High pressures and asymmetrical stresses in the scoliotic disc in the absence of muscle loading

Background

Loads acting on scoliotic spines are thought to be asymmetric and involved in progression of the scoliotic deformity; abnormal loading patterns lead to changes in bone and disc cell activity and hence to vertebral body and disc wedging. At present however there are no direct measurements of intradiscal stresses or pressures in scoliotic spines. The aim of this study was to obtain quantitative measurements of the intradiscal stress environment in scoliotic intervertebral discs and to determine if loads acting across the scoliotic spine are asymmetric. We performed in vivo measurements of stresses across the intervertebral disc in patients with scoliosis, both parallel (termed horizontal) and perpendicular (termed vertical) to the end plate, using a side mounted pressure transducer (stress profilometry)

Methods

Stress profilometry was used to measure horizontal and vertical stresses at 5 mm intervals across 25 intervertebral discs of 7 scoliotic patients during anterior reconstructive surgery. A state of hydrostatic pressure was defined by identical horizontal and vertical stresses for at least two consecutive readings. Results were compared with similar stress profiles measured during surgery across 10 discs of 4 spines with no lateral curvature and with data from the literature.

Results

Profiles across scoliotic discs were very different from those of normal, young, healthy discs of equivalent age previously presented in the literature. Hydrostatic pressure regions were only seen in 14/25 discs, extended only over a short distance. Non-scoliotic discs of equivalent age would be expected to show large centrally placed hydrostatic nuclear regions in all discs. Mean pressures were significantly greater (0.25 MPa) than those measured in other anaesthetised patients (<0.07 MPa). A stress peak was seen in the concave annulus in 13/25 discs. Stresses in the concave annulus were greater than in the convex annulus indicating asymmetric loading in these anaesthetised, recumbent patients.

Conclusion

Intradiscal pressures and stresses in scoliotic discs are abnormal, asymmetrical and high in magnitude even in the absence of significant applied muscle loading. The origin of these abnormal stresses is unclear.     

Sampling Rhyparida nitida Clark (Coleoptera: Chrysomelidae) and symptoms of their damage on sugarcane

Sampling statistics were determined for larvae, pupae and adults of the chrysomelid Rhyparida nitida associated with sugarcane in Australia and for symptoms of their damage. Iwao's patchiness regression was inappropriate for modelling the mean–variance relationships of the insect counts. Taylor's power law was used to model these data and relationships were developed for counts of small, medium and large larvae, all larvae combined, pupae and adults. The mean–variance relationships of counts of live shoots and shoots killed by larvae of R. nitida were modelled using Iwao's patchiness regression; Taylor's power law was not appropriate to either data set. Relationships to determine sample sizes for fixed levels of precision and fixed-precision-level stop lines for sequential sampling of the different stages and live and dead shoots were also developed. Neither the ln(x + 1) transformation nor the Healy and Taylor transformation consistently standardised the mean–variance relationships of insect counts and the appropriate transformation should be selected on a case-by-case basis. Counts of both live and dead shoots were adequately transformed by the Iwao and Kuno transformation.     

Sequencing of 15 622 gene‐bearing BACs clarifies the gene‐dense regions of the barley genome

Barley (Hordeum vulgare L.) possesses a large and highly repetitive genome of 5.1 Gb that has hindered the development of a complete sequence. In 2012, the International Barley Sequencing Consortium released a resource integrating whole‐genome shotgun sequences with a physical and genetic framework. However, because only 6278 bacterial artificial chromosome (BACs) in the physical map were sequenced, fine structure was limited. To gain access to the gene‐containing portion of the barley genome at high resolution, we identified and sequenced 15 622 BACs representing the minimal tiling path of 72 052 physical‐mapped gene‐bearing BACs. This generated ~1.7 Gb of genomic sequence containing an estimated 2/3 of all Morex barley genes. Exploration of these sequenced BACs revealed that although distal ends of chromosomes contain most of the gene‐enriched BACs and are characterized by high recombination rates, there are also gene‐dense regions with suppressed recombination. We made use of published map‐anchored sequence data from Aegilops tauschii to develop a synteny viewer between barley and the ancestor of the wheat D‐genome. Except for some notable inversions, there is a high level of collinearity between the two species. The software HarvEST:Barley provides facile access to BAC sequences and their annotations, along with the barley–Ae. tauschii synteny viewer. These BAC sequences constitute a resource to improve the efficiency of marker development, map‐based cloning, and comparative genomics in barley and related crops. Additional knowledge about regions of the barley genome that are gene‐dense but low recombination is particularly relevant.     

