Solid volume fraction estimation of bone:marrow replica models using ultrasound transit time spectroscopy

The acceptance of broadband ultrasound attenuation (BUA) for the assessment of osteoporosis suffers from a limited understanding of both ultrasound wave propagation through cancellous bone and its exact dependence upon the material and structural properties. It has recently been proposed that ultrasound wave propagation in cancellous bone may be described by a concept of parallel sonic rays; the transit time of each ray defined by the proportion of bone and marrow propagated. A Transit Time Spectrum (TTS) describes the proportion of sonic rays having a particular transit time, effectively describing the lateral inhomogeneity of transit times over the surface aperture of the receive ultrasound transducer. The aim of this study was to test the hypothesis that the solid volume fraction (SVF) of simplified bone:marrow replica models may be reliably estimated from the corresponding ultrasound transit time spectrum. Transit time spectra were derived via digital deconvolution of the experimentally measured input and output ultrasonic signals, and compared to predicted TTS based on the parallel sonic ray concept, demonstrating agreement in both position and amplitude of spectral peaks. Solid volume fraction was calculated from the TTS; agreement between true (geometric calculation) with predicted (computer simulation) and experimentally-derived values were R2=99.9% and R2=97.3% respectively. It is therefore envisaged that ultrasound transit time spectroscopy (UTTS) offers the potential to reliably estimate bone mineral density and hence the established T-score parameter for clinical osteoporosis assessment.

Functionally associated molecular genetic marker map construction in perennial ryegrass (Lolium perenne L.)

A molecular marker-based map of perennial ryegrass (Lolium perenne L.) has been constructed through the use of polymorphisms associated with expressed sequence tags (ESTs). A pair-cross between genotypes from a North African ecotype and the cultivar Aurora was used to generate a two-way pseudo-testcross population. A selection of 157 cDNAs assigned to eight different functional categories associated with agronomically important biological processes was used to detect polymorphic EST–RFLP loci in the F1(NA6 × AU6) population. A comprehensive set of EST–SSR markers was developed from the analysis of 14,767 unigenes, with 310 primer pairs showing efficient amplification and detecting 113 polymorphic loci. Two parental genetic maps were produced: the NA6 genetic map contains 88 EST–RFLP and 71 EST–SSR loci with a total map length of 963 cM, while the AU6 genetic map contains 67 EST–RFLP and 58 EST–SSR loci with a total map length of 757 cM. Bridging loci permitted the alignment of homologous chromosomes between the parental maps, and a sub-set of genomic DNA-derived SSRs was used to relate linkage groups to the perennial ryegrass reference map. Regions of segregation distortion were identified, in some instances in common with other perennial ryegrass maps. The EST-derived marker-based map provides the basis for in silico comparative genetic mapping, as well as the evaluation of co-location between QTLs and functionally associated genetic loci.

A universal test to determine the integrity of RNA, and its suitability for reverse transcription, in animal tissue laboratory specimens

Degradation of RNA in diagnostic specimens can cause false-negative test results and potential misdiagnosis when tests rely on the detection of specific RNA sequence. Current molecular methods of checking RNA integrity tend to be host species or group specific, necessitating libraries of primers and reaction conditions. The objective here was to develop a universal (multi-species) quality assurance tool for determining the integrity of RNA in animal tissues submitted to a laboratory for analyses. Ribosomal RNA (16S rRNA) transcribed from the mitochondrial 16S rDNA was used as template material for reverse transcription to cDNA and was amplified using polymerase chain reaction (PCR). As mitochondrial DNA has a high level of conservation, the primers used were shown to reverse transcribe and amplify RNA from every animal species tested. Deliberate degradation of rRNA template through temperature abuse of samples resulted in no reverse transcription and amplification. Samples spiked with viruses showed that single-stranded viral RNA and rRNA in the same sample degraded at similar rates, hence reverse transcription and PCR amplification of 16S rRNA could be used as a test of sample integrity and suitability for analysis that required the sample's RNA, including viral RNA. This test will be an invaluable quality assurance tool for determination of RNA integrity from tissue samples, thus avoiding erroneous test results that might occur if degraded target RNA is used unknowingly as template material for reverse transcription and subsequent PCR amplification.

Polymorphic microsatellite loci for the zebra shark Stegostoma fasciatum

We report on the development and characterization of 14 polymorphic microsatellite loci in the zebra shark (Stegostoma fasciatum). Five tetranucleotide and nine dinucleotide loci were polymorphic with heterozygosities ranging from 0.400 to 0.967 and from three to 22 alleles per locus. Cross-species amplification of these zebra shark primers on four other species of orectolobid sharks was not successful.

