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

Untersuchungen zur Funktion der Fettsäure-2-Hydroxylase (FA2H) in der Haut- und Haarfollikel-Homöostase der Maus

Die Fettsäure-2-Hydroxylase (FA2H) ist das einzige bisher bekannte Enzym, das die α-Hydroxylierung von unverzweigten Fettsäuren katalysiert. Letztere sind Bestandteile von α-hydroxylierten Sphingolipiden, die in der Haut in großen Mengen vorkommen. Sie spielen dort vermutlich eine entscheidende Rolle bei der Ausbildung der Hautbarriere. Die Funktion der FA2H in der Haut- und Haarfollikel-Homöostase wurde in der vorliegenden Arbeit anhand von FA2H-KO-Mäusen untersucht. Die Mäuse wiesen während der Morphogenese ein um zwei Tage verzögertes Fellwachstum auf und verloren in der ersten telogenen Phase ihr gesamtes Rückenfell. Der Haarverlust wurde in jeder weiteren telogenen Phase beobachtet und manifestierte sich in einer zyklischen Alopezie. Histologische Untersuchungen der Haut ließen weit offene, zum Teil mit lipidhaltigen Substanzen verstopfte Haarkanäle sowie Verformungen des Haarfollikels erkennen, was auf eine Haarfollikeldystrophie hindeutete. Die Analyse der epidermalen Lipide konnte überraschenderweise zeigen, dass die FA2H-KO-Mäuse trotz der FA2H-Defizienz noch immer über normale Mengen an α-hydroxylierten Lipiden verfügten. Untersuchungen zur Lokalisation der FA2H haben nachgewiesen, dass das Enzym in der murinen Haut ausschließlich in den Talgdrüsen exprimiert wird. Folglich muss in Keratinozyten ein zweites, bisher unbekanntes Enzym existieren, welches dort die α-Hydroxylierungs- Reaktion katalysiert. In Sebozyten führte die FA2H-Defizienz zu einer veränderten Zusammensetzung des Sebums, wobei vor allem die Menge an Typ II-Wachsdiestern signifikant reduziert war. Daraus resultierte eine deutliche Schmelzpunkterhöhung und vermutlich eine höhere Viskosität des Sebums. Ein normaler Sebumfluss durch den Haarkanal an die Hautoberfläche war damit nicht mehr möglich. Das Sebum verstopfte die Haarkanäle und behinderte somit das Herauswachsen der Haare. Die FA2H-Defizienz bedingte weiterhin eine Sebozyten-Hyperproliferation mit deutlich vergrößerten Talgdrüsen. Dies ging mit einer signifikanten Hochregulation des epidermalen Wachstumsfaktor- bzw. des ErbB-Rezeptor-Liganden Epigen einher. Vor kurzem im Menschen nachgewiesene Mutationen im Fa2h-Gen sind mit drei neurodegenerativen Funktionsstörungen assoziiert: Leukodystrophie, komplizierte Form der spastischen Paraplegie sowie Neurodegeneration mit Eisenablagerungen im Gehirn. Der durch die verschiedenen Mutationen hervorgerufene vollständige oder partielle Aktivitätsverlust der FA2H stellt vermutlich die Ursache der Erkrankungen dar. Die vorliegende Arbeit liefert wichtige Ergebnisse zur Rolle der FA2H in verschiedenen Organen der Maus und des Menschen und bildet die Grundlage für weitere Untersuchungen, die zum Verständnis der Funktion von α-hydroxylierten Sphingolipiden beitragen können

Complete DNA sequences of the plastid genomes of two parasitic flowering plant species, Cuscuta reflexa and Cuscuta gronovii

