Hypertrophic cardiomyopathy mutations in the calponin-homology domain of ACTN2 affect actin binding and cardiomyocyte Z-disc incorporation

α-Actinin-2 (ACTN2) is the only muscle isoform of α-actinin expressed in cardiac muscle. Mutations in this protein have been implicated in mild to moderate forms of hypertrophic cardiomyopathy (HCM). We have investigated the effects of two mutations identified from HCM patients, A119T and G111V, on the secondary and tertiary structure of a purified actin binding domain (ABD) of ACTN2 by circular dichroism and X-ray crystallography, and show small but distinct changes for both mutations. We also find that both mutants have reduced F-actin binding affinity, although the differences are not significant. The full length mEos2 tagged protein expressed in adult cardiomyocytes shows that both mutations additionally affect Z-disc localization and dynamic behaviour. Overall, these two mutations have small effects on structure, function and behaviour, which may contribute to a mild phenotype for this disease

Application of direct bioautography and SPME-GC-MS for the study of antibacterial chamomile ingredients

The isolation and characterization of antibacterial chamomile components were performed by the use of direct bioautography and solid phase microextraction (SPME)-GC-MS. Four ingredients, active against Vibrio fischeri, were identified as the polyacetylene geometric isomers cis- and trans-spiroethers, the coumarin related herniarin, and the sesquiterpene alcohol (-)-alpha-bisabolol

Inferring Phylogenies from RAD Sequence Data

Reduced-representation genome sequencing represents a new source of data for systematics, and its potential utility in interspecific phylogeny reconstruction has not yet been explored. One approach that seems especially promising is the use of inexpensive short-read technologies (e.g., Illumina, SOLiD) to sequence restriction-site associated DNA (RAD) – the regions of the genome that flank the recognition sites of restriction enzymes. In this study, we simulated the collection of RAD sequences from sequenced genomes of different taxa (Drosophila, mammals, and yeasts) and developed a proof-of-concept workflow to test whether informative data could be extracted and used to accurately reconstruct “known” phylogenies of species within each group. The workflow consists of three basic steps: first, sequences are clustered by similarity to estimate orthology; second, clusters are filtered by taxonomic coverage; and third, they are aligned and concatenated for “total evidence” phylogenetic analysis. We evaluated the performance of clustering and filtering parameters by comparing the resulting topologies with well-supported reference trees and we were able to identify conditions under which the reference tree was inferred with high support. For Drosophila, whole genome alignments allowed us to directly evaluate which parameters most consistently recovered orthologous sequences. For the parameter ranges explored, we recovered the best results at the low ends of sequence similarity and taxonomic representation of loci; these generated the largest supermatrices with the highest proportion of missing data. Applications of the method to mammals and yeasts were less successful, which we suggest may be due partly to their much deeper evolutionary divergence times compared to Drosophila (crown ages of approximately 100 and 300 versus 60 Mya, respectively). RAD sequences thus appear to hold promise for reconstructing phylogenetic relationships in younger clades in which sufficient numbers of orthologous restriction sites are retained across species

Nanoscale imaging of clinical specimens using pathology-optimized expansion microscopy

Expansion microscopy (ExM), a method for improving the resolution of light microscopy by physically expanding the specimen, has not been applied to clinical tissue samples. Here we report a clinically optimized form of ExM that supports nanoscale imaging of human tissue specimens that have been fixed with formalin, embedded in paraffin, stained with hematoxylin and eosin (H&E), and/or fresh frozen. The method, which we call expansion pathology (ExPath), converts clinical samples into an ExM-compatible state, then applies an ExM protocol with protein anchoring and mechanical homogenization steps optimized for clinical samples. ExPath enables ~70 nm resolution imaging of diverse biomolecules in intact tissues using conventional diffraction-limited microscopes, and standard antibody and fluorescent DNA in situ hybridization reagents. We use ExPath for optical diagnosis of kidney minimal-change disease, which previously required electron microscopy (EM), and demonstrate high-fidelity computational discrimination between early breast neoplastic lesions that to date have challenged human judgment. ExPath may enable the routine use of nanoscale imaging in pathology and clinical research

Genetics of focal segmental glomerulosclerosis

The recent advances in understanding the pathophysiology of focal segmental glomerulosclerosis (FSGS) and molecular function of glomerular filtration barrier come directly from genetic linkage and positional cloning studies. The exact role and function of the newly discovered genes and proteins are being investigated by in vitro and in vivo mechanistic studies. Those genes and proteins interactions seem to change susceptibility to kidney disease progression. Better understanding of their exact role in the development of FSGS may influence future therapies and outcomes in this complex disease

Tests of multiple molecular markers for the identification of Great Spotted and Syrian Woodpeckers and their hybrids

Great Spotted and Syrian Woodpeckers (Dendrocopos major and D. syriacus) are known to hybridize in nature; however, the extent of this phenomenon is not known due to difficulties in hybrid detection based on plumage analyses. Here, we tested five markers (one mitochondrial and four nuclear) and a set of six microsatellite loci for the identification of these two Woodpeckers and their hybrids. Sequencing of DNA from 26 individuals of both Woodpeckers from different parts of their ranges: one allopatric (D. major; Norway) and two sympatric (Poland and Bulgaria) showed that both species can be clearly separated based on all sequence markers. The highest number of fixed nucleotide sites were found in the mtDNA control region and intron 5 of the transforming growth factor. Analyses of microsatellite data distinguished the two species, but all loci showed a large number of common alleles and their utility in identifying hybrids is therefore doubtful. According to the DNA sequence analyses, 2 out of 18 specimens within the sympatric range in Poland were identified as possible hybrids, most probably paternal backcrosses. Moreover, both hybrids are from synantropic populations (settled in cities), whereas none of the D. major sampled in forests and in its allopatric range (Norway) showed signs of an intermixed genotype. Further research on hybridization and introgression in woodpeckers is undoubtedly needed and could be useful for understanding ecological and ethological interactions among these species, particularly for D. syriacus, which is relatively rare in Europe