Approaches in molecular biology, particularly those that deal with high-throughput sequencing of entire microbial communities (the field of metagenomics), are rapidly advancing our understanding of ...the composition and functional content of microbial communities involved in climate change, environmental pollution, human health, biotechnology, etc. Metagenomics provides researchers with the most complete picture of the taxonomic (i.e., what organisms are there) and functional (i.e., what are those organisms doing) composition of natively sampled microbial communities, making it possible to perform investigations that include organisms that were previously intractable to laboratory-controlled culturing; currently, these constitute the vast majority of all microbes on the planet. All organisms contained in environmental samples are sequenced in a culture-independent manner, most often with 16S ribosomal amplicon methods to investigate the taxonomic or whole-genome shotgun-based methods to investigate the functional content of sampled communities. Metagenomics allows researchers to characterize the community composition and functional content of microbial communities, but it cannot show which functional processes are active; however, near parallel developments in transcriptomics promise a dramatic increase in our knowledge in this area as well. Since 2008, MG-RAST (Meyer et al., BMC Bioinformatics 9:386, 2008) has served as a public resource for annotation and analysis of metagenomic sequence data, providing a repository that currently houses more than 150,000 data sets (containing 60+ tera-base-pairs) with more than 23,000 publically available. MG-RAST, or the metagenomics RAST (rapid annotation using subsystems technology) server makes it possible for users to upload raw metagenomic sequence data in (preferably) fastq or fasta format. Assessments of sequence quality, annotation with respect to multiple reference databases, are performed automatically with minimal input from the user (see Subheading 4 at the end of this chapter for more details). Post-annotation analysis and visualization are also possible, directly through the web interface, or with tools like matR (metagenomic analysis tools for R, covered later in this chapter) that utilize the MG-RAST API ( http://api.metagenomics.anl.gov/api.html ) to easily download data from any stage in the MG-RAST processing pipeline. Over the years, MG-RAST has undergone substantial revisions to keep pace with the dramatic growth in the number, size, and types of sequence data that accompany constantly evolving developments in metagenomics and related -omic sciences (e.g., metatranscriptomics).
Critics of this simple approach claim that to detect lameness one must specifically and simultaneously observe for many other signs, including movement of the limbs during the swing phase of the ...stride, cadence of footfall, orientation and placement of the limbs during weight bearing, the apparent ease, comfort, or ability to move in certain gaits, or to transition from one gait to another and other, even more difficult-to-define, signs like head orientation and facial expression.25 I have also observed for most of these signs. In a study tracking the eye movement of veterinary students evaluating videos of lame horses, it was shown that experienced students who concentrated on head and pelvic movement scored significantly better at picking the correct lame limb than inexperienced students who looked at head, pelvis and limb movement and who named a greater number of visual lameness features that they thought were important to track.26 When veterinary trainees are told specifically what to observe, in language clear enough to propose a way to measure it, they can learn to be proficient. Assessment of mild hindlimb lameness during over ground locomotion using linear discriminant analysis of inertial sensor data. Disease-specific changes in equine ground reaction force data documented by use of principal component analysis.
MG-RAST (http://metagenomics.anl.gov) is an open-submission data portal for processing, analyzing, sharing and disseminating metagenomic datasets. The system currently hosts over 200,000 datasets and ...is continuously updated. The volume of submissions has increased 4-fold over the past 24 months, now averaging 4 terabasepairs per month. In addition to several new features, we report changes to the analysis workflow and the technologies used to scale the pipeline up to the required throughput levels. To show possible uses for the data from MG-RAST, we present several examples integrating data and analyses from MG-RAST into popular third-party analysis tools or sequence alignment tools.