Summative evaluation of climate application activities with pastoralists in western Queensland

Survey methods were engaged to measure the change in use and knowledge of climate information by pastoralists in western Queensland. The initial mail survey was undertaken in 2000-01 (n=43) and provided a useful benchmark of pastoralists climate knowledge. Two years of climate applications activities were completed and clients were re-surveyed in 2003 (n=49) to measure the change in knowledge and assess the effectiveness of the climate applications activities. Two methods were used to assess changes in client knowledge, viz., self-assessment and test questions. We found that the use of seasonal climate forecasts in decision making increased from 36% in 2001 (n=42) to 51% in 2003 (n=49) (P=0.07). The self-assessment technique was unsatisfactory as a measure of changing knowledge over short periods (1-3 years), but the test question technique was successful and indicated an improvement in climate knowledge among respondents. The increased levels of use of seasonal climate forecasts in management and improved knowledge was partly attributed to the climate applications activities of the project. Further, those who used seasonal forecasting (n=25) didn't understand key components of forecasts (e.g. probability, median) better than those who didn't use seasonal forecasts (n=24) (P>0.05). This identifies the potential for misunderstanding and misinterpretation of forecasts among users and highlights the need for providers of forecasts to understand the difficulties and prepare simply written descriptions of forecasts and disseminate these with the maps showing probabilities. The most preferred means of accessing climate information were internet, email, 'The Season Ahead' newsletter and newspaper. The least preferred were direct contact with extension officers and attending field days and group meetings. Eighty-six percent of respondents used the internet and 67% used ADSL broadband internet (April 2003). Despite these findings, extension officers play a key role in preparing and publishing the information on the web, in emails and newsletters. We also believe that direct contact with extension officers trained in climate applications is desirable in workshop-like events to improve knowledge of the difficult concepts underpinning climate forecasts, which may then stimulate further adoption.

Effect of the puroindoline locus and environment on Chinese fresh noodle texture

Grain produced from doubled-haploid (DH) wheat lines, developed from a hard- and a soft-grained wheat cultivar, were bulked according to Pinb (puroindoline b) genotypes for an assessment of Chinese fresh noodle texture by a trained taste panel. Each DH line was designated as 'soft' or 'hard' grained, based on a PCR amplification of the wildtype, soft allele, or the mutant, hard allele. Theoretically, the soft and hard grain bulks represented respective Pinb alleles and an independent assortment of unlinked alleles from the parents, Sunco and Chuanyu 12. Grains from the parents and DH lines were grown at 2 locations in Queensland, Australia, and one in Sichuan, China. The grains were milled and processed for a taste panel evaluation in Chengdu, Sichuan. Results suggest the Pinb alleles had a significant effect on noodle softness and explained 30% of the variation; the 'soft' Pinb allele conferred a softer noodle texture. Location had a significant effect on noodle smoothness; wheat grain grown at Biloela, Queensland, produced a smoother noodle texture than grain grown in Sichuan. The effect of location confirms the importance of environment as a variable for this quality character. This investigation exemplifies the utility of Pinb markers for specifically altering Chinese Fresh Noodle texture.

Estrogen modulation of retinol-binding protein in immature chicks: Comparison with riboflavin carrier protein

The kinetics of estrogen (E) modulation of retinol-binding protein (RBP) production in the liver of immature chicks were compared with those governing de novo induction of riboflavin carrier protein (RCP) in the same tissue. A single dose of E markedly enhanced the plasma levels of RBP without any detectable lag period to reach peak value by 24 h and this was followed by a decline to attain the baseline by 4 days. There was no amplification of the response during secondary stimulation unlike the case with RCP induction. With multiple E administration, the 4-fold increased plasma RBP concentrations were sustained at a steady state during both primary and secondary stimulations, whereas concomitant RCP concentration progressively increased with each hormone administration and this response was further amplified during secondary stimulation. Unlike RCP induction, enhanced RBP accumulation was not strictly E dose dependent although a minimal threshold level of the steroid was required to elicit measurable response. Progesterone (P) could neither modulate nor substitute for E in enhancing plasma levels of either of the 2 proteins while the anti-estrogens, en- and zuclomifene citrate severely suppressed the production of both the proteins. RCP induction was completely inhibited by both α-amanitin and cycloheximide for prolonged periods while E-stimulated RBP production was affected only partially by α-amanitin. Likewise, cycloheximide inhibition of RBP accumulation followed a pattern similar to that of hepatic general protein synthesis.

