Selection of reference genes for gene expression analysis by real-time qPCR in avian cells infected with infectious bronchitis virus.

Infectious bronchitis virus (IBV) causes infectious bronchitis in poultry, a respiratory disease that is a source of major economic loss to the poultry industry. Detection and the study of the molecular pathogenesis of the virus often involve the use of real-time quantitative PCR assays (qPCR). To account for error within the experiments, the levels of target gene transcription are normalized to that of suitable reference genes. Despite publication of the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines in 2009, single un-tested reference genes are often used for normalization of qPCR assays in avian research studies. Here, we use the geNorm algorithm to identify suitable reference genes in different avian cell types during infection with apathogenic and pathogenic strains of IBV. We discuss the importance of selecting an appropriate experimental sample subset for geNorm analysis, and show the effect that this selection can have on resultant reference gene selection. The effects of inappropriate normalization on the transcription pattern of a cellular signalling gene, AKT1, and the interferon-inducible, MX1, were studied. We identify the possibility of the misinterpretation of qPCR data when an inappropriate normalization strategy is employed. This is most notable when measuring the transcription of AKT1, where changes are minimal during infection

Once Daily Valacyclovir for Reducing Viral Shedding in Subjects Newly Diagnosed with Genital Herpes

Objective. Genital herpes (GH) recurrences and viral shedding are more frequent in the first year after initial HSV-2 infection. The objective of this study was to provide the first evaluation of valacyclovir 1 g once daily compared to placebo in reducing viral shedding in subjects newly diagnosed with GH. Methods. 70 subjects were randomized to receive valacyclovir 1 g daily or placebo in a crossover design for 60 days with a 7-day washout period. A daily swab of the genital/anal-rectal area was self-collected for HSV-2 detection by PCR. Subjects attended the clinic for routine study visits and GH recurrence visits. Treatment differences were assessed using a nonparametric crossover analysis. Results. 52 subjects had at least one PCR measurement in both treatment periods and comprised the primary efficacy population. Valacyclovir significantly reduced HSV-2 shedding during all days compared to placebo (mean 2.9% versus 13.5% of all days (P < .01), a 78% reduction). Valacyclovir significantly reduced subclinical HSV-2 shedding during all days compared to placebo (mean 2.4% versus 11.0% of all days (P < .01), a 78% reduction). However, 79% of subjects had no GH recurrences while receiving valacyclovir compared to 52% of subjects receiving placebo (P < .01). Conclusion. In this study, the frequency of total and subclinical HSV-2 shedding was greater than reported in earlier studies involving subjects with a history of symptomatic genital recurrences. Our study is the first to demonstrate a significant reduction in viral shedding with valacyclovir 1 g daily compared to placebo in a population of subjects newly diagnosed with HSV-2 infection

A maximum likelihood QTL analysis reveals common genome regions controlling resistance to Salmonella colonization and carrier-state

<p>Abstract</p> <p>Background</p> <p>The serovars Enteritidis and Typhimurium of the Gram-negative bacterium <it>Salmonella enterica</it> are significant causes of human food poisoning. Fowl carrying these bacteria often show no clinical disease, with detection only established post-mortem. Increased resistance to the carrier state in commercial poultry could be a way to improve food safety by reducing the spread of these bacteria in poultry flocks. Previous studies identified QTLs for both resistance to carrier state and resistance to <it>Salmonella</it> colonization in the same White Leghorn inbred lines. Until now, none of the QTLs identified was common to the two types of resistance. All these analyses were performed using the F2 inbred or backcross option of the QTLExpress software based on linear regression. In the present study, QTL analysis was achieved using Maximum Likelihood with QTLMap software, in order to test the effect of the QTL analysis method on QTL detection. We analyzed the same phenotypic and genotypic data as those used in previous studies, which were collected on 378 animals genotyped with 480 genome-wide SNP markers. To enrich these data, we added eleven SNP markers located within QTLs controlling resistance to colonization and we looked for potential candidate genes co-localizing with QTLs.</p> <p>Results</p> <p>In our case the QTL analysis method had an important impact on QTL detection. We were able to identify new genomic regions controlling resistance to carrier-state, in particular by testing the existence of two segregating QTLs. But some of the previously identified QTLs were not confirmed. Interestingly, two QTLs were detected on chromosomes 2 and 3, close to the locations of the major QTLs controlling resistance to colonization and to candidate genes involved in the immune response identified in other, independent studies.</p> <p>Conclusions</p> <p>Due to the lack of stability of the QTLs detected, we suggest that interesting regions for further studies are those that were identified in several independent studies, which is the case of the QTL regions on chromosomes 2 and 3, involved in resistance to both <it>Salmonella</it> colonization and carrier state. These observations provide evidence of common genes controlling <it>S.</it> Typhimurium colonization and <it>S</it>. Enteritidis carrier-state in chickens.</p

