Origin and diffusion of mtDNA haplogroup X

A maximum parsimony tree of 21 complete mitochondrial DNA (mtDNA) sequences belonging to haplogroup X and the survey of the haplogroup-associated polymorphisms in 13,589 mtDNAs from Eurasia and Africa revealed that haplogroup X is subdivided into two major branches, here defined as “X1” and “X2.” The first is restricted to the populations of North and East Africa and the Near East, whereas X2 encompasses all X mtDNAs from Europe, western and Central Asia, Siberia, and the great majority of the Near East, as well as some North African samples. Subhaplogroup X1 diversity indicates an early coalescence time, whereas X2 has apparently undergone a more recent population expansion in Eurasia, most likely around or after the last glacial maximum. It is notable that X2 includes the two complete Native American X sequences that constitute the distinctive X2a clade, a clade that lacks close relatives in the entire Old World, including Siberia. The position of X2a in the phylogenetic tree suggests an early split from the other X2 clades, likely at the very beginning of their expansion and spread from the Near East.     

Menkes copper-translocating P-type ATPase (ATP7A): biochemical and cell biology properties,and role in Menkes disease

The Menkes copper-translocating P-type ATPase (ATP7A; MNK) is a ubiquitous protein that regulates the absorption of copper in the gastrointestinal tract. Inside cells the protein has a dual function: it delivers copper to cuproenzymes in the Golgi compartment and effluxes excess copper. The latter property is achieved through copper-dependent vesicular trafficking of the Menkes protein to the plasma membrane of the cell. The trafficking mechanism and catalytic activity combine to facilitate absorption and intercellular transport of copper. The mechanism of catalysis and copper-dependent trafficking of the Menkes protein are the subjects of this review. Menkes disease, a systemic copper deficiency disorder, is caused by mutations in the gene encoding the Menkes protein. The effect of these mutations on the catalytic cycle and the cell biology of the Menkes protein, as well as predictions of the effect of particular mutant MNKs on observed Menkes disease symptoms will also be discussed.     

Cryogenic absorption spectra of hydroperoxo-ferric heme oxygenase,the active intermediate of enzymatic heme oxygenation

Using radiolysis with (32)P enriched phosphate as an internal source of ionizing radiation, the formation of hydroperoxo-ferric complex from oxy-ferrous precursor with a high yield was monitored at 77 K in heme oxygenase (HO) by means of optical absorption spectroscopy. Well-resolved absorption spectra (maxima at 421 nm, 530 nm, 557 nm) of hydroperoxo-ferric intermediate of this heme enzyme were measured in 70% glycerol/buffer frozen glasses. After annealing at 210-215 K this complex converts to the product complex, alpha-meso hydroxyheme-HO. No heme degradation products were formed in control experiments with ferric HO or other heme proteins.     

Androgen receptor expression and DNA content of paraffin-embedded archival human prostate tumors

BACKGROUND: Androgen receptors (AR) are expressed in human prostate cells and immunohistochemistry has been used for qualitative analysis of AR expression in prostate tumor cells. Quantitative and multiparametric analysis of receptor expression could be of diagnostic and prognostic value in the management of patients on antiandrogen therapy. Multiparametric flow cytometric methods have been developed for analysis of hormone receptor expression and DNA content in nuclei isolated from formalin-fixed/paraffin-embedded human solid tumors. The present study was undertaken for analysis of AR expression and DNA content in archival human prostate tumors. METHODS: AR expression and DNA content were measured in nuclei isolated by enzyme digestion from thick sections cut from 51 paraffin-embedded human prostate tumors. AR expression in different subpopulations was studied by gated analysis. The relationship among AR activity, DNA content, and histopathological grade was analyzed. RESULTS: Distinct aneuploid populations were observed in 23% of tumors examined. AR activity was observed in all the specimens and the percentage of AR- positive nuclei in the 48 samples analyzed was <10% (n = 4), 11-50% (n = 39), and >51% (n = 5). Tumor subpopulations with aneuploid DNA content had higher AR expression (percent AR-positive cells and mean log fluorescence) than the diploid subpopulations. No strong correlation was seen between AR expression and histopathological grade of the tumors. CONCLUSIONS: Flow cytometric analysis of archival prostate tumor can be used for rapid determination of aneuploid DNA content and AR expression in subpopulations of nuclei isolated from formalin-fixed/paraffin-embedded prostate tumor blocks.     