The development of an improved PCR-based marker system for Sw-5, an important TSWV resistance gene of tomato

Sw-5 is an important disease resistance gene of tomato, providing broad resistance to Tomato spotted wilt virus (TSWV). A cleaved amplified polymorphic sequence (CAPS) marker, closely linked to the gene, has been reported. Although the Sw-5 locus has been characterised, a gene-specific marker has not been developed. This paper presents a PCR-based marker-system that consists of the co-amplification of a dominant marker representing the Sw-5 gene sequence, and the modified CAPS marker as a positive control and indicator of genotype.

A one-tube fluorescent assay for the quarantine detection and identification of Tilletia indica and other grass bunts in wheat

A molecular assay with enhanced specificity and sensitivity has been developed to assist in the surveillance of Karnal bunt, a quarantineable disease with a significant impact on international trade. The protocol involves the release of DNA from spores, PCR amplification to enrich Tilletia-specific templates from released DNA and a five-plex, real-time PCR assay to detect, identify and distinguish T. indica and other Tilletia species (T. walkeri, T. ehrhartae, T. horrida and a group comprising T. caries, T. laevis, T. contraversa, T. bromi and T. fusca) in wheat grains. This fluorescent molecular tool has a detection sensitivity of one spore and thus bypasses the germination step, which in the current protocol is required for confirmation when only a few spores have been found in grain samples. The assay contains five dual-labelled, species-specific probes and associated species-specific primer pairs in a PCR mix in one tube. The different amplification products are detected simultaneously by five different fluorescence spectra. This specific and sensitive assay with reduced labour and reagent requirements makes it an effective and economically sustainable tool to be used in a Karnal bunt surveillance program. This protocol will also be valuable for the identification of some contaminant Tilletia sp. in wheat grains.

Campylobacter jejuni and Campylobacter coli Genotyping by High-Resolution Melting Analysis of a flaA Fragment.

The highly variable flagellin-encoding flaA gene has long been used for genotyping Campylobacter jejuni and Campylobacter coli. High-resolution melting (HRM) analysis is emerging as an efficient and robust method for discriminating DNA sequence variants. The objective of this study was to apply HRM analysis to flaA-based genotyping. The initial aim was to identify a suitable flaA fragment. It was found that the PCR primers commonly used to amplify the flaA short variable repeat (SVR) yielded a mixed PCR product unsuitable for HRM analysis. However, a PCR primer set composed of the upstream primer used to amplify the fragment used for flaA restriction fragment length polymorphism (RFLP) analysis and the downstream primer used for flaA SVR amplification generated a very pure PCR product, and this primer set was used for the remainder of the study. Eighty-seven C. jejuni and 15 C. coli isolates were analyzed by flaA HRM and also partial flaA sequencing. There were 47 flaA sequence variants, and all were resolved by HRM analysis. The isolates used had previously also been genotyped using single-nucleotide polymorphisms (SNPs), binary markers, CRISPR HRM, and flaA RFLP.flaA HRM analysis provided resolving power multiplicative to the SNPs, binary markers, and CRISPR HRM and largely concordant with the flaA RFLP. It was concluded that HRM analysis is a promising approach to genotyping based on highly variable genes.

