Novel high-resolution characterization of ancient DNA reveals C > U-type base modification events as the sole cause of post mortem miscoding lesions

Ancient DNA (aDNA) research has long depended on the power of PCR to amplify trace amounts of surviving genetic material from preserved specimens. While PCR permits specific loci to be targeted and amplified, in many ways it can be intrinsically unsuited to damaged and degraded aDNA templates. PCR amplification of aDNA can produce highly-skewed distributions with significant contributions from miscoding lesion damage and non-authentic sequence artefacts. As traditional PCR-based approaches have been unable to fully resolve the molecular nature of aDNA damage over many years, we have developed a novel single primer extension (SPEX)-based approach to generate more accurate sequence information. SPEX targets selected template strands at defined loci and can generate a quantifiable redundancy of coverage; providing new insights into the molecular nature of aDNA damage and fragmentation. SPEX sequence data reveals inherent limitations in both traditional and metagenomic PCR-based approaches to aDNA, which can make current damage analyses and correct genotyping of ancient specimens problematic. In contrast to previous aDNA studies, SPEX provides strong quantitative evidence that C > U-type base modifications are the sole cause of authentic endogenous damage-derived miscoding lesions. This new approach could allow ancient specimens to be genotyped with unprecedented accuracy.     

Effect of adrenocorticoid receptors on potassium and sodium flux in rat C6 glioma cells

C6 glioma cells contain two types of receptors for adrenocorticoids. Glucocorticoid (Type II) receptors are present at higher density and mediate increases in glycerol phosphate dehydrogenase and glutamine synthetase activity. The function of mineralocorticoid (Type I) receptors present at low density in C6 cells is unknown. Since mineralocorticoid (Type I) receptors in renal epithelial cells regulate cation transport, we sought to determine whether adrenocorticoid receptors located in glioma cells are similarly linked to electrolyte transporting activity. Occupation of mineralocorticoid receptors in C6 glioma by adrenocorticoids did not alter Na+ or K+ transport, in contrast to their effects on renal epithelial and vascular smooth muscle cells. Occupation of glucocorticoid receptors produced a 20-25% decrease in K+ uptake into C6 cells, but did not alter Na+ influx. Stimulation of Na+ influx with the ionophore monensin produced a large ouabain-sensitive increase in glucose utilization, as measured by 2-deoxyglucose uptake. However, mineralocorticoid receptor occupation did not alter glucose utilization, providing further evidence that these receptors do not influence Na+ transport in C6 cells. These studies provide evidence that mineralocorticoid receptors in glioma cells do not regulate Na+ or K+ transport. Glial glucocorticoid receptors have an inhibitory effect on glial K+ influx, which may contribute to glucocorticoid hormone effects on brain excitability.     

Hereditary hepatic porphyria with delta aminolevulinate dehydrase deficiency: Immunologic characterization of the non-catalytic enzyme

Summary Immunoreactive -aminolevulinate dehydrase (ALA-D) was measured in lysates from two porphyric patients with ALA-D deficiency (enzyme activities were below 2% of the normal level). By using two different immunologic methods, we found a cross-reactive immunologic material (CRIM+) which corresponded to 20% and 33% of the control level. Therefore the molecular basis that accounts for the deficiency of ALA-D in these patients is a structurally modified enzyme. The methods used to determine the molecular weight (by Western blotting) and the isoelectric point (by chromatofocusing) of the mutants did not show any difference by comparison with the normal enzyme.     

The small-subunit ribosomal RNA gene sequences from the hypotrichous ciliates Oxytricha nova and Stylonychia pustulata

We have determined the complete nucleotide sequence of the small- subunit ribosomal RNA genes for the ciliate protozoans Stylonychia pustulata and Oxytricha nova. The sequences are homologous and sufficiently similar that these organisms must be closely related. In a phylogeny inferred from comparisons of several eukaryotic small-subunit ribosomal RNAs, the divergence of the ciliates from the eukaryotic line of descent is seen to coincide with the radiation of the plants, the animals, and the fungi. This radiation is preceded by the divergence of the slime mold, Dictyostelium discoideum.      

Donor milk intake and infant growth in a South African neonatal unit: a cohort study

Background

Implications of donor milk feedings on infant growth in resource limited settings remain uncertain. This knowledge gap includes the impact of donor milk availability on infant intake of mother’s own milk. Therefore, this investigation aimed to measure intake and growth in infants receiving donor milk when born to women from resource limited backgrounds with high rates of human immunodeficiency virus (HIV).

Methods

A retrospective cohort study enrolled eligible infants admitted to a South African combined neonatal intensive and secondary high care unit, within a one year admission period during 2015, with signed consent for donor milk feedings. A certified milk bank provided donor milk. Daily nutritional intake during the first month was recorded. Details included proportional intake of donor milk, mother’s own milk and infant formula. The primary outcome of infant growth velocity from day back to birth weight to discharge was calculated when length of stay was ≥14 days. Analyses primarily used T-tests; mixed effects models compared weekly calorie intake.

Results

One hundred five infants with donor milk consent were born at 30.9?±?3.6 weeks of gestation, weighing 1389?±?708 g. Forty percent of mothers had HIV. Infant growth velocity did not differ based on percent of feedings as donor milk (≥ 50%: 11.8?±?4.9 g/kg/d; <?50%: 13.5?±?5.3 g/kg/d; p?=?0.3). Percent of feedings from donor milk was similar based on maternal HIV status (positive: 31?±?25%; negative: 36?±?29%; p?=?0.4), as was percent of feedings as mother’s milk (positive: 53?±?35%; negative: 58?±?30%; p?=?0.4). Calorie intake increased markedly during the first two weeks and then plateaued (p?<?0.0001).

Conclusions

Donor milk feedings in higher proportions did not further impair growth of infants managed in a South African combined neonatal intensive and secondary high care unit with growth rates already below reference ranges. The provision of donor milk contributed to feedings being composed of primarily human milk during the first month. Increasing early calorie intake may improve infant growth in this center.

     

Modulation of the phenotype in dominant erythropoietic protoporphyria by a low expression of the normal ferrochelatase allele.

Erythropoietic protoporphyria (EPP) is a monogenic inherited disorder of the heme biosynthetic pathway due to ferrochelatase (FC) deficiency. EPP is generally considered to be transmitted as an autosomal dominant disease with incomplete penetrance, although autosomal recessive inheritance has been documented at the enzymatic and molecular level in some families. In the dominant form of EPP, statistical analysis of FC activities documented a significantly lower mean value in patients than in asymptomatic carriers, suggesting a more complex mode of inheritance. To account for these findings, we tested a multiallelic inheritance model in one EPP family in which the enzymatic data were compatible with this hypothesis. In this EPP family, the specific FC gene mutation was an exon 10 skipping (delta Ex10), resulting from a G deletion within the exon 10 consensus splice donor site. The segregation of all FC alleles within the family was followed using the delta Ex10 mutation and a new intragenic dimorphism (1520 C/T). mRNAs transcribed from each FC allele were then subjected to relative quantification by a primer extension assay and to absolute quantification by a ribonuclease protection assay. The data support the hypothesis that in this family the EPP phenotype results from the coinheritance of a low output normal FC allele and a mutant delta Ex10 allele.