Half Of Sequence Research Articles

MiR-146a wild-type 3' sequence identity is dispensable for proper innate immune function in vivo.

The prevailing model of microRNA function is that the "seed region" (nt 2-8) is sufficient to mediate target recognition and repression. However, numerous recent studies have challenged this model, either by demonstrating extensive 3' pairing between physically defined miRNA-mRNA pairs or by showing in Caenorhabditis elegans that disrupted 3' pairing can result in impaired function in vivo. To test the importance of miRNA 3' pairing in a mammalian system in vivo, we engineered a mutant murine mir-146a allele in which the 5' half of the mature microRNA retains its wild-type sequence, but the 3' half's sequence has been altered to robustly disrupt predicted pairing to this latter region. Mice homozygous or hemizygous for this mutant allele are phenotypically indistinguishable from wild-type controls and do not recapitulate any of the immunopathology previously described for mir-146a-null mice. Our results indicate that 3' pairing is dispensable for the established myeloid function of this key mammalian microRNA.

Sequence analysis and identification of new isoform of EP4 receptors in different atlantic salmon tissues (Salmo salar L.) and its role in PGE2 induced immunomodulation in vitro.

PGE2 plays an important role in a broad spectrum of physiological and pathological processes mediated through a membrane-bound G protein-coupled receptor (GPCR) called EP receptor. In mammals, four subtypes of EP receptor (EP 1-4) are identified and each of them functions through different signal transduction pathways. Orthologous EP receptors have also been identified in other non-mammalian species, such as chicken and zebrafish. EP4 is the only identified PGE2 receptor to date in Atlantic salmon but its tissue distribution and function have not been studied in any detail. In this study, we first sequenced EP4 receptor in different tissues and found that the presence of the 3nt deletion in the 5’ untranslated region was accompanied by silent mutation at nt 668. While attempting to amplify the same sequence in TO cells (an Atlantic salmon macrophage-like cell line), we failed to obtain the full-length product. Further investigation revealed different isoform of EP4 receptor in TO cells and we subsequently documented its presence in different Atlantic salmon tissues. These two isoforms of EP4 receptor share high homology in their first half of sequence but differ in the second half part with several deletion segments though the final length of coding sequence is the same for two isoforms. We further studied the immunomodulation effect of PGE2 in TO cells and found that PGE2 inhibited the induction of CXCL-10, CCL-4, IL-8 and IL-1β genes expression in a time dependent manner and without cAMP upregulation.

Successional changes in the chicken cecal microbiome during 42 days of growth are independent of organic acid feed additives.

BackgroundPoultry remains a major source of foodborne bacterial infections. A variety of additives with presumed anti-microbial and/or growth-promoting effects are commonly added to poultry feed during commercial grow-out, yet the effects of these additives on the gastrointestinal microbial community (the GI microbiome) as the bird matures remain largely unknown. Here we compared temporal changes in the cecal microbiome to the effects of formic acid, propionic acid, and medium-chain fatty acids (MCFA) added to feed and/or drinking water.ResultsCecal bacterial communities at day of hatch (n = 5 birds), 7d (n = 32), 21d (n = 27), and 42d (n = 36) post-hatch were surveyed using direct 454 sequencing of 16S rRNA gene amplicons from each bird in combination with cultivation-based recovery of a Salmonella Typhimurium marker strain and quantitative-PCR targeting Clostridium perfringens. Treatment effects on specific pathogens were generally non-significant. S. Typhimurium introduced by oral gavage at day of hatch was recovered by cultivation from nearly all birds sampled across treatments at 7d and 21d, but by 42d, S. Typhimurium was only recovered from ca. 25% of birds, regardless of treatment. Sequencing data also revealed non-significant treatment effects on genera containing known pathogens and on the cecal microbiome as a whole. In contrast, temporal changes in the cecal microbiome were dramatic, highly significant, and consistent across treatments. At 7d, the cecal community was dominated by three genera (Flavonifractor, Pseudoflavonifractor, and a Lachnospiracea sequence type) that accounted for more than half of sequences. By 21d post-hatch, a single genus (Faecalibacterium) accounted for 23-55% of sequences, and the number of Clostridium 16S rRNA gene copies detected by quantitative-PCR reached a maximum.ConclusionsOver the 42 d experiment, the cecal bacterial community changed significantly as measured by a variety of ecological metrics and increases in the complexity of co-occurrence networks. Management of poultry to improve animal health, nutrition, or food safety may need to consider the interactive effects of any treatments with the dramatic temporal shifts in the taxonomic composition of the cecal microbiome as described here.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-014-0282-8) contains supplementary material, which is available to authorized users.

Citations

Citations

A Nanoparticle-Based Solution DNA Sandwich Assay Using ICP-AES for Readout

We report herein a nanoparticle-based methodology for detecting DNA in solution using inductively coupled plasma atomic emission spectrometry (ICP-AES) as a readout tool. This represents the first homogeneous solution assay of biologically significant targets by employing ICP-related techniques. Two types of particles are employed: silica nanoparticles or gold nanoparticles functionalized with oligonucleotides that are capable of hybridizing with half of the target DNA sequence as signal readout components, and magnetic microparticles functionalized with oligonucleotides that are capable of hybridizing with the other half of the sequence as capture components. In the presence of target DNA, three components form typical sandwich structures, and the application of a magnetic field could effectively separate them from the rest of the solution. Subsequent application of ICP-AES effectively provides an inorganic elemental readout for the diagnosis of target DNA.

Dynamic T1 estimation of brain tumors using double-echo dynamic MR imaging.

To assess the clinical utility of a new method for real-time estimation of T1 during the first pass of contrast agent by using this method to examine brain tumors. The multi-phase spoiled gradient-echo pulse sequence using the double-echo magnetic resonance (MR) technique was modified. In the first half of the pulse sequence, the flip angle was varied systematically. Then, static T1 values were calculated using differences in MR signal intensities between different flip angles. In the latter half of this sequence, changes in absolute T1 were calculated using differences in signal intensities before and after injection of contrast agent. The double-echo MR data were used to minimize the T2* effect. Five cases of neurinoma and seven cases of meningioma were examined. Changes in T1 during the first pass of contrast agent were compared between neurinoma and meningioma. Changes in absolute T1 were clearly demonstrated on the parametric map. Although the changes in absolute T1 during the first pass of contrast agent did not allow differentiation between the two types of tumors, the mean gradient after the first pass was statistically higher for neurinoma than for meningioma (P < 0.05; meningioma, 0.011 +/- 0.012 second(-1)/second; neurinoma, 0.034 +/- 0.020 second(-1)/second). The present method appears to be useful for estimation of dynamic T1 changes in brain tumors in clinical settings.

Dynamic contrast-enhanced T(1) measuring MRI using variable flip angle SPGR

We modified the multi-phase spoiled gradient recalled echo (SPGR) pulse sequence using the double-echo MR technique for estimation of T(1) during the first pass of contrast agent, and examined its precision. In the first half of the pulse sequence, the flip angle was varied systematically to calculate static T(1) values. It was necessary to choose optimal flip angles to minimize the calculation error of static T(1) values. In the latter half of this sequence, changes in absolute T(1) were calculated using differences in signal intensities before and after the injection of contrast agent. The optimal flip angle was 20 degrees for precise conversion to T(1) values under the short TR (33.3 ms) condition. Double echo MR data were used to minimize the T(2)* effect. The present method appears to be useful for quantitative estimation of dynamic contrast-enhanced MRI.