Asymmetric introgression between sympatric molestus and pipiens forms of Culex pipiens (Diptera: Culicidae) in the Comporta region, Portugal

Background  

Culex pipiens L. is the most widespread mosquito vector in temperate regions. This species consists of two forms, denoted molestus and pipiens, that exhibit important behavioural and physiological differences. The evolutionary relationships and taxonomic status of these forms remain unclear. In northern European latitudes molestus and pipiens populations occupy different habitats (underground vs. aboveground), a separation that most likely promotes genetic isolation between forms. However, the same does not hold in southern Europe where both forms occur aboveground in sympatry. In these southern habitats, the extent of hybridisation and its impact on the extent of genetic divergence between forms under sympatric conditions has not been clarified. For this purpose, we have used phenotypic and genetic data to characterise Cx. pipiens collected aboveground in Portugal. Our aims were to determine levels of genetic differentiation and the degree of hybridisation between forms occurring in sympatry, and to relate these with both evolutionary and epidemiological tenets of this biological group.     

Transposon-mediated single-copy gene delivery leads to increased transgene expression stability in barley

Instability of transgene expression in plants is often associated with complex multicopy patterns of transgene integration at the same locus, as well as position effects due to random integration. Based on maize transposable elements Activator (Ac) and Dissociation (Ds), we developed a method to generate large numbers of transgenic barley (Hordeum vulgare var Golden Promise) plants, each carrying a single transgene copy at different locations. Plants expressing Ac transposase (AcTPase) were crossed with plants containing one or more copies of bar, a selectable herbicide (Basta) resistance gene, located between inverted-repeat Ds ends (Ds-bar). F(1) plants were self-pollinated and the F(2) generation was analyzed to identify plants segregating for transposed Ds-bar elements. Of Ds-bar transpositions, 25% were in unlinked sites that segregated from vector sequences, other Ds-bar copies, and the AcTPase gene, resulting in numerous single-copy Ds-bar plants carrying the transgene at different locations. Transgene expression in F(2) plants with transposed Ds-bar was 100% stable, whereas only 23% of F(2) plants carrying Ds-bar at the original site expressed the transgene product stably. In F(3) and F(4) populations, transgene expression in 81.5% of plants from progeny of F(2) plants with single-copy, transposed Ds-bar remained completely stable. Analysis of the integration site in single-copy plants showed that transposed Ds-bar inserted into single- or low-copy regions of the genome, whereas silenced Ds-bar elements at their original location were inserted into redundant or highly repetitive genomic regions. Methylation of the non-transposed transgene and its promoter, as well as a higher condensation of the chromatin around the original integration site, was associated with plants exhibiting transgene silencing.     

Mapping Ds insertions in barley using a sequence-based approach

A transposon tagging system, based upon maize Ac/Ds elements, was developed in barley (Hordeum vulgare subsp. vulgare). The long-term objective of this project is to identify a set of lines with Ds insertions dispersed throughout the genome as a comprehensive tool for gene discovery and reverse genetics. AcTPase and Ds-bar elements were introduced into immature embryos of Golden Promise by biolistic transformation. Subsequent transposition and segregation of Ds away from AcTPase and the original site of integration resulted in new lines, each containing a stabilized Ds element in a new location. The sequence of the genomic DNA flanking the Ds elements was obtained by inverse PCR and TAIL-PCR. Using a sequence-based mapping strategy, we determined the genome locations of the Ds insertions in 19 independent lines using primarily restriction digest-based assays of PCR-amplified single nucleotide polymorphisms and PCR-based assays of insertions or deletions.The proncipal strategy was to identify and map sequence polymorphisms in the regions corresponding to the flanking DNA using the Oregon Wolfe Barley mapping population. The mapping results obtained by the sequence-based approach were confirmed by RFLP analyses in four of the lines. In addition, cloned DNA sequences corresponding to the flanking DNA were used to assign map locations to Morex-derived genomic BAC library inserts, thus integrating genetic and physical maps of barley. BLAST search results indicate that the majority of the transposed Ds elements are found within predicted or known coding sequences. Transposon tagging in barley using Ac/Ds thus promises to provide a useful tool for studies on the functional genomics of the Triticeae.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by M.-A. GrandbastienThe first three authors contributed equally to this work