Stability of nuclear and mitochondrial reference genes in selected tissues of the Ambrosia beetle Xylosandrus germanus

SIMPLE SUMMARY: The ambrosia beetle Xylosandrus germanus (Blandford) is a destructive wood-boring insect of horticultural tree crops. A fungal mutualist is cultivated within host trees that provides the sole source of nutrition for the larvae and adults of this beetle. Female X. germanus adults use a pouch-like structure (i.e., mycangium) to maintain and transport spores of their fungal mutualist. To facilitate future studies examining gene expression of X. germanus’ mycangium, the identification of stable genes unaffected by experimental treatments is needed to provide a standard reference during gene expression studies. Selected tissue types were dissected from laboratory-reared and field-collected specimens of the ambrosia beetle X. germanus to evaluate the stability of five reference genes, namely, 28S ribosomal RNA (28S rRNA), arginine kinase (AK), carbamoyl-phosphate synthetase 2-aspartate transcarbamylase-dihydroorotase (CAD), mitochondrial cytochrome oxidase 1 (CO1), and elongation factor-1α (EF1α). The reference genes CO1 and AK were identified as primary and secondary reference genes. By contrast, EF1α was considered unsuitable for use as a reference gene during gene expression studies with X. germanus. These results will aid in normalizing the expression of target genes during studies with X. germanus. ABSTRACT: The fungus-farming ambrosia beetle Xylosandrus germanus (Blandford) uses a pouch-like structure (i.e., mycangium) to transport spores of its nutritional fungal mutualist. Our current study sought to identify reference genes necessary for future transcriptome analyses aimed at characterizing gene expression within the mycangium. Complementary DNA was synthesized using selected tissue types from laboratory-reared and field-collected X. germanus consisting of the whole body, head + thorax, deflated or inflated mycangium + scutellum, inflated mycangium, and thorax + abdomen. Quantitative reverse-transcription PCR reactions were performed using primers for 28S ribosomal RNA (28S rRNA), arginine kinase (AK), carbamoyl-phosphate synthetase 2-aspartate transcarbamylase-dihydroorotase (CAD), mitochondrial cytochrome oxidase 1 (CO1), and elongation factor-1α (EF1α). Reference gene stability was analyzed using GeNorm, NormFinder, BestKeeper, ΔCt, and a comprehensive final ranking by RefFinder. The gene CO1 was identified as the primary reference gene since it was generally ranked in first or second position among the tissue types containing the mycangium. Reference gene AK was identified as a secondary reference gene. In contrast, EF1α was generally ranked in the last or penultimate place. Identification of two stable reference genes will aid in normalizing the expression of target genes for subsequent gene expression studies of X. germanus’ mycangium

Greenhouse Evaluation of Air-Assisted Delivery Parameters for Mature Poinsettias

Understanding the performance characteristics of application equipment is important for helping make the most efficacious applications. While handguns making high volume applications are common in greenhouse production, it is difficult to achieve uniform distribution of product in a timely manner. Broadcast applications made using air-assistance can help aid canopy penetration and the volume of carrier required to make applications. The objectives of this research were to determine how air-assist sprayer application parameters influence spray deposits on the undersides of leaves in a mature poinsettia canopy. Bench-top trials were conducted using a motorized boom inside a greenhouse to treat a mature and dense poinsettia canopy. Sprayer treatments applied a tank mix of water and fluorescent tracer. Nylon screen targets were secured to the underside surfaces of leaves in the upper and lower elevation of target plants. A five-port, air-assist nozzle with flat fan nozzle tips was used to make the applications. Three air outlet speeds, two travel speeds, and three nozzle flow rates were evaluated. Each treatment was replicated three times. Spray deposits were highly variable. Upper elevation spray deposits were significantly greater than lower elevation deposits. Individually, higher air outlet speed (36.0 m s(-1)), slower travel speed (3.2 km h(-1)), and higher nozzle flow rate (1.17 L min(-1)) tended to produce higher sprayer deposits on the underside surfaces of leaves. The combination of travel speed and nozzle flow rate that produced the highest application rate (900 L ha(-1)) also produced the highest deposits. There was a 500% increase in underside leaf surface deposits in the lower canopy area for a corresponding 500% increase in application rate. However, the main effects produced no significant differences in spray deposits in the lower canopy area. Further improvements in directing sprays or providing canopy turbulence are necessary to improve deposition and management of insect pests feeding on the underside of poinsettia leaves

The Nuclear Factor of Activated T Cells (Nfat) Transcription Factor Nfatp (Nfatc2) Is a Repressor of Chondrogenesis

