Selective enrichment and sequencing of whole mitochondrial genomes in the presence of nuclear encoded mitochondrial pseudogenes (numts)

Numts are an integral component of many eukaryote genomes offering a snapshot of the evolutionary process that led from the incorporation of an α-proteobacterium into a larger eukaryotic cell some 1.8 billion years ago. Although numt sequence can be harnessed as molecular marker, these sequences often remain unidentified and are mistaken for genuine mtDNA leading to erroneous interpretation of mtDNA data sets. It is therefore indispensable that during the process of amplifying and sequencing mitochondrial genes, preventive measures are taken to ensure the exclusion of numts to guarantee the recovery of genuine mtDNA. This applies to mtDNA analyses in general but especially to studies where mtDNAs are sequenced de novo as the launch pad for subsequent mtDNA-based research. By using a combination of dilution series and nested rolling circle amplification (RCA), we present a novel strategy to selectively amplify mtDNA and exclude the amplification of numt sequence. We have successfully applied this strategy to de novo sequence the mtDNA of the Black Field Cricket Teleogryllus commodus, a species known to contain numts. Aligning our assembled sequence to the reference genome of Teleogryllus emma (GenBank EU557269.1) led to the identification of a numt sequence in the reference sequence. This unexpected result further highlights the need of a reliable and accessible strategy to eliminate this source of error.     

Phenetic analysis shows conspecificity between Hispaniolan Viola domingensis Urban and North American Viola macloskeyi sensu lato (Violaceae)

Phenetic investigations were undertaken to evaluate differences between the cloud forest endemicViola domingensis of the Dominican Republic and the morphologically similarV. macloskeyi of continental North America. Cluster, principal components (PCA), and canonical variates analyses (CVA) of leaf characters and of ratios derived from them consistently separateV. macloskeyi subsp.macloskeyi of the Pacific states fromV. domingensis and transcontinentalV. macloskeyi subsp.pallens. However, all analyses portray the latter two as extensively intergradient extremes of a continuum, withV. domingensis plants tending to be smaller. Phenetic analyses of capsule and seed features, which are often divergent among other species of subsect.Stolonosae, showV. domingensis to be nested within a broaderV. macloskeyi subsp.pallens. Similarly, floral morphology does not provide observable differences between the pair. Results support the synonymy ofV. domingensis underV. macloskeyi subsp.pallens and reveal that it represents a disjunct outlier of an otherwise continental North American violet.     

Effect of eccentric contraction-induced injury on force and intracellular pH in rat skeletal muscles.

The effect of eccentric contraction on force generation and intracellular pH (pH(i)) regulation was investigated in rat soleus muscle. Eccentric muscle damage was induced by stretching muscle bundles by 30% of the optimal length for a series of 10 tetani. After eccentric contractions, there was reduction in force at all stimulation frequencies and a greater reduction in relative force at low-stimulus frequencies. There was also a shift of optimal length to longer lengths. pH(i) was measured with a pH-sensitive probe, 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein AM. pH(i) regulation was studied by inducing an acute acid load with the removal of 20-40 mM ammonium chloride, and the rate of pH(i) recovery was monitored. The acid extrusion rate was obtained by multiplying the rate of pH(i) recovery by the buffering power. The resting pH(i) after eccentric contractions was more acidic, and the rate of recovery from acid load post-eccentric contractions was slower than that from postisometric controls. This is further supported by the slower acid extrusion rate. Amiloride slowed the recovery from an acid load in control experiments. Because the Na(+)/H(+) exchanger is the dominant mechanism for the recovery of pH(i), this suggests that the impairment in the ability of the muscle to regulate pH(i) after eccentric contractions is caused by decreased activity of the Na(+)/H(+) exchanger.     

A mouse model of Haemophilus ducreyi infection (chancroid)

Abstract Six- to 8-week-old CBA mice were inoculated intradermally and/or subcutaneously on each flank with one of 3 strains of Haemophilus ducreyi , 2 of these being recent isolates and the other a reference strain. Pustular nodules developed at all sites the day after 10⁷ organisms had been given. Severe ulceration similar to chancroid occurred in about half of the lesions which took at least 13 days to heal. All three strains of H. ducreyi were isolated from the mice during the first week. The model is invaluable in helping to understand the pathogenicity of H. ducreyi as well as the immunopathogenesis and other aspects of chancroid.