Validation of DNA methylation profiling in formalin-fixed paraffin-embedded samples using the Infinium HumanMethylation450 Microarray

A formalin-fixed paraffin-embedded (FFPE) sample usually yields highly degraded DNA, which limits the use of techniques requiring high-quality DNA, such as Infinium Methylation microarrays. To overcome this restriction, we have applied an FFPE restoration procedure consisting of DNA repair and ligation processes in a set of paired fresh-frozen (FF) and FFPE samples. We validated the FFPE results in comparison with matched FF samples, enabling us to use FFPE samples on the Infinium HumanMethylation450 Methylation array.     

Genetic diversity in the endangered Sicilian endemic Brassica rupestris: Proposals for a conservation strategy

Abstract

Brassica rupestris Raf. is a chasmophyte species that includes two subspecies, both endemic to Central-Western Sicily (Italy). Inter-Simple Sequence Repeat (ISSR) markers were used to detect genetic diversity within and among eight populations representative of the species' distribution range. High levels of genetic diversity were revealed both at the population (PPB = 53.88%, H _S = 0.212, Sh = 0.309) and at the species level (PPB = 96.55%, H _T = 0.307, Sh = 0.464). The correlation between genetic and geographical distances was negative (Mantel test, r = ?0.06, P < 0.95). The two subspecies of B. rupestris, subsp. rupestris and subsp. hispida, showed remarkable genetic similarity and molecular data did not unequivocally support their distinctness. The pattern of genetic variation revealed by our study bears important consequences for conservation management: It is desirable to preserve B. rupestris populations in situ with a “dynamic” strategy, while, ex situ conservation programmes might be improved to safeguard maximum genetic diversity.     

A nonradioisotope biomedical assay for intact oligonucleotide and its chain-shortened metabolites used for determination of exposure and elimination half-life of antisense drugs in tissue.

Rigorous extraction methods coupled with capillary gel electrophoresis (CGE) provide a basis for a nonradiolabel assay for quantitation of intact antisense drug and its numerous chain-shortened metabolites. As part of the validation of the CGE method, we compared the quantitation of unlabeled ISIS 3521 (ISI 641A) and its chain-shortened metabolites with total radioactivity of [(35)S]-ISIS 3521. ISIS 3521 was labeled on the fifth nucleotide linkage from the 5'-end with (35)S by well-established methods. Multiple tissues collected from rats after administration of [(35)S]-ISIS 3521 were assayed by both radiolabel (liquid scintillation spectroscopy) and CGE methods. The CGE method provided accurate quantitation of the drug and its metabolites in kidney cortex and liver tissues. The correlation between methods for multiple tissues over time was excellent with 88.5% of the measurements being statistically equivalent. These data suggest that CGE is an accurate means of quantitating oligonucleotide in tissue and that it compares favorably with traditional radiochemical techniques. Clearance half-lives for total measurable oligonucleotides were equivalent to clearance of total radioactivity in both liver and kidney with the longest clearance half-life associated with the kidney.     

Rapid, simple and sensitive high-performance liquid chromatographic method for detection and determination of acyclovir in human plasma and its use in bioavailability studies

A rapid, simple and sensitive reversed-phase high-performance liquid chromatographic (HPLC) method has been developed for the measurement of acyclovir concentrations in human plasma and its use in bioavailability studies is evaluated. Unchanged acyclovir has been quantified without the introduction of an internal standard using the present method. Human plasma proteins were selectively precipitated by the addition of 7% perchloric acid to spiked plasma samples or to the plasma samples obtained after acyclovir administration to human volunteers and the mixture was spun at 1000 g for 10 min. The supernatant was directly injected into a Novaflex C₁₈ column and detected at 254 nm. The mobile phase consisted of octane sulfonic acid buffer (pH 2.5) and methanol (92:08). The limit of quantitation for acyclovir in plasma was 20 ng/ml, which enabled the determination of the area under the curve (AUC) more precisely, that is, it is much closer to its extrapolated value. The present method has been successfully applied to samples from bioavailability studies.     