<p>Abstract</p> <p>Background</p> <p>The holoparasitic plant genus <it>Cuscuta </it>comprises species with photosynthetic capacity and functional chloroplasts as well as achlorophyllous and intermediate forms with restricted photosynthetic activity and degenerated chloroplasts. Previous data indicated significant differences with respect to the plastid genome coding capacity in different <it>Cuscuta </it>species that could correlate with their photosynthetic activity. In order to shed light on the molecular changes accompanying the parasitic lifestyle, we sequenced the plastid chromosomes of the two species <it>Cuscuta reflexa </it>and <it>Cuscuta gronovii</it>. Both species are capable of performing photosynthesis, albeit with varying efficiencies. Together with the plastid genome of <it>Epifagus virginiana</it>, an achlorophyllous parasitic plant whose plastid genome has been sequenced, these species represent a series of progression towards total dependency on the host plant, ranging from reduced levels of photosynthesis in <it>C. reflexa </it>to a restricted photosynthetic activity and degenerated chloroplasts in <it>C. gronovii </it>to an achlorophyllous state in <it>E. virginiana</it>.</p> <p>Results</p> <p>The newly sequenced plastid genomes of <it>C. reflexa </it>and <it>C. gronovii </it>reveal that the chromosome structures are generally very similar to that of non-parasitic plants, although a number of species-specific insertions, deletions (indels) and sequence inversions were identified. However, we observed a gradual adaptation of the plastid genome to the different degrees of parasitism. The changes are particularly evident in <it>C. gronovii </it>and include (a) the parallel losses of genes for the subunits of the plastid-encoded RNA polymerase and the corresponding promoters from the plastid genome, (b) the first documented loss of the gene for a putative splicing factor, MatK, from the plastid genome and (c) a significant reduction of RNA editing.</p> <p>Conclusion</p> <p>Overall, the comparative genomic analysis of plastid DNA from parasitic plants indicates a bias towards a simplification of the plastid gene expression machinery as a consequence of an increasing dependency on the host plant. A tentative assignment of the successive events in the adaptation of the plastid genomes to parasitism can be inferred from the current data set. This includes (1) a loss of non-coding regions in photosynthetic <it>Cuscuta </it>species that has resulted in a condensation of the plastid genome, (2) the simplification of plastid gene expression in species with largely impaired photosynthetic capacity and (3) the deletion of a significant part of the genetic information, including the information for the photosynthetic apparatus, in non-photosynthetic parasitic plants.</p

Protein Kinase D regulates several aspects of development in Drosophila melanogaster

<p>Abstract</p> <p>Background</p> <p>Protein Kinase D (PKD) is an effector of diacylglycerol-regulated signaling pathways. Three isoforms are known in mammals that have been linked to diverse cellular functions including regulation of cell proliferation, differentiation, motility and secretory transport from the trans-Golgi network to the plasma membrane. In <it>Drosophila</it>, there is a single PKD orthologue, whose broad expression implicates a more general role in development.</p> <p>Results</p> <p>We have employed tissue specific overexpression of various PKD variants as well as tissue specific RNAi, in order to investigate the function of the PKD gene in <it>Drosophila</it>. Apart from a wild type (WT), a kinase dead (kd) and constitutively active (SE) <it>Drosophila </it>PKD variant, we also analyzed two human isoforms hPKD2 and hPKD3 for their capacity to substitute PKD activity in the fly. Overexpression of either WT or kd-PKD variants affected primarily wing vein development. However, overexpression of SE-PKD and PKD RNAi was deleterious. We observed tissue loss, wing defects and degeneration of the retina. The latter phenotype conforms to a role of PKD in the regulation of cytoskeletal dynamics. Strongest phenotypes were larval to pupal lethality. RNAi induced phenotypes could be rescued by a concurrent overexpression of <it>Drosophila </it>wild type PKD or either human isoform hPKD2 and hPKD3.</p> <p>Conclusion</p> <p>Our data confirm the hypothesis that <it>Drosophila </it>PKD is a multifunctional kinase involved in diverse processes such as regulation of the cytoskeleton, cell proliferation and death as well as differentiation of various fly tissues.</p

Infectious bronchitis virus nonstructural protein 4 alone induces membrane pairing

Positive-strand RNA viruses, such as coronaviruses, induce cellular membrane rearrangements during replication to form replication organelles allowing for efficient viral RNA synthesis. Infectious bronchitis virus (IBV), a pathogenic avian of significant importance to the global poultry industry, has been shown to induce the formation of double membrane vesicles (DMVs), zippered endoplasmic reticulum (zER) and tethered vesicles, known as spherules. These membrane rearrangements are virally induced; however, it remains unclear which viral proteins are responsible. In this study, membrane rearrangements induced when expressing viral non-structural proteins (nsps) from two different strains of IBV were compared. Three non-structural transmembrane proteins, nsp3, nsp4, and nsp6, were expressed in cells singularly or in combination and the effects on cellular membranes investigated using electron microscopy and electron tomography. In contrast to previously studied coronaviruses, IBV nsp4 alone is necessary and sufficient to induce membrane pairing; however, expression of the transmembrane proteins together was not sufficient to fully recapitulate DMVs. This indicates that although nsp4 is able to singularly induce membrane pairing, further viral or host factors are required in order to fully assemble IBV replicative structures. This study highlights further differences in the mechanism of membrane rearrangements between members of the coronavirus family

