Impaired 17,20-lyase activity in male mice lacking cytochrome b5 in Leydig cells

Androgen and estrogen biosynthesis in mammals requires the 17,20-lyase activity of cytochrome P450 17A1 (steroid 17-hydroxylase/17,20-lyase). Maximal 17,20-lyase activity in vitro requires the presence of cytochrome b5 (b5), and rare cases of b5 deficiency in human beings causes isolated 17,20-lyase deficiency. To study the consequences of conditional b5 removal from testicular Leydig cells in an animal model, we generated Cyb5(flox/flox):Sf1-Cre (LeyKO) mice. The LeyKO male mice had normal body weights, testis and sex organ weights, and fertility compared with littermates. Basal serum and urine steroid profiles of LeyKO males were not significantly different than littermates. In contrast, marked 17-hydroxyprogesterone accumulation (100-fold basal) and reduced testosterone synthesis (27% of littermates) were observed after human chorionic gonadotropin stimulation in LeyKO animals. Testis homogenates from LeyKO mice showed reduced 17,20-lyase activity and a 3-fold increased 17-hydroxylase to 17,20-lyase activity ratio, which were restored to normal upon addition of recombinant b5. We conclude that Leydig cell b5 is required for maximal androgen synthesis and to prevent 17-hydroxyprogesterone accumulation in the mouse testis; however, the b5-independent 17,20-lyase activity of mouse steroid 17-hydroxylase/17,20-lyase is sufficient for normal male genital development and fertility. LeyKO male mice are a good model for the biochemistry but not the physiology of isolated 17,20-lyase deficiency in human beings

Molecular detection of the Luteoviridae

The Luteoviridae is a family of single stranded positive sense RNA plant viruses which cause yield losses in many important food crops worldwide and are therefore of significant economic concern for some countries. Fast and accurate detection and identification is important for strategies designed to control the spread of Luteoviridae species and to reduce their economic impact. This study offers significant advances on current molecular protocols for their detection and differentiation by being taxonomically broad-ranging, time-efficient, sensitive to asymptomatic detection and with the potential to pick up unknown sequence isolates. Current molecular detection and identification tools for the Luteoviridae are mainly species specific, which limits their application for fast and accurate detection and identification. Firstly, the development of new generic primers for the family was investigated. Using the two-step reverse transcription polymerase chain reaction (RT-PCR), thirteen out of sixteen Luteoviridae species analysed were detected using three separate combinations of low-degeneracy generic primers, targeting the coat protein gene region. A synthetic positive control containing all primer sequence priming sites was designed as a generic tool for use with a variety of host plants and the Luteoviridae species. The Luteoviridae primers described in this study present a simple infection-detection tool which will be of benefit to biosecurity authorities in nursery-stock surveillance, disease management or outbreak prevention, and may also be useful in detection of as-yet undiscovered species within the family. Secondly, the suitability of a two-step reverse transcription real time PCR (RT-qPCR) plus melting curve analysis (MCA) as a tool for the rapid detection and discrimination of Luteoviridae species was investigated. Melting temperature and shape of the melting peak were analysed for 13 Luteoviridae species using SYBR® GreenERTM fluorescent dye. Specific melting peaks were observed for all isolates investigated, however due to the high variability of sequences for some members of this family, different melting temperatures were also observed between different isolates of some species. Nevertheless, discrimination was achieved among 6 species. MCA, in this study, was demonstrated to be a faster and more discriminatory alternative to gel electrophoresis of end-point PCR products for the detection of Luteoviridae infection. Thirdly, this study evaluated two combinations of generic Luteoviridae primers for the detection of an early double-infection with BYDV-PAV and CYDV-RPV in barley and oats via RT-qPCR at the early stage of virus infection (3-15 days post inoculation (dpi)). The distribution of viruses in young and old leaves for optimising plant tissue collection strategies was also considered. Quantitative data from this study indicate that in some plants the titre of both luteoviruses is comparable to the expression of the plant mitochondrial gene nad5 (used as an internal RNA control) as early as 3 dpi and that titre differs greatly between individual plants. This study also suggests that virus distribution in different parts of the plant is probably host dependent; while young barley leaves at 9 dpi had a higher titre of both BYDV-PAV and CYDV-RPV viruses than old leaves, no such trend was observed in oats over the experimental period. Lastly, this study investigated the performance of the generic primers in several multiplex situations using both RT-PCR and RT-qPCR-MCA. Multiplex RT-PCR using seven generic primers resulted in some non-specific amplification, which although of non-viral origin, significantly impacted on the use of such an assay. Contrary to RT-PCR, multiplex RT-qPCR was shown to be a good solution for detection and discrimination of BYDV-PAV and CYDV-RPV infection in a range of samples and has the potential to be used in diagnostics. Using this work as a model, similar assays based on more versatile generic primers could be designed for other plant virus groups or other pathogens