Bloodmeal analysis in Culicoides midges collected near horses,donkeys and zebras in the Eastern Cape,South Africa

An upsurge in African horse sickness (AHS) in the Eastern Cape, South Africa, from 2006 led to an epidemiological reassessment of the disease there. Light trapping surveys carried out near horses, donkeys and zebras in 2014–2016 collected 39 species of Culicoides midge (Diptera: Ceratopogonidae) that are potential vectors of AHS. To establish if these midges fed on equids, DNA sequences were obtained from the gut contents of 52 female midges (35 freshly blood‐fed, 13 gravid and four parous), representing 11 species collected across 11 sites. Culicoides leucostictus fed on all three equids. Culicoides bolitinos, Culicoides imicola and Culicoides magnus fed on both horses and donkeys. Culicoides onderstepoortensis fed on donkeys, and Culicoides similis and Culicoides pycnostictus fed on zebras. Bloodmeals from cows, pigs, warthogs, impalas and a domestic dog were also identified in various species, but none of the midges tested had fed on birds. These results contribute to knowledge of the vectorial capacity of several species of Culicoides with regard to AHS in the Eastern Cape and point to potential reservoir hosts, of which donkeys, zebras and domestic dogs have previously been found to harbour AHS. Blood‐fed midges were also obtained throughout winter, indicating the potential for endemic AHS in the province.     

Patterns of Sleeping Site and Sleeping Tree Selection by Black-and-Gold Howler Monkeys (Alouatta caraya) in Northern Argentina

International Journal of Primatology - The selection of sleeping sites and sleeping trees in nonhuman primates is related to social and ecological factors. We investigate the role of body...     

Ontogeny of the hyobranchial apparatus in the salamanders Ambystoma talpoideum (Ambystomatidae) and Notophthalmus viridescens (Salamandridae): The ecological morphology of two neotenic strategies

Comparison of metamorphosis of skull and hyobranchial system in two species of neotenic salamanders reveals two different types of neoteny. Ambystoma talpoideum is completely neotenic owing to delayed metamorphosis. Notophthalmus viridescens exhibits limited neoteny as a result of incomplete metamorphosis. Morphological details of neoteny are compared to life history in both species in order to discuss the ecological morphology of the two neotenic strategies. Comparisons to Taricha granulosa, Triturus vulgaris, and Ambystoma gracile indicate that these two strategies are widely employed and may represent familial patterns.     

Molecular genetics of peripheral populations of Nova Scotian Unionidae (Mollusca: Bivalvia)

Peripheral populations of eight species of freshwater bivalves (Unionidae.) extending their geographic ranges into Nova Scotia, Canada, were examined electrophoretically to determine both the extent of genetic variability within such populations, and whether the hypothesized pathway of colonization across the Isthmus of Chignecto is reflected in patterns of genetic resemblance among these populations. The Nova Scotian species examined could be separated into two groups based on levels of observed heterozygosity and levels of variability in allele frequencies. The first group is characterized by low levels of heterozygosity and polymorphism compared with north-eastern American populations, and in the case of one species, Elliptio complanala, considerable variability in allele frequencies among populations occurring in similar habitats in different drainages. Populations of E. complanata from Nova Scotia can be differentiated from conspecific populations on the southern Atlantic Slope by possession of fast alleles at two loci. Multivariate analyses define subgroups within populations of E. complanata consistent with hypothesis that the species invaded Nova Scotia by way of the Isthmus of Chignecto, and then split into two groups, one of which colonized Cape Breton to the north and the other of which colonized southern areas of the Province. The second group of Nova Scotian species is characterized by little reduction in heterozygosity and polymorphism compared with values observed among north-eastern American conspecifics or congeners, little variability in allele frequencies from population to population, and little evidence to suggest that these species were dependent on the land bridge to invade the Province. The type of dispersal is hypothesized to be responsible, in part, for these differences: larvae of species in the first group rely on a parasitic attachment to fish with territorial habits limited to fresh water, and are thus likely to invade new drainages separated by salt water by chance, in small numbers, and in stepping-stone fashion. Species in the second group parasitize anadromous or saltwater tolerant hosts, are likely to be introduced into new habitats in greater numbers and/or receive greater amounts of gene flow subsequent to colonization, and seem less dependent on land-bridges to colonize new habitats.