Flow over a cylinder with a hinged-splitter plate

Previous work on rigid splitter plates in the wake of a bluff body has shown that the primary vortex shedding can be suppressed for sufficiently long splitter plates. In the present work, we study the problem of a hinged-splitter plate in the wake of a circular cylinder. The splitter plate can rotate about the hinge at the base of the cylinder due to the unsteady fluid forces acting on it, and hence the communication between the two sides of the wake is not totally disrupted as in the rigid splitter plate case. In our study, we investigate this problem in the limit where the stiffness and internal damping associated with the hinge are negligible, and the mass ratio of the splitter plate is small. The experiments show that the splitter plate oscillations increase with Reynolds numbers at low values of Re, and are found to reach a saturation amplitude level at higher Re, Re>4000. This type of saturation amplitude level that appears to continue indefinitely with Re, appears to be related to the fact that there is no structural restoring force, and has been observed previously for transversely oscillating cylinders with no restorin force. In the present case, the saturation tip amplitude level can be tip to 0.45D, where D is the cylinder diameter. For this hinged-rigid splitter plate case, it is found that the splitter plate length to cylinder diameter ratio (L/D) is crucial in determining the character and magnitude of the oscillations. For small splitter plate length (L/D <= 3.0), the oscillations appear to be nearly periodic with tip amplitudes of about 0.45D nearly independent of L/D. The nondiinensional oscillation frequencies (fD/U) on the other hand are found to continuously vary with L/D from fD/U approximate to 0.2 at L/D = 1 to fD/U approximate to 0.1 at L/D = 3. As the splitter plate length is further increased beyond L/D >= 4.0, the character of the splitter plate oscillations suddenly changes. The oscillations become aperiodic with much smaller amplitudes. In this long splitter plate regime, the spectra of the oscillations become broadband, and are reminiscent of the change in character of the wake oscillations seen in the earlier fixed-rigid splitter plate case for L/D >= 5.0. In the present case of the hinged-splitter plate, the sudden transition seen as the splitter plate length (L/D) is increased from 3 to 4 may be attributed to the fact that the wake vortices are no longer able to synchronize with the plate motions for larger splitter plate lengths. Hence, as observed in other vortex-induced vibration problems, the oscillations becomeaperiodic and the amplitude reduces dramatically.

Estrogen Induction Of Biotin-Binding Protein In Immature Chicks - Kinetics, Hormonal Specificity And Modulation

Employing a specific radioimmunoassay for quantification, the kinetics of estrogen-induced elevation in the plasma concentration of biotin-binding protein (BBP) in immature male chicks was investigated. A single injection of the steroid hormone enhanced the plasma BBP content several-fold at 6 h, reaching peak levels around 48 h and declining thereafter. A 2-fold amplification of the response was evident during secondary stimulation with the hormone. The magnitude of the response was hormonal dose-dependent while the initial lag phase and the time of peak protein accumulation were unaltered within the hormonal doses tested. The circulatory half-life of the specific protein in normal and estrogenized birds was 10 h. Hyperthyroidism markedly decreased the hormonal response while the opposite effect was seen during hypothyroidism. The antiestrogens E- and Z-clomiphene citrate effectively blocked the protein induction whereas progesterone, either alone or in combination with estrogen, was ineffective in modulating the induction. Cycloheximide administration drastically inhibited the inductive response. The above observations clearly suggest that the genes corresponding to the two isofunctional proteins of chicken egg, viz. BBP and avidin, are differentially regulated.

Characterization of highly informative cross-species microsatellite panels for the Australian dugong (Dugong dugon) and Florida manatee (Trichechus manatus latirostris) including five novel primers

The Australian dugong (Dugong dugon) and Florida manatee (Trichechus manatus latirostris) are threatened species of aquatic mammals in the order Sirenia. Sirenian conservation and management actions would benefit from a more complete understanding of genetic diversity and population structure. Generally, species-specific microsatellite markers are employed in conservation genetic studies; however, robust markers can be difficult and costly to isolate. To increase the number of available markers, dugong and manatee microsatellite primers were evaluated for cross-species amplification. Furthermore, one manatee and four dugong novel primers are reported. After polymerase chain reaction optimization, 23 (92%) manatee primers successfully amplified dugong DNA, of which 11 (48%) were polymorphic. Of the 32 dugong primers tested, 27 (84%) yielded product in the manatee, of which 17 (63%) were polymorphic. Dugong and manatee primers were compared and the most informative markers were selected to create robust and informative marker-panels for each species. These cross-species microsatellite marker-panels can be employed to assess other sirenian populations and can provide beneficial information for the protection and management of these unique mammals.

Phenotypic and genotypic variation within populations of kikuyu (Pennisetum clandestinum) in Australia.