This study has investigated the immediate effect of induced hindlimb length difference on hindlimb lameness measured as differences in minimum (Pmin) and maximum (Pmax) pelvic heights in 16 horses ...trotting in a straight line and lungeing on both hard and soft surfaces with body-mounted inertial sensors. Hindlimb length differences were induced by applying an Easyboot Glue-on shoe to one hindlimb. Changes in Pmin and Pmax with induced hindlimb length difference were assessed with a two-way repeated-measures ANOVA with trial (straight, lunge with inside limb elevation, lunge with outside limb elevation) and surface (hard, soft) as within-subject factors. Change in Pmin, indicating an impact-type lameness, in the hind limb with the elevation, was significant in both the straight line and while lunging on both hard and soft surfaces. Change in Pmax, indicating pushoff-type lameness, in the opposite, non-elevated hind limb, was significant when trotting in a straight line but not while lunging.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The fruitfly, Drosophila melanogaster, exhibits many of the cardinal features of sleep, yet little is known about the neural circuits governing its sleep. Here we have performed a screen of GAL4 ...lines expressing a temperature-sensitive synaptic blocker shibirets1 in a range of discrete neural circuits, and assayed the amount of sleep at different temperatures. We identified three short-sleep lines at the restrictive temperature with shared expression in the mushroom bodies, a neural locus central to learning and memory. Chemical ablation of the mushroom bodies also resulted in reduced sleep. These studies highlight a central role for the mushroom bodies in sleep regulation.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
We provide a novel method, DRISEE (duplicate read inferred sequencing error estimation), to assess sequencing quality (alternatively referred to as "noise" or "error") within and/or between ...sequencing samples. DRISEE provides positional error estimates that can be used to inform read trimming within a sample. It also provides global (whole sample) error estimates that can be used to identify samples with high or varying levels of sequencing error that may confound downstream analyses, particularly in the case of studies that utilize data from multiple sequencing samples. For shotgun metagenomic data, we believe that DRISEE provides estimates of sequencing error that are more accurate and less constrained by technical limitations than existing methods that rely on reference genomes or the use of scores (e.g. Phred). Here, DRISEE is applied to (non amplicon) data sets from both the 454 and Illumina platforms. The DRISEE error estimate is obtained by analyzing sets of artifactual duplicate reads (ADRs), a known by-product of both sequencing platforms. We present DRISEE as an open-source, platform-independent method to assess sequencing error in shotgun metagenomic data, and utilize it to discover previously uncharacterized error in de novo sequence data from the 454 and Illumina sequencing platforms.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The soil ecosystem is critical for human health, affecting aspects of the environment from key agricultural and edaphic parameters to critical influence on climate change. Soil has more unknown ...biodiversity than any other ecosystem. We have applied diverse DNA extraction methods coupled with high throughput pyrosequencing to explore 4.88 × 10(9) bp of metagenomic sequence data from the longest continually studied soil environment (Park Grass experiment at Rothamsted Research in the UK). Results emphasize important DNA extraction biases and unexpectedly low seasonal and vertical soil metagenomic functional class variations. Clustering-based subsystems and carbohydrate metabolism had the largest quantity of annotated reads assigned although <50% of reads were assigned at an E value cutoff of 10(-5). In addition, with the more detailed subsystems, cAMP signaling in bacteria (3.24±0.27% of the annotated reads) and the Ton and Tol transport systems (1.69±0.11%) were relatively highly represented. The most highly represented genome from the database was that for a Bradyrhizobium species. The metagenomic variance created by integrating natural and methodological fluctuations represents a global picture of the Rothamsted soil metagenome that can be used for specific questions and future inter-environmental metagenomic comparisons. However, only 1% of annotated sequences correspond to already sequenced genomes at 96% similarity and E values of <10(-5), thus, considerable genomic reconstructions efforts still have to be performed.
To compare results for initial body-mounted inertial sensor (BMIS) measurement of lameness in equids trotting in a straight line with definitive findings after full lameness evaluation.
1,224 equids.
...Lameness measured with BMIS equipment while trotting in a straight line was classified into categories of none, forelimb only, hind limb only, and 8 patterns of combined forelimb and hind limb lameness (CFHL). Definitive findings after full lameness evaluation were established in most horses and classified into types (no lameness, forelimb- or hind limb-only lameness, CFHL, or lameness not localized to the limbs). Observed proportions of lameness type in equids with definitive findings for each initial BMIS-assessed category were compared with hypothetical expected proportions through χ
goodness-of-fit analysis.