Predation by Ostenia robusta on Costelytra zealandica pupae

Soil sampling in a cereal crop near Southbridge, Canterbury, revealed dipteran larvae attacking Costelytra zealandica (White) pupae. Approximately 50% of the pupae had larvae associated with them. DNA sequencing analysis of larval specimens indicated they most likely belonged to the family Dolichopodidae. Larval specimens were reared through to adults and were identified as Ostenia robusta (Hutton) (Diptera: Dolichopodidae). This is the first record of the association of O. robusta larvae with C. zealandica pupae. A general description of the adult and larva of this species is provided, along with DNA sequencing data and observations on its association with C. zealandica pupae. The potential role of O. robusta in regulating populations of C. zealandica is briefly considered

Population structure of Bactrocera dorsalis s.s., B. papayae and B. philippinensis (Diptera: Tephritidae) in southeast Asia: evidence for a single species hypothesis using mitochondrial DNA and wingshape data

Background Bactrocera dorsalis s.s. is a pestiferous tephritid fruit fly distributed from Pakistan to the Pacific, with the Thai/Malay peninsula its southern limit. Sister pest taxa, B. papayae and B. philippinensis, occur in the southeast Asian archipelago and the Philippines, respectively. The relationship among these species is unclear due to their high molecular and morphological similarity. This study analysed population structure of these three species within a southeast Asian biogeographical context to assess potential dispersal patterns and the validity of their current taxonomic status. Results Geometric morphometric results generated from 15 landmarks for wings of 169 flies revealed significant differences in wing shape between almost all sites following canonical variate analysis. For the combined data set there was a greater isolation-by-distance (IBD) effect under a ‘non-Euclidean’ scenario which used geographical distances within a biogeographical ‘Sundaland context’ (r2 = 0.772, P Conclusions Bactrocera dorsalis s.s., B. papayae and B. philippinensis likely represent one species structured around the South China Sea, having migrated from northern Thailand into the southeast Asian archipelago and across into the Philippines. No migration is apparent between the Philippines and Taiwan. This information has implications for quarantine, trade and pest management

A Multi-Cylinder Airflow & amp; Residual Gas Estimation Tool Applied to a Vehicle Demonstrator

Copyright © 2010 SAE International In a gasoline engine, the cycle-by-cycle fresh trapped charge, and corresponding unswept residual gas fraction (RGF) are critical parameters of interest for maintaining the desired air-fuel ratio (AFR). Accurate fueling is a key precursor to improved engine fuel economy, and reduced engine out emissions. Asymmetric flow paths to cylinders in certain engines can cause differences in the gas exchange process, which in turn cause imbalances in trapped fresh charge and RGF. Variable cam timing (VCT) can make the gas exchange process even more complex. Due to the reasons stated above, simplified models can result in significant estimation errors for fresh trapped charge and RGF if they are not gas dynamics-based or detailed enough to handle features such as variable valve timing, duration, or lift. In this paper, a new air flow and RGF measurement tool is introduced. The tool is a combination of 1-D gas dynamics modeling and imposed, measured dynamic pressure signals in the intake and exhaust manifolds. With this method, the components to be modeled in the gas exchange system are reduced to a minimum, making it possible to model the gas exchange process with adequate detail and still maintain reasonable tool execution time. In this approach, the accuracy of the air flow and RGF estimation are not influenced by the location of pressure transducers and it is possible to “track ” EGR flow from the exhaust to the intake system. The tool is demonstrated on a 4-cylinder gasoline engine powered vehicle with dual intake and exhaust cam phasing. Predictions from the tool are compared to AFR and other real-time signals measured during a variety of different real-world transient drive cycles. The instantaneous fresh trapped charge, RGF across all cylinders, as well as the influence of VCT actuation is discussed

Detection and discrimination of members of the family Luteoviridae by real-time PCR and SYBR® GreenER™ melting curve analysis

This study investigated the suitability of a two step real-time RT-PCR melting curve analysis as a tool for the detection and discrimination of nine species in the plant virus family Luteoviridae, being Soybean dwarf virus [SbDV], Bean leafroll virus [BLRV], Beet chlorosis virus [BChV], Beet mild yellowing virus [BMYV], Beet western yellows virus [BWYV], Cereal yellow dwarf virus-RPV [CYDV-RPV], Cucurbit aphid-borne yel-lows virus [CABYV], Potato leafroll virus [PLRV] and Turnip yellows virus [TuYV]. Melting temperature and shape of the melting peak were analysed for 68 bp and 148 bp coat protein gene amplicons using SYBR® GreenERTM fluorescent dye. Specific melting peaks with unique melting temperature were observed for the various species of the family Luteoviridae using the 68 bp amplicon, but not with the 148 bp amplicon. Due to the high variability of sequences for some members of this family, different melting temperatures were also observed between different isolates of the species CYDV-RPV and TuYV. Nevertheless, discrim-ination between species was achieved for SbDV, BLRV, BChV, BMYV, CABYV and either PLRV or BWYV. Melting curve analysis, in this study, is a faster and more discriminatory alternative to gel electrophoresis of end-point PCR products for the detection of Luteoviridae infection