Alkylcobalt chelates with Schiff bases derived from a β-diketone bearing both alkyl and aryl groups

Organocobalt(III) complexes with Schiff bases derived from a β-diketone bearing both an alkyl and an aryl group have been prepared. The template syntheses using benzoylacetone and ethylenediamine as complexing agents provide a route to alkylcobalt chelates with the corresponding tri- and tetradentate Schiff bases. However, if a β-diketone with two aryl groups, e.g. dibenzoylmethane, was employed as the starting ketoenol component, no organometallic products were detected; a new mixed-ligand ‘inorganic’ chelate of cobalt(II), [Co{O=C(Ph)CH=C(Ph)O}₂(en)], was isolated instead. Its structure as well as that of one of the alkylcobalt complexes with a tridentate Schiff base composed of benzoylacetone and ethylenediamine have been established by X-ray techniques. The current scope of the template synthesis of alkylcobalt complexes with Schiff bases is summarized.     

In Depth Characterization of Repetitive DNA in 23 Plant Genomes Reveals Sources of Genome Size Variation in the Legume Tribe Fabeae

The differential accumulation and elimination of repetitive DNA are key drivers of genome size variation in flowering plants, yet there have been few studies which have analysed how different types of repeats in related species contribute to genome size evolution within a phylogenetic context. This question is addressed here by conducting large-scale comparative analysis of repeats in 23 species from four genera of the monophyletic legume tribe Fabeae, representing a 7.6-fold variation in genome size. Phylogenetic analysis and genome size reconstruction revealed that this diversity arose from genome size expansions and contractions in different lineages during the evolution of Fabeae. Employing a combination of low-pass genome sequencing with novel bioinformatic approaches resulted in identification and quantification of repeats making up 55–83% of the investigated genomes. In turn, this enabled an analysis of how each major repeat type contributed to the genome size variation encountered. Differential accumulation of repetitive DNA was found to account for 85% of the genome size differences between the species, and most (57%) of this variation was found to be driven by a single lineage of Ty3/gypsy LTR-retrotransposons, the Ogre elements. Although the amounts of several other lineages of LTR-retrotransposons and the total amount of satellite DNA were also positively correlated with genome size, their contributions to genome size variation were much smaller (up to 6%). Repeat analysis within a phylogenetic framework also revealed profound differences in the extent of sequence conservation between different repeat types across Fabeae. In addition to these findings, the study has provided a proof of concept for the approach combining recent developments in sequencing and bioinformatics to perform comparative analyses of repetitive DNAs in a large number of non-model species without the need to assemble their genomes.     

Reproducibility of In Vivo Corneal Confocal Microscopy Using an Automated Analysis Program for Detection of Diabetic Sensorimotor Polyneuropathy

Objective

In vivo Corneal Confocal Microscopy (IVCCM) is a validated, non-invasive test for diabetic sensorimotor polyneuropathy (DSP) detection, but its utility is limited by the image analysis time and expertise required. We aimed to determine the inter- and intra-observer reproducibility of a novel automated analysis program compared to manual analysis.

Methods

In a cross-sectional diagnostic study, 20 non-diabetes controls (mean age 41.4±17.3y, HbA1c 5.5±0.4%) and 26 participants with type 1 diabetes (42.8±16.9y, 8.0±1.9%) underwent two separate IVCCM examinations by one observer and a third by an independent observer. Along with nerve density and branch density, corneal nerve fibre length (CNFL) was obtained by manual analysis (CNFL_MANUAL), a protocol in which images were manually selected for automated analysis (CNFL_{SEMI-AUTOMATED}), and one in which selection and analysis were performed electronically (CNFL_{FULLY-AUTOMATED}). Reproducibility of each protocol was determined using intraclass correlation coefficients (ICC) and, as a secondary objective, the method of Bland and Altman was used to explore agreement between protocols.

