers grains diets on beef cattle fecal bacterial community structure

<p>Abstract</p> <p>Background</p> <p>The high demand for ethanol in the U.S. has generated large stocks of wet distillers grains (DG), a byproduct from the manufacture of ethanol from corn and sorghum grains. Little is known, however, about the potential influence of dietary DG on fecal microbial community structure. A better understanding of the microbial population in beef cattle feces could be an important monitoring tool to facilitate goals of improving nutrient management, increasing animal growth performance and decreasing odors and/or shedding of pathogens. Five diets consisting of a traditional diet fed to finishing beef cattle in the Southern High Plains of Texas-CON (steam-flaked corn control with 0% DG), and four concentrations of DG in the dietary dry matter; 10 C (10% corn-based DG), 5S (5% sorghum-based DG), 10S (10% sorghum DG), and 15S (15% sorghum DG) were fed to steers at the Texas Tech University Burnett Animal Center. Diets were essentially isonitrogenous with a formulated crude protein value of 13.5%.</p> <p>Results</p> <p>Fecal grab samples were obtained from 20 steers (n = 4 per diet) and the barcoded DNA pyrosequencing method was used to generate 127,530 16S operational taxonomic units (OTUs). A total of 24 phyla were observed, distributed amongst all beef cattle on all diets, revealing considerable animal to animal variation, however only six phyla (core set) were observed in all animals regardless of dietary treatment. The average abundance and range of abundance, respectively of the core phyla were as follows: Firmicutes (61%, 19 to 83%), Bacteroidetes (28%, 11 to 63%), Proteobacteria (3%, 0.34 to 17.5%), Tenericutes (0.15%, 0.0 to 0.35%), Nitrospirae (0.11%, 0.03 to 0.22%), and Fusobacteria (0.086%, 0.017 to 0.38%). Feeding DG-based diets resulted in significant shifts in the fecal microbial community structure compared with the traditional CON. Four low abundance phyla significantly responded to dietary treatments: Synergistetes (<it>p </it>= 0.01), WS3 (<it>p </it>= 0.054), Actinobacteria (<it>p </it>= 0.06), and Spirochaetes (<it>p </it>= 0.06).</p> <p>Conclusions</p> <p>This is, to our knowledge, the first study using this method to survey the fecal microbiome of beef cattle fed various concentrations of wet DG. Comparison of our results with other cattle DNA sequencing studies of beef and dairy cattle feces from a variety of geographical locations and different management practices identifies a core set of three phyla shared across all cattle. These three phyla, in order of relative abundance are; Firmicutes, Bacteroidetes, and Proteobacteria. The presence of large animal-to-animal variation in cattle microbiome was noted in our study as well as by others.</p

Biological expressions of early life trauma in the immune system of older adults

Background Poor immune function is associated with increased risk for a number of age-related diseases, however, little is known about the impact of early life trauma on immune function in late-life. Methods Using nationally representative data from the Health and Retirement Study (n = 5,823), we examined the association between experiencing parental/caregiver death or separation before age 16 and four indicators of immune function in late-life: C-reactive Protein (CRP), Interleukin-6 (IL-6), soluble Tumor Necrosis Factor (sTNFR), and Immunoglobulin G (IgG) response to cytomegalovirus (CMV). We also examined racial/ethnic differences. Findings Individuals that identified as racial/ethnic minorities were more likely to experience parental/caregiver loss and parental separation in early life compared to Non-Hispanic Whites, and had poorer immune function in late-life. We found consistent associations between experiencing parental/caregiver loss and separation and poor immune function measured by CMV IgG levels and IL-6 across all racial/ethnic subgroups. For example, among Non-Hispanic Blacks, those that experienced parental/caregiver death before age 16 had a 26% increase in CMV IgG antibodies in late-life (β = 1.26; 95% CI: 1.17, 1.34) compared to a 3% increase in CMV antibodies among Non-Hispanic Whites (β = 1.03; 95% CI: 0.99, 1.07) controlling for age, gender, and parental education. Interpretation Our results suggest a durable association between experiencing early life trauma and immune health in late-life, and that structural forces may shape the ways in which these relationships unfold over the life course

Income and Markers of Immunological Cellular Aging

Socioeconomic disadvantage may contribute to poor health through immune-related biological mechanisms. We examined the associations between socioeconomic status, as measured by annual household income, and T-cell markers of aging, including the ratios of CD4 and CD8 effector cells to naïve cells (E:N ratio) and the CD4:CD8 T-cell ratio. We hypothesized that participants with a lower income would have higher E:N ratios and lower CD4:CD8 ratios compared to participants with a higher income, and that these associations would be partially mediated by elevated cytomegalovirus (CMV) IgG antibody levels, a virus implicated in aging and clonal expansion of T-cells