Nuclear factor of activated T cells (NFAT) transcription factors regulate gene expression in lymphocytes and control cardiac valve formation. Here, we report that NFATp regulates chondrogenesis in the adult animal. In mice lacking NFATp, resident cells in the extraarticular connective tissues spontaneously differentiate to cartilage. These cartilage cells progressively differentiate and the tissue undergoes endochondral ossification, recapitulating the development of endochondral bone. Proliferation of already existing articular cartilage cells also occurs in some older animals. At both sites, neoplastic changes in the cartilage cells occur. Consistent with these data, NFATp expression is regulated in mesenchymal stem cells induced to differentiate along a chondrogenic pathway. Lack of NFATp in articular cartilage cells results in increased expression of cartilage markers, whereas overexpression of NFATp in cartilage cell lines extinguishes the cartilage phenotype. Thus, NFATp is a repressor of cartilage cell growth and differentiation and also has the properties of a tumor suppressor

Genetic Diversity of PCR-Positive, Culture-Negative and Culture-Positive Mycobacterium ulcerans Isolated from Buruli Ulcer Patients in Ghana.

Culture of Mycobacterium ulcerans from Buruli ulcer patients has very low sensitivity. Thus confirmation of M. ulcerans infection is primarily based on PCR directed against IS2404. In this study we compare the genotypes obtained by variable number of tandem repeat analysis of DNA from IS2404-PCR positive cultures with that obtained from IS2404 positive, culture-negative tissue. A significantly greater genetic heterogeneity was found among culture-negative samples compared with that found in cultured strains but a single genotype is over-represented in both sample sets. This study provides evidence that both the focal location of bacteria in a lesion as well as differences in the ability to culture a particular genotype may underlie the low sensitivity of culture. Though preliminary, data from this work also suggests that mycobacteria previously associated with fish disease (M. pseudoshottsii) may be pathogenic for humans

MicroRNA expression profiles in pediatric dysembryoplastic neuroepithelial tumors.

© Springer Science+Business Media New York 2015Among noncoding RNAs, microRNAs (miRNAs) have been most extensively studied, and their biology has repeatedly been proven critical for central nervous system pathological conditions. The diagnostic value of several miRNAs was appraised in pediatric dysembryoplastic neuroepithelial tumors (DNETs) using miRNA microarrays and receiving operating characteristic curves analyses. Overall, five pediatric DNETs were studied. As controls, 17 samples were used: the FirstChoice Human Brain Reference RNA and 16 samples from deceased children who underwent autopsy and were not present with any brain malignancy. The miRNA extraction was carried out using the mirVANA miRNA Isolation Kit, while the experimental approach included miRNA microarrays covering 1211 miRNAs. Quantitative real-time polymerase chain reaction was performed to validate the expression profiles of miR-1909* and miR-3138 in all samples initially screened with miRNA microarrays. Our findings indicated that miR-3138 might act as a tumor suppressor gene when down-regulated and miR-1909* as a putative oncogenic molecule when up-regulated in pediatric DNETs compared to the control cohort. Subsequently, both miRNA signatures might serve as putative diagnostic biomarkers for pediatric DNETs.Peer reviewedFinal Accepted Versio

Hormone replacement therapy and cancer mortality in women with site specific cancers : A cohort study using linked medical records.

Acknowledgements We would like to acknowledge the support of the eDRIS team (Public Health Scotland) for their involvement in obtaining approvals, provisioning and linking data and the secure analytical platform within the National Safe Haven. We would also like to acknowledge support of SAIL Databank for facilitating access to the dataset from Wales. We acknowledge the contribution of EMIS practices who contribute to the QResearch database and the Chancellor, Masters and Scholars of the University of Oxford for continuing to develop and support the QResearch database. The Hospital Episode Statistics data used in the English portion of this analysis are re438 used by permission from NHS Digital who retain the copyright. We thank the Office for National Statistics (ONS) for providing the mortality data for the English analyses. The ONS bears no responsibility for the analysis or interpretation of the data. The authors would also like to thank the PPI representatives for providing a patient and public perspective on the study design, findings, interpretation of the study and lay summary materials.Peer reviewe

The behaviour of lubricated EHD contacts subjected to vibrations

Machine components containing contacts working in elastohydrodynamic (EHD) conditions are often subjected to vibrations. These may be originated from the mechanism or machine the contact is part of, the surrounding environment and within the contact itself. The influence of vibrations upon the behaviour of elastohydrodynamic films has been studied experimentally in a number of papers, but a comprehensive study of the effect of the parameters of the oscillatory motion upon the film thickness has not been carried out yet. In this study the authors evaluate the effect of the frequency of the oscillatory motion upon the EHD film thickness. Optical interferometry is used to measure lubricant film thickness in a ball-on-flat disc arrangement. A high – speed camera records the interferometric images for later analysis and conversion into film thickness maps. The disc runs at a constant angular velocity while the ball is driven by the traction forces developed in the EHD film. In steady state conditions, this would ensure pure rolling conditions, however in the present investigation the ball is subjected to harmonic vibrations in a direction perpendicular to the plane of the film. The contact under study is lubricated by basic oils and the temperature is kept at a constant value of 60°C. The aim of this paper is to understand how vibrations influence the lubricant film formation