Results

Mean CNFL_Manual was 16.7±4.0, 13.9±4.2 mm/mm² for non-diabetes controls and diabetes participants, while CNFL_{Semi-Automated} was 10.2±3.3, 8.6±3.0 mm/mm² and CNFL_{Fully-Automated} was 12.5±2.8, 10.9 ± 2.9 mm/mm². Inter-observer ICC and 95% confidence intervals (95%CI) were 0.73(0.56, 0.84), 0.75(0.59, 0.85), and 0.78(0.63, 0.87), respectively (p = NS for all comparisons). Intra-observer ICC and 95%CI were 0.72(0.55, 0.83), 0.74(0.57, 0.85), and 0.84(0.73, 0.91), respectively (p<0.05 for CNFL_{Fully-Automated} compared to others). The other IVCCM parameters had substantially lower ICC compared to those for CNFL. CNFL_{Semi-Automated} and CNFL_{Fully-Automated} underestimated CNFL_Manual by mean and 95%CI of 35.1(-4.5, 67.5)% and 21.0(-21.6, 46.1)%, respectively.

Conclusions

Despite an apparent measurement (underestimation) bias in comparison to the manual strategy of image analysis, fully-automated analysis preserves CNFL reproducibility. Future work must determine the diagnostic thresholds specific to the fully-automated measure of CNFL.     

Potential to exploit postmortem enzyme degradation for evaluating arthropod viability

Inspecting for live organisms is the main method used to verify efficacy of phytosanitary treatments. Evaluating whether small, immobile organisms such as eggs, pupae and scale insects are alive or dead usually involves either checking morphological criteria or rearing them to observe development. These methods can be inaccurate, impractical and time consuming; thus, better methods are needed. To evaluate the potential for developing enzyme-based viability assays, we used electrophoretic gels to evaluate postmortem degradation of ten enzymes in Musca domestica L. (Diptera: Muscidae), four in Bemisia flocculosa Gill and Holder (Hemiptera: Aleyrodidae), and seven in Listronotus bonariensis (Kuschel) (Coleoptera: Curculionidae). Fresh insects displayed strong enzyme activity and distinct bands, but dead insects exhibited either no activity or weakened activity with reduced band resolution and increased migration of stained areas. Of ten enzymes investigated, seven showed clear indications of degradation just 1 day postmortem. Polyacrylamide gel electrophoresis of enzymes can be used to evaluate organism viability and has potential for estimating postmortem intervals. We also measured postmortem degradation rates of five M. domestica enzymes by assaying them in solution; these showed constant or gradually declining activity for 28 days postmortem, so live and dead specimens were less easily distinguished. By assaying enzymes in solution, it is possible to develop quick, easily operated tests that can be used outside the laboratory for a variety of quarantine-related purposes.     

Protease-Sensitive Synthetic Prions

Prions arise when the cellular prion protein (PrP^C) undergoes a self-propagating conformational change; the resulting infectious conformer is designated PrP^Sc. Frequently, PrP^Sc is protease-resistant but protease-sensitive (s) prions have been isolated in humans and other animals. We report here that protease-sensitive, synthetic prions were generated in vitro during polymerization of recombinant (rec) PrP into amyloid fibers. In 22 independent experiments, recPrP amyloid preparations, but not recPrP monomers or oligomers, transmitted disease to transgenic mice (n = 164), denoted Tg9949 mice, that overexpress N-terminally truncated PrP. Tg9949 control mice (n = 174) did not spontaneously generate prions although they were prone to late-onset spontaneous neurological dysfunction. When synthetic prion isolates from infected Tg9949 mice were serially transmitted in the same line of mice, they exhibited sPrP^Sc and caused neurodegeneration. Interestingly, these protease-sensitive prions did not shorten the life span of Tg9949 mice despite causing extensive neurodegeneration. We inoculated three synthetic prion isolates into Tg4053 mice that overexpress full-length PrP; Tg4053 mice are not prone to developing spontaneous neurological dysfunction. The synthetic prion isolates caused disease in 600–750 days in Tg4053 mice, which exhibited sPrP^Sc. These novel synthetic prions demonstrate that conformational changes in wild-type PrP can produce mouse prions composed exclusively of sPrP^Sc.