Black Box Chimera Check (B2C2): a Windows-Based Software for Batch Depletion of Chimeras from Bacterial 16S rRNA Gene Datasets

The existing chimera detection programs are not specifically designed for "next generation" sequence data. Technologies like Roche 454 FLX and Titanium have been adapted over the past years especially with the introduction of bacterial tag-encoded FLX/Titanium amplicon pyrosequencing methodologies to produce over one million 250-600 bp 16S rRNA gene reads that need to be depleted of chimeras prior to downstream analysis. Meeting the needs of basic scientists who are venturing into high-throughput microbial diversity studies such as those based upon pyrosequencing and specifically providing a solution for Windows users, the B2C2 software is designed to be able to accept files containing large multi-FASTA formatted sequences and screen for possible chimeras in a high throughput fashion. The graphical user interface (GUI) is also able to batch process multiple files. When compared to popular chimera screening software the B2C2 performed as well or better while dramatically decreasing the amount of time required generating and screening results. Even average computer users are able to interact with the Windows .Net GUI-based application and define the stringency to which the analysis should be done. B2C2 may be downloaded from http://www.researchandtesting.com/B2C2

Untying a nanoscale knotted polymer structure to linear chains for efficient gene delivery in vitro and to the brain

The purpose of this study was to develop a platform transfection technology, for applications in the brain, which could transfect astrocytes without requiring cell specific functionalization and without the common cause of toxicity through high charge density. Here we show that a simple and scalable preparation technique can be used to produce a “knot” structured cationic polymer, where single growing chains can crosslink together via disulphide intramolecular crosslinks (internal cyclizations). This well-defined knot structure can thus “untie” under reducing conditions, showing a more favorable transfection profile for astrocytes compared to 25 kDa-PEI (48-fold), SuperFect® (39-fold) and Lipofectamine®2000 (18-fold) whilst maintaining neural cell viability at over 80% after four days of culture. The high transfection/lack of toxicity of this knot structured polymer in vitro, combined with its ability to mediate luciferase transgene expression in the adult rat brain, demonstrates its use as a platform transfection technology which should be investigated further for neurodegenerative disease therapies

First validation of high-resolution satellite-derived methane emissions from an active gas leak in the UK

Atmospheric methane (CH4) is the second-most-important anthropogenic greenhouse gas and has a 20-year global warming potential 82 times greater than carbon dioxide (CO2). Anthropogenic sources account for g1/4g% of global CH4 emissions, of which 20g% come from oil and gas exploration, production and distribution. High-resolution satellite-based imaging spectrometers are becoming important tools for detecting and monitoring CH4 point source emissions, aiding mitigation. However, validation of these satellite measurements, such as those from the commercial GHGSat satellite constellation, has so far not been documented for active leaks. Here we present the monitoring and quantification, by GHGSat's satellites, of the CH4 emissions from an active gas leak from a downstream natural gas distribution pipeline near Cheltenham, UK, in the spring and summer of 2023 and provide the first validation of the satellite-derived emission estimates using surface-based mobile greenhouse gas surveys. We also use a Lagrangian transport model, the UK Met Office's Numerical Atmospheric-dispersion Modelling Environment (NAME), to estimate the flux from both satellite-and ground-based observation methods and assess the leak's contribution to observed concentrations at a local tall tower site (30gkm away). We find GHGSat's emission estimates to be in broad agreement with those made from the in situ measurements. During the study period (March-June 2023) GHGSat's emission estimates are 236-1357gkggCH4gh-1, whereas the mobile surface measurements are 634-846gkggCH4gh-1. The large variability is likely down to variations in flow through the pipe and engineering works across the 11-week period. Modelled flux estimates in NAME are 181-1243gkggCH4gh-1, which are lower than the satellite-and mobile-survey-derived fluxes but are within the uncertainty. After detecting the leak in March 2023, the local utility company was contacted, and the leak was fixed by mid-June 2023. Our results demonstrate that GHGSat's observations can produce flux estimates that broadly agree with surface-based mobile measurements. Validating the accuracy of the information provided by targeted, high-resolution satellite monitoring shows how it can play an important role in identifying emission sources, including unplanned fugitive releases that are inherently challenging to identify, track, and estimate their impact and duration. Rapid, widespread access to such data to inform local action to address fugitive emission sources across the oil and gas supply chain could play a significant role in reducing anthropogenic contributions to climate change.</p