Selective miRNA disruption in T reg cells leads to uncontrolled autoimmunity

A new regulatory T (T reg) cell–specific, FoxP3-GFP-hCre bacterial artificial chromosome transgenic mouse was crossed to a conditional Dicer knockout (KO) mouse strain to analyze the role of microRNAs (miRNAs) in the development and function of T reg cells. Although thymic T reg cells developed normally in this setting, the cells showed evidence of altered differentiation and dysfunction in the periphery. Dicer-deficient T reg lineage cells failed to remain stable, as a subset of cells down-regulated the T reg cell–specific transcription factor FoxP3, whereas the majority expressed altered levels of multiple genes and proteins (including Neuropilin 1, glucocorticoid-induced tumor necrosis factor receptor, and cytotoxic T lymphocyte antigen 4) associated with the T reg cell fingerprint. In fact, a significant percentage of the T reg lineage cells took on a T helper cell memory phenotype including increased levels of CD127, interleukin 4, and interferon γ. Importantly, Dicer-deficient T reg cells lost suppression activity in vivo; the mice rapidly developed fatal systemic autoimmune disease resembling the FoxP3 KO phenotype. These results support a central role for miRNAs in maintaining the stability of differentiated T reg cell function in vivo and homeostasis of the adaptive immune system

Case Report: The Effect of Automated Manual Lymphatic Drainage Therapy on Lymphatic Contractility in 4 Distinct Cases

INTRODUCTION: Automated manual lymphatic drainage therapy (AMLDT) is available for home use in the form of a pneumatic mat of 16 pressurized air channels that inflate and deflate to mimic the stretch and release action of manual lymphatic drainage therapy. Four cases (a patient with complex regional pain syndrome and lymphedema, a healthy patient, a breast cancer survivor with chronic pain, and a patient with a history of abdominal surgery) underwent near-infrared fluorescence lymphatic imaging (NIRFLI) with AMLDT to evaluate the effect of AMLDT on lymphatic pumping and pain. METHODS: Each patient received 32-36 injections of 25 μg indocyanine green (ICG) on the anterior and posterior sides of their body and underwent 1 h of NIRFLI to assess the drainage of ICG laden lymph toward regional nodal basins at baseline. Each patient lay supine on the mat for 1 h of AMLDT with NIRFLI to assess lymphatic flow during treatment. A final NIFRFLI assessment was done 30-60 min posttreatment with the patient in the supine and prone position. Patients reported baseline and posttreatment pain using the Visual Analogue Scale. An imager analyzed NIRFLI images using ImageJ (US National Institutes of Health). Using time stamps of the first and last images to determine time lapsed and the number of pulses observed in a timeframe, pulsing frequency (pulses/min) was obtained to assess lymphatic function. RESULTS: All 4 cases completed the NIRFLI and AMLDT without complications; all 3 patients with baseline pain reported reduced pain posttreatment. AMLDT appeared to alter lymphatic contractility, with both increased and decreased pulsing frequencies observed, including in nonaffected limbs. Pulsing frequencies were very heterogeneous among patients and varied within anatomic regions of the same patient. DISCUSSION: This proof-of-concept study suggests that AMLDT may impact lymphatic contractility. Further research on its effect on lymphatic function is warranted

Large scale variation in DNA copy number in chicken breeds

Background Detecting genetic variation is a critical step in elucidating the molecular mechanisms underlying phenotypic diversity. Until recently, such detection has mostly focused on single nucleotide polymorphisms (SNPs) because of the ease in screening complete genomes. Another type of variant, copy number variation (CNV), is emerging as a significant contributor to phenotypic variation in many species. Here we describe a genome-wide CNV study using array comparative genomic hybridization (aCGH) in a wide variety of chicken breeds. Results We identified 3,154 CNVs, grouped into 1,556 CNV regions (CNVRs). Thirty percent of the CNVs were detected in at least 2 individuals. The average size of the CNVs detected was 46.3 kb with the largest CNV, located on GGAZ, being 4.3 Mb. Approximately 75% of the CNVs are copy number losses relatively to the Red Jungle Fowl reference genome. The genome coverage of CNVRs in this study is 60 Mb, which represents almost 5.4% of the chicken genome. In particular large gene families such as the keratin gene family and the MHC show extensive CNV. Conclusions A relative large group of the CNVs are line-specific, several of which were previously shown to be related to the causative mutation for a number of phenotypic variants. The chance that inter-specific CNVs fall into CNVRs detected in chicken is related to the evolutionary distance between the species. Our results provide a valuable resource for the study of genetic and phenotypic variation in this phenotypically diverse species

A prescription for the Epley maneuver: www.youtube.com?