The most common initial BMIS-assessed lameness category was CFHL (693/1,224 56.6%), but this was the least common definitive finding (94/ 862 10.9%). The observed frequency of no lameness after full lameness evaluation was greater than expected only when initial BMIS measurements indicated no lameness. The observed frequency of forelimb-only lameness was greater than expected when initially measured as forelimb-only lameness and for CFHL categories consistent with the diagonal movement principle of compensatory lameness. Observed frequency of hind limb-only lameness was greater than expected when initially measured as hind limb-only lameness and for CFHL categories consistent with the sagittal movement principle of compensatory lameness. Equids initially assessed as having no lameness had the highest (103/112 92%) and those assessed as CFHL pattern 7 (forelimb with contralateral hind limb impact-only lameness) had the lowest (36/66 55%) rates of definitive findings.
In equids, results of initial straight-line trotting evaluations with a BMIS system did not necessarily match definitive findings but may be useful in planning the remaining lameness evaluation.
Soluble epoxide hydrolase (sEH) inhibitors are novel anti-inflammatory and analgesic agents that could improve pain management in horses. The objective of the present study was to evaluate the ...anti-nociceptive effect of a single-dose intravenous administration of the sEH inhibitor trans-4-{4-3-(4-trifluro-methoxy-phenyl)-ureido-cyclohexyloxy}-benzoic acid (t-TUCB) using an adjustable heart bar shoe (a-HBS) model of lameness. We hypothesized that t-TUCB would improve objective and subjective lameness measures compared to the control.
Reversible lameness was induced in 8 horses for 24 h using an a-HBS in a randomized, crossover design. A vehicle-control placebo (DMSO) or t-TUCB (1 mg/kg) was intravenously administered at time 0 following a baseline induced lameness evaluation. Blood was collected and lameness was objectively measured with an inertial sensor system at 0-, 1-, 3-, 6-, 12-, and 24-h time points. Front-facing videos were obtained at each time point for subjective evaluation by three blinded evaluators using a visual analog scale (VAS).
Treatment with t-TUCB significantly decreased (i.e. improved) lameness compared to placebo at 1-h and compared to baseline at 1-, 3-, and 6-h following administration. Lameness significantly increased (i.e. worsened) from baseline in placebo-treated horses 12 h after administration. All horses returned to baseline soundness within 24 h of reversing lameness.
Treatment with single-dose IV administration of t-TUCB improved lameness induced by the a-HSB, suggesting that t-TUCB has anti-nociceptive effects in horses.
The soluble epoxide hydrolase inhibitor, t-TUCB, is a promising novel analgesic for horses.
•Soluble epoxide hydrolase inhibitors attenuate nociceptive pain in horses.•No adverse reactions were observed following intravenous administration of t-TUCB.•Plasma concentrations correlated with clinical effect of t-TUCB.
Preterm infants represent a unique patient population that is born functionally immature and must accomplish development under the influence of a hospital environment. Neonatal necrotizing ...enterocolitis (NEC) is an inflammatory intestinal disorder affecting preterm infants. The purpose of this study was to evaluate the progression of intestinal microbiota community development between preterm infants who remained healthy compared to preterm infants who developed NEC.
Weekly fecal samples from ten preterm infants, five with NEC and five matched healthy controls were obtained. Bacterial DNA from individual fecal samples was subjected to sequencing of 16S rRNA-based inventories using the 454 GS-FLX platform. Fecal samples from control infants demonstrated a temporal pattern in their microbiota, which converged toward that of a healthy full term breast-fed infant. Microbiota development in NEC patients diverged from controls beginning three weeks prior to diagnosis. Shotgun metagenomic sequencing was performed to identify functional differences in the respective microbiota of fecal samples from a set of twins in which one twin developed NEC and one did not. The majority of the differentially abundant genes in the NEC patient were associated with carbohydrate metabolism and mapped to members of the family Enterobacteriaceae. This may indicate an adaptation of the community to an altered profile of substrate availability for specific members as a first step towards the development of NEC. We propose that the microbial communities as a whole may metabolize milk differently, resulting in differential substrate availability for specific microbial groups. Additional differentially represented gene sets of interest were related to antibiotic resistance and vitamin biosynthesis.
Our results suggest that there is a temporal component to microbiome development in healthy preterm infants. Thus, bacteriotherapy for the treatment or prevention of NEC must consider this temporal component of the microbial community in addition to its taxonomic composition and functional content.
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