Development of a Multiple Loci Variable Number of Tandem Repeats Analysis (MLVA) to Unravel the Intra-Pathovar Structure of Pseudomonas syringae pv. actinidiae Populations Worldwide

The bacterial canker of kiwifruit by Pseudomonas syringae pv. actinidiae is an emblematic example of a catastrophic disease of fruit crops. In 2008 a new, extremely virulent form of the pathogen emerged and rapidly devastated many Actinidia spp. orchards all over the world. In order to understand differences in populations within this pathovar and to elucidate their diffusion and movements on world scale, it is necessary to be able to quickly and on a routine basis compare new isolates with previous records. In this report a worldwide collection of 142 strains was analyzed by MLVA, chosen as investigative technique for its efficacy, reproducibility, simplicity and low cost. A panel of 13 Variable Number of Tandem Repeats (VNTR) loci was identified and used to describe the pathogen population. The MLVA clustering is highly congruent with the population structure as previously established by other molecular approaches including whole genome sequencing and correlates with geographic origin, time of isolation and virulence. For convenience, we divided the VNTR loci in two panels. Panel 1 assay, using six loci, recognizes 23 different haplotypes, clustered into ten complexes with highest congruence with previous classifications. Panel 2, with seven VNTR loci, provides discriminatory power. Using the total set of 13 VNTR loci, 58 haplotypes can be distinguished. The recent hypervirulent type shows very limited diversity and includes, beside the strains from Europe, New Zealand and Chile, a few strains from Shaanxi, China. A broad genetic variability is observed in China, but different types are also retrievable in Japan and Korea. The low virulent strains cluster together and are very different from the other MLVA genotypes. Data were used to generate a public database in MLVAbank. MLVA represents a very promising first-line assay for large-scale routine genotyping, prior to whole genome sequencing of only the most relevant samples.     

αII-Spectrin Regulates Invadosome Stability and Extracellular Matrix Degradation

Invadosomes are actin-rich adhesion structures involved in tissue invasion and extracellular matrix (ECM) remodelling. αII-Spectrin, an ubiquitous scaffolding component of the membrane skeleton and a partner of actin regulators (ABI1, VASP and WASL), accumulates highly and specifically in the invadosomes of multiple cell types, such as mouse embryonic fibroblasts (MEFs) expressing SrcY527F, the constitutively active form of Src or activated HMEC-1 endothelial cells. FRAP and live-imaging analysis revealed that αII-spectrin is a highly dynamic component of invadosomes as actin present in the structures core. Knockdown of αII-spectrin expression destabilizes invadosomes and reduces the ability of the remaining invadosomes to digest the ECM and to promote invasion. The ECM degradation defect observed in spectrin-depleted-cells is associated with highly dynamic and unstable invadosome rings. Moreover, FRAP measurement showed the specific involvement of αII-spectrin in the regulation of the mobile/immobile β3-integrin ratio in invadosomes. Our findings suggest that spectrin could regulate invadosome function and maturation by modulating integrin mobility in the membrane, allowing the normal processes of adhesion, invasion and matrix degradation. Altogether, these data highlight a new function for spectrins in the stability of invadosomes and the coupling between actin regulation and ECM degradation.     

A Non-Synonymous HMGA2 Variant Decreases Height in Shetland Ponies and Other Small Horses

The identification of quantitative trait loci (QTL) such as height and their underlying causative variants is still challenging and often requires large sample sizes. In humans hundreds of loci with small effects control the heritable portion of height variability. In domestic animals, typically only a few loci with comparatively large effects explain a major fraction of the heritability. We investigated height at withers in Shetland ponies and mapped a QTL to ECA 6 by genome-wide association (GWAS) using a small cohort of only 48 animals and the Illumina equine SNP70 BeadChip. Fine-mapping revealed a shared haplotype block of 793 kb in small Shetland ponies. The HMGA2 gene, known to be associated with height in horses and many other species, was located in the associated haplotype. After closing a gap in the equine reference genome we identified a non-synonymous variant in the first exon of HMGA2 in small Shetland ponies. The variant was predicted to affect the functionally important first AT-hook DNA binding domain of the HMGA2 protein (c.83G>A; p.G28E). We assessed the functional impact and found impaired DNA binding of a peptide with the mutant sequence in an electrophoretic mobility shift assay. This suggests that the HMGA2 variant also affects DNA binding in vivo and thus leads to reduced growth and a smaller stature in Shetland ponies. The identified HMGA2 variant also segregates in several other pony breeds but was not found in regular-sized horse breeds. We therefore conclude that we identified a quantitative trait nucleotide for height in horses.     

Trachypogon plumosus (Poaceae,Andropogoneae): ancient thatch and more from the ceren site,El Salvador

Paleoethnobotanical studies in the Zapotitdn Valley of north-central El Salvador revealed that a species of grass,Trachypogon plumosus, was in common use as a thatch material at the Ceren site, a small mesoamerican farming community inundated by volcanic ash circaa.d. 590. Although the grass must have been common in Precolumbian times, repeated attempts to collect fresh specimens from the modern valley were unsuccessful. A survey of the major herbaria with collections from El Salvador likewise demonstrated a lack of modern specimens from the Zapotitdn Valley. The elimination of theT. plumosus from this area and its characteristic savanna habitat probably occurred as a result of the introduction of competing Old World grasses and excessive herbivory. This finding demonstrates why conservationists working in the tropics should be concerned not only with the extinction of rain forest habitats, but with the elimination of other habitats as well.     

Long-chain Acyl-CoA Dehydrogenase Deficiency as a Cause of Pulmonary Surfactant Dysfunction

Long-chain acyl-CoA dehydrogenase (LCAD) is a mitochondrial fatty acid oxidation enzyme whose expression in humans is low or absent in organs known to utilize fatty acids for energy such as heart, muscle, and liver. This study demonstrates localization of LCAD to human alveolar type II pneumocytes, which synthesize and secrete pulmonary surfactant. The physiological role of LCAD and the fatty acid oxidation pathway in lung was subsequently studied using LCAD knock-out mice. Lung fatty acid oxidation was reduced in LCAD^−/− mice. LCAD^−/− mice demonstrated reduced pulmonary compliance, but histological examination of lung tissue revealed no obvious signs of inflammation or pathology. The changes in lung mechanics were found to be due to pulmonary surfactant dysfunction. Large aggregate surfactant isolated from LCAD^−/− mouse lavage fluid had significantly reduced phospholipid content as well as alterations in the acyl chain composition of phosphatidylcholine and phosphatidylglycerol. LCAD^−/− surfactant demonstrated functional abnormalities when subjected to dynamic compression-expansion cycling on a constrained drop surfactometer. Serum albumin, which has been shown to degrade and inactivate pulmonary surfactant, was significantly increased in LCAD^−/− lavage fluid, suggesting increased epithelial permeability. Finally, we identified two cases of sudden unexplained infant death where no lung LCAD antigen was detectable. Both infants were homozygous for an amino acid changing polymorphism (K333Q). These findings for the first time identify the fatty acid oxidation pathway and LCAD in particular as factors contributing to the pathophysiology of pulmonary disease.