Evaluating whole transcriptome amplification for gene profiling experiments using RNA-Seq

BACKGROUND: RNA-Seq has enabled high-throughput gene expression profiling to provide insight into the functional link between genotype and phenotype. Low quantities of starting RNA can be a severe hindrance for studies that aim to utilize RNA-Seq. To mitigate this bottleneck, whole transcriptome amplification (WTA) technologies have been developed to generate sufficient sequencing targets from minute amounts of RNA. Successful WTA requires accurate replication of transcript abundance without the loss or distortion of specific mRNAs. Here, we test the efficacy of NuGEN’s Ovation RNA-Seq V2 system, which uses linear isothermal amplification with a unique chimeric primer for amplification, using white adipose tissue from standard laboratory rats (Rattus norvegicus). Our goal was to investigate potential biological artifacts introduced through WTA approaches by establishing comparisons between matched raw and amplified RNA libraries derived from biological replicates. RESULTS: We found that 93% of expressed genes were identical between all unamplified versus matched amplified comparisons, also finding that gene density is similar across all comparisons. Our sequencing experiment and downstream bioinformatic analyses using the Tuxedo analysis pipeline resulted in the assembly of 25,543 high-quality transcripts. Libraries constructed from raw RNA and WTA samples averaged 15,298 and 15,253 expressed genes, respectively. Although significant differentially expressed genes (P < 0.05) were identified in all matched samples, each of these represents less than 0.15% of all shared genes for each comparison. CONCLUSIONS: Transcriptome amplification is efficient at maintaining relative transcript frequencies with no significant bias when using this NuGEN linear isothermal amplification kit under ideal laboratory conditions as presented in this study. This methodology has broad applications, from clinical and diagnostic, to field-based studies when sample acquisition, or sample preservation, methods prove challenging. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12896-015-0155-7) contains supplementary material, which is available to authorized users

Infectious Complications in Obese Patients Following Trauma

Background Obesity is a public health concern in the United States due to its increasing prevalence, especially in younger age groups. Trauma is the most common cause of death for people under aged 40 y. The purpose of this study is to determine the association between obesity and specific infectious complications after traumatic injury. Materials and methods A retrospective analysis was conducted using data from the 2012 National Trauma Data Bank. The National Trauma Data Bank defined obesity as having a body mass index of 30 or greater. Descriptive statistics were calculated and stratified by obesity status. A hierarchical regression model was used to determine the odds of experiencing an infectious complication in patients with obesity while controlling for age, gender, diabetes, number of comorbidities, injury severity, injury mechanism, head injury, and surgical procedure. Results Patients with a body mass index of 30 or greater compared with nonobese patients had increased odds of having an infectious complication (Odds Ratio, 1.59; 1.49-1.69). In addition to obesity, injury severity score greater than 29, age 40 y or older, diabetes, comorbid conditions, and having a surgical procedure were also predictive of an infectious complication. Conclusions Our results indicate that trauma patients with obesity are nearly 60% more likely to develop an infectious complication in the hospital. Infection prevention and control measures should be implemented soon after hospital arrival for patients with obesity, particularly those with operative trauma

The Single-Nucleotide Resolution Transcriptome of Pseudomonas aeruginosa Grown in Body Temperature

One of the hallmarks of opportunistic pathogens is their ability to adjust and respond to a wide range of environmental and host-associated conditions. The human pathogen Pseudomonas aeruginosa has an ability to thrive in a variety of hosts and cause a range of acute and chronic infections in individuals with impaired host defenses or cystic fibrosis. Here we report an in-depth transcriptional profiling of this organism when grown at host-related temperatures. Using RNA-seq of samples from P. aeruginosa grown at 28°C and 37°C we detected genes preferentially expressed at the body temperature of mammalian hosts, suggesting that they play a role during infection. These temperature-induced genes included the type III secretion system (T3SS) genes and effectors, as well as the genes responsible for phenazines biosynthesis. Using genome-wide transcription start site (TSS) mapping by RNA-seq we were able to accurately define the promoters and cis-acting RNA elements of many genes, and uncovered new genes and previously unrecognized non-coding RNAs directly controlled by the LasR quorum sensing regulator. Overall we identified 165 small RNAs and over 380 cis-antisense RNAs, some of which predicted to perform regulatory functions, and found that non-coding RNAs are preferentially localized in pathogenicity islands and horizontally transferred regions. Our work identifies regulatory features of P. aeruginosa genes whose products play a role in environmental adaption during infection and provides a reference transcriptional landscape for this pathogen

An inducible CiliaGFP mouse model for in vivo visualization and analysis of cilia in live tissue

BACKGROUND: Cilia are found on nearly every cell type in the mammalian body, and have been historically classified as either motile or immotile. Motile cilia are important for fluid and cellular movement; however, the roles of non-motile or primary cilia in most tissues remain unknown. Several genetic syndromes, called the ciliopathies, are associated with defects in cilia structure or function and have a wide range of clinical presentations. Much of what we know about the formation and maintenance of cilia comes from model systems like C. elegans and Chalmydomonas. Studies of mammalian cilia in live tissues have been hampered by difficulty visualizing them. RESULTS: To facilitate analyses of mammalian cilia function we generated an inducible Cilia(GFP) mouse by targeting mouse cDNA encoding a cilia-localized protein somatostatin receptor 3 fused to GFP (Sstr3::GFP) into the ROSA26 locus. In this system, Sstr3::GFP is expressed from the ubiquitous ROSA26 promoter after Cre mediated deletion of an upstream Neo cassette flanked by lox P sites. Fluorescent cilia labeling was observed in a variety of live tissues and after fixation. Both cell-type specific and temporally regulated cilia labeling were obtained using multiple Cre lines. The analysis of renal cilia in anesthetized live mice demonstrates that cilia commonly lay nearly parallel to the apical surface of the tubule. In contrast, in more deeply anesthetized mice the cilia display a synchronized, repetitive oscillation that ceases upon death, suggesting a relationship to heart beat, blood pressure or glomerular filtration. CONCLUSIONS: The ability to visualize cilia in live samples within the Cilia(GFP) mouse will greatly aid studies of ciliary function. This mouse will be useful for in vivo genetic and pharmacological screens to assess pathways regulating cilia motility, signaling, assembly, trafficking, resorption and length control and to study cilia regulated physiology in relation to ciliopathy phenotypes

