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.     

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.     

Systematic analysis of the long-chain components of Eubacterium lentum

The cellular long-chain component patterns of 33 strains of Eubacterium lentum were determined by gas chromatography. Two main types of long-chain component patterns were distinguished. The first (26 strains) was characterized by saturated branched-chain fatty acids (br14:0, br15:0, br16:0 and br17:0). The second (7 strains) did not contain branched-chain fatty acids and was characterized by saturated straight-chain fatty acids (11:0, 12:0, 14:0 and 16:0). Both types contained fatty aldehydes and their respective dimethyl acetals (14ald and 14dma, 16ald and 16dma). br16dma was only found in the first type. The G + C content of the DNA (Tm) of the 33 strains varied between 63.7 and 69.1 mol %. Canonical correlation analysis distinguished three subtypes within the first main type.     

Phase determination of the circadian rhythm of conidiation in heterocaryons between two out-of-phase mycelia in Neurospora crassa

Neurospora grows vegetatively as a syncytium in which multiple nuclei exist within a connected cytoplasm. Because of the ability of separate and distinct mycelia to fuse, the possibility exists of generating heterocaryotic cultures in which the nuclei and cytoplasms of two different strains are comingled into the same syncytium. We have used such heterocaryons, in which the component parts differed with respect to their circadian clock phase, to examine whether or not clock-dominant phases exist in the circadian cycle. To this end, the phase subsequent to the formation of heterocaryons by pairs of mycelial discs that are initially at different circadian phases was examined in Neurospora crassa. The resulting phase was an average of the parent phases in many cases, but was sometimes observed to correspond more closely to just one of the original parental phases. In these cases, we did not observe any dominant phases in the circadian cycle; the phase of a particular parent disc was more dominant in the heterocaryon when the proportion of the nuclei from that parent was greater in the heterocaryon. In some instances, which occurred mostly when the difference in phase of the parental discs was large, the resultant phase could not be related in a simple way to the parental phases. An interpretation based on a limit cycle model of the circadian oscillation is possible.     

The effect of hemoglobin ligands on the kinetics of human hemoglobin A1c formation

HbA1c is the most prevalent of the minor human hemoglobins. It is formed by the nonenzymatic addition of glucose to the alpha-amino group of the beta chain by an initial condensation reaction and a subsequent intermolecular Amadori rearrangement. We have developed a method of analysis which utilizes high performance liquid chromatography to follow the formation of HbA1c and greatly simplifies the determination of the kinetic parameters associated with this reaction. This has allowed us to study the effects of several Hb ligands, including the hydrogen ion, on the kinetics of this glycosylation reaction. Both the initial condensation reaction and the subsequent rearrangement are shown to exhibit acid catalysis, but the rate of the condensation step is limited by the extent of protonation of the alpha-amino group. The variation in kinetic parameters as a function of hydrogen ion concentration has allowed us to determine the probable reaction mechanism of HbA1c formation by comparison to previously reported model systems of Schiff base formation and Amadori rearrangement. The formation of pre-HbA1c from deoxy-Hb shows an increased forward rate when compared to oxy-Hb. The presence of physiologic concentrations of CO2 causes a proportional decrease in both k1 and k-1. 2,3-Diphosphoglycerate causes a significant increase in the keq of the formation reaction. The effects of CO and the substitution of L-glucose for D-glucose are not significant.