OBJECTIVES: Video-sharing Web sites are being used for information about common conditions including dizziness. The Epley maneuver (EM) is a simple and effective treatment for benign paroxysmal positional vertigo (BPPV) of the posterior canal. However, the maneuver is underused in routine care. In this study, we aimed to describe and analyze the available information about the EM on youtube.com. METHODS: A YouTube search was performed on August 31, 2011, for videos that demonstrated the entire EM. Detailed data were abstracted from each video and corresponding Web site. Videos were rated on the accuracy of the maneuver by 2 authors, with differences resolved by adjudication. Comments posted by viewers were assessed for themes regarding video use. RESULTS: Of the 3,319 videos identified, 33 demonstrated the EM. The total number of hits for all videos was 2,755,607. The video with the most hits (802,471) was produced by the American Academy of Neurology. Five of the videos accounted for 85% of all the hits. The maneuver demonstration was rated as accurate in 64% (21) of the videos. Themes derived from the 424 posted comments included patients self-treating with the maneuver after reviewing the videos, and providers using the videos as a prescribed treatment or for educational purposes. CONCLUSION: Accurate video demonstration of the Epley maneuver is available and widely viewed on YouTube. Video-sharing media may be an important way to disseminate effective interventions such as the EM. The impact of video Web sites on outcomes and costs of care is not known and warrants future study

Novel IL10 gene family associations with systemic juvenile idiopathic arthritis

Juvenile idiopathic arthritis (JIA) is the most common cause of chronic childhood disability and encompasses a number of disease subgroups. In this study we have focused on systemic JIA (sJIA), which accounts for approximately 11% of UK JIA cases. This study reports the investigation of three members of the IL10 gene family as candidate susceptibility loci in children with sJIA. DNA from 473 unaffected controls and 172 patients with sJIA was genotyped for a single nucleotide polymorphism (SNP) in IL19 and IL20 and two SNPs in IL10. We examined evidence for association of the four SNPs by single marker and haplotype analysis. Significant differences in allele frequency were observed between cases and controls, for both IL10-1082 (p = 0.031) and IL20-468 (p = 0.028). Furthermore, examination of the haplotypes of IL10-1082 and IL20-468 revealed greater evidence for association (global p = 0.0006). This study demonstrates a significant increased prevalence of the low expressing IL10-1082 genotype in patients with sJIA. In addition, we show a separate association with an IL20 polymorphism, and the IL10-1082A/IL20-468T haplotype. The two marker 'A-T' haplotype confers an odds ratio of 2.24 for sJIA. This positive association suggests an important role for these cytokines in sJIA pathogenesis

A four-dimensional X-ray tomographic microscopy study of bubble growth in basaltic foam

Understanding the influence of bubble foams on magma permeability and strength is critical to investigations of volcanic eruption mechanisms. Increasing foam porosity decreases strength, enhancing the probability of an eruption. However, higher porosities lead to larger permeabilities, which can lessen the eruption hazard. Here we measure bubble size and wall thickness distributions, as well as connectivity, and calculate permeabilities and tensile strengths of basaltic foams imaged by synchrotron X-ray tomographic microscopy during bubble growth in hydrated basaltic melts. Rapid vesiculation produces porous foams whose fragmentation thresholds are only 5-6 MPa and whose permeabilities increase from approximately 1x10(-10) to 1x10(-9) m(2) between 10 and 14 s despite decreasing connectivity between bubbles. These results indicate that basaltic magmas are most susceptible to failure immediately upon vesiculation and at later times, perhaps only 10's of seconds later, permeability increases may lessen the hazard of explosive, basaltic, Plinian eruptions

Practice Guideline: Cervical and Ocular Vestibular Evokedmyogenic Potential Testing: Report of the Guideline Development Dissemination and Implementation Subcommittee of the American Academy of Neurology

Objective: To systematically review the evidence and make recommendations with regard to diagnostic utility of cervical and ocular vestibular evoked myogenic potentials (cVEMP and oVEMP, respectively). Four questions were asked: Does cVEMP accurately identify superior canal dehiscence syndrome (SCDS)? Does oVEMP accurately identify SCDS? For suspected vestibular symptoms, does cVEMP/oVEMP accurately identify vestibular dysfunction related to the saccule/ utricle? For vestibular symptoms, does cVEMP/oVEMP accurately and substantively aid diagnosis of any specific vestibular disorder besides SCDS? Methods: The guideline panel identified and classified relevant published studies (January 1980- December 2016) according to the 2004 American Academy of Neurology process. Results and Recommendations: Level C positive: Clinicians may use cVEMP stimulus threshold values to distinguish SCDS from controls (2 Class III studies) (sensitivity 86%-91%, specificity 90%-96%). Corrected cVEMP amplitude may be used to distinguish SCDS from controls (2 Class III studies) (sensitivity 100%, specificity 93%). Clinicians may use oVEMP amplitude to distinguish SCDS from normal controls (3 Class III studies) (sensitivity 77%-100%, specificity 98%-100%). oVEMP threshold may be used to aid in distinguishing SCDS from controls (3 Class III studies) (sensitivity 70%-100%, specificity 77%-100%). Level U: Evidence is insufficient to determine whether cVEMP and oVEMP can accurately identify vestibular function specifically related to the saccule/utricle, or whether cVEMP or oVEMP is useful in diagnosing vestibular neuritis or M