The proteome of the infectious bronchitis virus Beau-R Virion

Infectious bronchitis is a highly contagious respiratory disease of poultry caused by the coronavirus IBV. It was thought that coronavirus virions were composed of three major viral structural proteins, until investigations of other coronaviruses showed that coronavirus virions also include viral non-structural and group specific proteins as well as host cell proteins. To study the proteome of IBV virions, virus was grown in embryonated chicken eggs and purified by sucrose gradient ultracentrifugation and analysed by mass spectrometry proteomic. Analysis of three preparations of purified IBV yielded the three expected structural proteins plus thirty-five additional virion-associated host proteins. Virion-associated host proteins had a diverse range of functional attributions, being involved in cytoskeleton formation, RNA binding and protein folding pathways. Some of these proteins were unique to this study, whilst others were found to be orthologous to proteins identified in SARS-CoV virions, and also virions from a number of other RNA and DNA viruses. Together these results demonstrate that coronaviruses have the capacity to incorporate a substantial variety of host protein, which may have implications for the disease process

Temperature Sensitivity: A Potential Method for the Generation of Vaccines against the Avian Coronavirus Infectious Bronchitis Virus.

The Gammacoronavirus infectious bronchitis virus (IBV) is a highly contagious economically important respiratory pathogen of domestic fowl. Reverse genetics allows for the molecular study of pathogenic determinants to enable rational vaccine design. The recombinant IBV (rIBV) Beau-R, a molecular clone of the apathogenic Beaudette strain, has previously been investigated as a vaccine platform. To determine tissues in which Beau-R could effectively deliver antigenic genes, an in vivo study in chickens, the natural host, was used to compare the pattern of viral dissemination of Beau-R to the pathogenic strain M41-CK. Replication of Beau-R was found to be restricted to soft tissue within the beak, whereas M41-CK was detected in beak tissue, trachea and eyelid up to seven days post infection. In vitro assays further identified that, unlike M41-CK, Beau-R could not replicate at 41 °C, the core body temperature of a chicken, but is able to replicate a 37 °C, a temperature relatable to the very upper respiratory tract. Using a panel of rIBVs with defined mutations in the structural and accessory genes, viral replication at permissive and non-permissive temperatures was investigated, identifying that the Beau-R replicase gene was a determinant of temperature sensitivity and that sub-genomic mRNA synthesis had been affected. The identification of temperature sensitive allelic lesions within the Beau-R replicase gene opens up the possibility of using this method of attenuation in other IBV strains for future vaccine development as well as a method to investigate the functions of the IBV replicase proteins

Verslag andijvierassenproef onder staand glas, 1956-1957

<p><b>Copyright information:</b></p><p>Taken from "Complete DNA sequences of the plastid genomes of two parasitic flowering plant species, and "</p><p>http://www.biomedcentral.com/1471-2229/7/45</p><p>BMC Plant Biology 2007;7():45-45.</p><p>Published online 22 Aug 2007</p><p>PMCID:PMC2089061.</p><p></p> for partial editing sites two chromatograms are shown in photosynthetic active tissue (top) and in pale tissue (bottom)

Targeting the Conserved Stem Loop 2 Motif in the SARS-CoV-2 Genome.

RNA structural elements occur in numerous single-stranded positive-sense RNA viruses. The stem-loop 2 motif (s2m) is one such element with an unusually high degree of sequence conservation, being found in the 3' untranslated region (UTR) in the genomes of many astroviruses, some picornaviruses and noroviruses, and a variety of coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. The evolutionary conservation and its occurrence in all viral subgenomic transcripts imply a key role for s2m in the viral infection cycle. Our findings indicate that the element, while stably folded, can nonetheless be invaded and remodeled spontaneously by antisense oligonucleotides (ASOs) that initiate pairing in exposed loops and trigger efficient sequence-specific RNA cleavage in reporter assays. ASOs also act to inhibit replication in an astrovirus replicon model system in a sequence-specific, dose-dependent manner and inhibit SARS-CoV-2 replication in cell culture. Our results thus permit us to suggest that the s2m element is readily targeted by ASOs, which show promise as antiviral agents. IMPORTANCE The highly conserved stem-loop 2 motif (s2m) is found in the genomes of many RNA viruses, including SARS-CoV-2. Our findings indicate that the s2m element can be targeted by antisense oligonucleotides. The antiviral potential of this element represents a promising start for further research into targeting conserved elements in RNA viruses.ERC, BBSR