Phylogenetic Signal Variation in the Genomes of Medicago (Fabaceae)

Genome-scale data offer the opportunity to clarify phylogenetic relationships that are difficult to resolve with few loci, but they can also identify genomic regions with evolutionary history distinct from that of the species history. We collected whole-genome sequence data from 29 taxa in the legume genus Medicago, then aligned these sequences to the Medicago truncatula reference genome to confidently identify 87 596 variable homologous sites. We used this data set to estimate phylogenetic relationships among Medicago species, to investigate the number of sites needed to provide robust phylogenetic estimates and to identify specific genomic regions supporting topologies in conflict with the genome-wide phylogeny. Our full genomic data set resolves relationships within the genus that were previously intractable. Subsampling the data reveals considerable variation in phylogenetic signal and power in smaller subsets of the data. Even when sampling 5000 sites, no random sample of the data supports a topology identical to that of the genome-wide phylogeny. Phylogenetic relationships estimated from 500-site sliding windows revealed genome regions supporting several alternative species relationships among recently diverged taxa, consistent with the expected effects of deep coalescence or introgression in the recent history of Medicago. [Medicago; phylogenomics; whole-genome resequencing.

“A very orderly retreat”: Democratic transition in East Germany, 1989-90

East Germany's 1989-90 democratisation is among the best known of East European transitions, but does not lend itself to comparative analysis, due to the singular way in which political reform and democratic consolidation were subsumed by Germany's unification process. Yet aspects of East Germany's democratisation have proved amenable to comparative approaches. This article reviews the comparative literature that refers to East Germany, and finds a schism between those who designate East Germany's transition “regime collapse” and others who contend that it exemplifies “transition through extrication”. It inquires into the merits of each position and finds in favour of the latter. Drawing on primary and secondary literature, as well as archival and interview sources, it portrays a communist elite that was, to a large extent, prepared to adapt to changing circumstances and capable of learning from “reference states” such as Poland. Although East Germany was the Soviet state in which the positions of existing elites were most threatened by democratic transition, here too a surprising number succeeded in maintaining their position while filing across the bridge to market society. A concluding section outlines the alchemy through which their bureaucratic power was transmuted into property and influence in the “new Germany”

Multi-Antenna Vision-and-Inertial-Aided CDGNSS for Micro Aerial Vehicle Pose Estimation

A system is presented for multi-antenna carrier phase differential GNSS (CDGNSS)-based pose (position and orientation) estimation aided by monocular visual measurements and a smartphone-grade inertial sensor. The system is designed for micro aerial vehicles, but can be applied generally for low-cost, lightweight, high-accuracy, geo-referenced pose estimation. Visual and inertial measurements enable robust operation despite GNSS degradation by constraining uncertainty in the dynamics propagation, which improves fixed-integer CDGNSS availability and reliability in areas with limited sky visibility. No prior work has demonstrated an increased CDGNSS integer fixing rate when incorporating visual measurements with smartphone-grade inertial sensing. A central pose estimation filter receives measurements from separate CDGNSS position and attitude estimators, visual feature measurements based on the ROVIO measurement model, and inertial measurements. The filter's pose estimates are fed back as a prior for CDGNSS integer fixing. A performance analysis under both simulated and real-world GNSS degradation shows that visual measurements greatly increase the availability and accuracy of low-cost inertial-aided CDGNSS pose estimation.Aerospace Engineering and Engineering Mechanic

Sanctions, partner recognition, and variation in mutualism

Mutualistic interactions can be stabilized against invasion by noncooperative individuals by putting such "cheaters" at a selective disadvantage. Selection against cheaters should eliminate genetic variation in partner quality—yet such variation is often found in natural populations. One explanation for this paradox is that mutualism outcomes are determined not only by responses to partner performance but also by partner signals. Here, we build a model of coevolution in a symbiotic mutualism, in which hosts’ ability to sanction noncooperative symbionts and recognition of symbiont signals are determined by separate loci, as are symbionts’ cooperation and expression of signals. In the model, variation persists without destabilizing the interaction, in part because coevolution of symbiont signals and host recognition is altered by the coevolution of sanctions and cooperation, and vice versa. Individual-based simulations incorporating population structure strongly corroborate these results. The dual systems of sanctions and partner recognition converge toward conditions similar to some economic models of mutualistic symbiosis, in which hosts offering the right incentives to potential symbionts can initiate symbiosis without screening for partner quality. These results predict that mutualists can maintain variation in recognition of partner signals or in the ability to sanction noncooperators without destabilizing mutualism, and they reinforce the notion that studies of mutualism should consider communication between partners as well as the exchange of benefits.The American Naturalist 190(4), 491-505. (2017)0003-014