Experiments at 2 sites in subtropical eastern Australia investigated the variation in agronomic attributes, quality and genetic structure existing within: naturally-occurring populations of kikuyu ( Pennisetum clandestinum) from within Australia; selections produced from the treatment of Whittet seed with mutagenic chemicals; and available cultivars. Runners were collected from coastal areas extending from Western Australia to the Atherton Tableland in north Queensland. One experiment evaluated 10 mutagenic selections and 4 cultivars in a lattice design and the other evaluated 12 ecotypes and 3 cultivars in a randomised block design. The experimental unit was single plants, which were sown on a 1.5 m grid into a weed-free seed-bed (Mutdapilly) or a killed kikuyu stand (Wollongbar), both of which were kept clear of weeds and other kikuyu plants for the duration of the experiments. Foliage height, forage production and runner yield were assessed. Leaf material was analysed for concentrations of crude protein (CP), acid detergent fibre (ADF) and neutral detergent fibre (NDF) and for in vitro dry matter digestibility (IVDDM) in autumn, winter and spring. DNA was extracted from each plant in the ecotype comparison and subjected to a modified DAF (DNA amplification fingerprinting) analysis to determine the level of genetic relatedness. In the first experiment, none of the mutagenic lines derived from Whittet yielded significantly more or was more digestible than commercial Whittet material, although some selections were superior to the other commercial kikuyu cultivars, Noonan and Crofts, and 'common' kikuyu. However, there were significant differences in plant height and runner expansion. In the second experiment, significant differences in plant height, foliage yield, runner development, and leaf CP, ADF, NDF and IVDDM concentrations were demonstrated between the ecotypes, mutagenic selections and cultivars. There was a 4- to 6-fold difference in plant yield and a 6- to 10-fold difference in runner production between the ecotypes at the 2 sites. Quality of the leaf ranged from 200 to 270 g/kg (CP), from 700 to 770 g/kg (IVDDM), from 170 to 250 g/kg (ADF) and from 470 to 550 g/kg (NDF). Improvements in quality and agronomic attributes were not mutually exclusive. Genetic fingerprint analysis of the kikuyu lines indicated that they formed 2 broad groupings. Most of the regional ecotypes were grouped with 'common' kikuyu as represented by the material collected from Wollongbar, and the Beechmont, Atherton Tableland and Gympie ecotypes were grouped with the registered cultivars Whittet, Noonan and Crofts. Two lines produced by mutagenesis from Whittet remained closely linked to Whittet. These results suggest that there was variation between populations of kikuyu in yield, quality and genetic diversity but that mutagenesis by treating seed with sodium azide and diethylene sulphide did not achieve a significant change in the digestibility of leaf over cv. Whittet.

Inflammation and Complement Activation in Intracranial Artery Aneurysms

Intracranial artery aneurysms (IAs) are estimated to be present in 2.3% of the population. A rupture of an IA causes subarachnoid hemorrhage, with up to 50% mortality. The annual low rupture risk of an IA indicates that most IAs never rupture. The current treatment options are invasive and somewhat risky. Thus rupture-prone IAs should be identified and this requires a better understanding of the IA wall pathobiology. Inflammatory cell infiltrations have been found to precede IA rupture, indicating the role of inflammation in IA wall degeneration and rupture. The complement system is a key mediator of inflammation and house-hold processing of injured tissue. This study aimed at identifying the role of complement activation in IA wall degeneration and the complement activators involved and determining how the complement system is regulated in the IA wall. In immunostainings, the end-product of complement activation, the terminal complement complex (TCC), was located mainly in the outer part of the IA wall, in areas that had also sustained loss of cells. In electron microscopy, the area of maximum TCC accumulation contained cellular debris and evidence of both apoptotic and necrotic cell death. Complement activation correlated with IA wall degeneration and rupture, de-endothelialization, and T-cell and CD163-positive macrophage infiltration. The complement system was found to become activated in all IAs by the classical pathway, with recruitment of alternative pathway amplification. Of the potential activators immunoglobulins G and M and oxidatively modified lipids were found in large areas. Lipid accumulation was observed to clearly colocalize with TCC and C-reactive protein. In the luminal parts of the IA wall, complement activation was limited by cellular expression of protectin (CD59) and extracellular matrix-bound inhibitors, C4b binding protein and factor H whereas the outer part of the wall lacked cells expressing protectin as well as matrix-bound factor H. In single nucleotide polymorphism-analysis, age-related macular degeneration-associated factor H Y402H polymorphism did not associate with the presence of IAs or their rupture The data suggest that complement activation and TCC formation are involved in IA wall degeneration and rupture. Complement seems to become activated by more than one specific activator. The association of complement with de-endothelialization and expression of several complement activators indicate a possible role of endothelial dysfunction and/or impaired clearance mechanisms. Impaired complement regulation seems to be associated with increased complement activation in IA walls. These results stress the role of chronic inflammation in IA wall pathobiology and the regulatory role of complement within this process. Imaging inflammation would possibly enhance the diagnostics of rupture-prone IAs, and targeting IA treatment to prevent chronic inflammation might improve IA treatment in the future.