Liver mtDNA content increases during development: a comparison of methods and the importance of age- and tissue-specific controls for the diagnosis of mtDNA depletion

BACKGROUND: The quantitative loss of mitochondrial DNA (mtDNA) known as mtDNA depletion, often gives rise to liver disease. The diagnosis of mtDNA depletion syndrome is frequently imprecise, both for technical reasons and because of the lack of established age-adjusted normal ranges. We aimed to refine quantitative methods for diagnosing the hepatic type of mtDNA depletion syndrome, firstly by establishing an age-matched reference range for mitochondrial to nuclear DNA ratio (henceforth "mtDNA content") and secondly by investigating mtDNA in fibroblasts. METHODS: By comparing realtime PCR with an established method for quantifying mtDNA content we established a reference range for young children using biopsy and post-mortem material from patients <15 years. In addition, we investigated the arrangement of mtDNA in nucleoids from fibroblasts using fluorescence microscopy. RESULTS: Both methods showed that the mtDNA content of liver increases rapidly over the perinatal period. In a patient whose liver mtDNA content fell, but remained within the reference range, early investigation and age-matched controls were essential, as we found a progressive increase in muscle mtDNA copy number, respiratory chain activity and muscle power with age. In three further patients, fluorescence microscopy of the fibroblasts proved diagnostic. In one case a movement disorder was an important pointer. CONCLUSIONS: These cases highlight the (i) need for comparing mtDNA copy number data generated from patients to DNA isolated from an age-matched normal range from the tissue of interest and (ii) the utility of mtDNA staining with PicoGreen as a method to detect aberrant nucleoid morphology in mtDNA depletion patient fibroblast lines when affected tissues are not available for measuring mtDNA copy number.     

The role of intraspecific competition in the dispersal of an invasive fish

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The influence of American mink odour on the spatial distribution and behaviour of water voles

The perception and assessment of predation risk often cause changes in the activities of animals and induce behavioural responses that may in turn affect their movements and distribution. To simulate high predation risk in a midfield pond riparian habitat, we used fresh faeces from ranch American mink Neovison vison and recorded behavioural responses of water voles Arvicola amphibius. In areas where mink odour was deployed, the numbers of captured vole individuals and their trappability were significantly lower than in control areas. Several voles migrated from the zones with deployed mink faeces to the areas without faeces, thus proving that increased predation risk affects the distribution of individuals in a population. The response to mink odour was much more pronounced in females than in males; in areas with deployed mink faeces, not a single female was trapped. We conclude that although American mink is a non‐native, invasive predator, water voles respond to mink odour by reducing their activity and/or by avoiding places with higher predation risk.     

Introduced cats Felis catus eating a continental fauna: inventory and traits of Australian mammal species killed

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No sitting on the fence: protecting wetlands from feral pig damage by exclusion fences requires effective fence maintenance

Feral pigs damage the significant ecological and cultural values of tropical Australian wetlands. Control measures such as culling, baiting, and trapping can reduce overall pig populations, but do not eliminate the substantial physical damage to wetlands that can occur from just a few individuals. Exclusion fences have been adopted as a potential technique to prevent damage to selected wetlands. To test the effectiveness of exclusion fences we measured the physical damage caused by pigs to multiple wetlands in the Archer River catchment of tropical northern Australia. Wetlands were fenced using a typical cattle exclusion fence, a specific pig exclusion fence or had no fence. Initial analyses of these fence treatments showed no significant difference in the intensity of physical pig damage to exposed wetland sediments and fringing vegetation. However, several of the pig exclusion fences were found to have been compromised. Reanalysis indicated wetlands with functioning pig exclusion fences had no physical pig damage and this was significantly less damage than in all other treatments. In contrast, wetlands with compromised pig exclusion fences had damage that was statistically equivalent to sites without fences or with cattle exclusion fences, but in individual cases had the worst damage recorded in any of the treatments. Compromised pig exclusion fencing of wetlands can thus be worse than having no fencing at all. This suggests that the successful prevention of pig damage to wetlands by exclusion fences requires ongoing and effective fence monitoring and maintenance regimes.

     

Ergosterol and Fusarium mycotoxins content in two maize cultivars under different forms of nitrogen fertilizers

Mycotoxins are secondary metabolites produced by many filamentous fungi, such as Fusarium, and are widespread in nature and can adversely affect almost all organisms. Mycotoxins can be formed in very different conditions such as when plants are growing or in crops that are stored in improper conditions. One of the methods to prevent the formation of mycotoxins in powdered feed is using varieties of appropriate quality and proper mineral fertilization, primarily nitrogen. In this study, the concentration of ergosterol and mycotoxins in the kernels of two maize cultivars from different maturity groups in Poland during the harvest of 2011 and 2012 was investigated using HPLC methods. We aimed to analyse the concentration of ergosterol as a fungal biomass indicator and mycotoxins from two maize cultivars under six different forms of nitrogen fertilizers. The “stay‐green” hybrid ES Paroli was characterized by a significantly lower ergosterol and the ability to accumulate the mycotoxins, compared to the classic cultivar ES Palazzo. The varieties of maize that remain green for a longer time period, the so‐called stay‐green type, are characterized by a different coefficient of nitrogen remobilization compared to the classic hybrids. Their dynamics of growth and accumulation of nutrients indicates a nitrogen fertilization system, indicating that slow‐release fertilizers are potentially more adapted to the cycles of maize vegetation. Hence, compared to the classic variant, the “stay‐green” variety uses nitrogen more effectively from mineral fertilizers. Such hybrids are healthier and remain in good condition for a longer time; therefore, they are characterized by a lower pathogen pressure. Thus, the cultivation of such varieties can be considered as one of the elements of integrated maize production. We observed identical reactions of the studied experimental factors for both vegetation seasons, which indicates the lack of environmental impact on the functioning and interaction of experimental factors.     

Mutant phosphatidate phosphatase Pah1-W637A exhibits altered phosphorylation,membrane association,and enzyme function in yeast

The Saccharomyces cerevisiae PAH1-encoded phosphatidate (PA) phosphatase, which catalyzes the dephosphorylation of PA to produce diacylglycerol, controls the bifurcation of PA into triacylglycerol synthesis and phospholipid synthesis. Pah1 is inactive in the cytosol as a phosphorylated form and becomes active on the membrane as a dephosphorylated form by the Nem1–Spo7 protein phosphatase. We show that the conserved Trp-637 residue of Pah1, located in the intrinsically disordered region, is required for normal synthesis of membrane phospholipids, sterols, triacylglycerol, and the formation of lipid droplets. Analysis of mutant Pah1-W637A showed that the tryptophan residue is involved in the phosphorylation-mediated/dephosphorylation-mediated membrane association of the enzyme and its catalytic activity. The endogenous phosphorylation of Pah1-W637A was increased at the sites of the N-terminal region but was decreased at the sites of the C-terminal region. The altered phosphorylation correlated with an increase in its membrane association. In addition, membrane-associated PA phosphatase activity in vitro was elevated in cells expressing Pah1-W637A as a result of the increased membrane association of the mutant enzyme. However, the inherent catalytic function of Pah1 was not affected by the W637A mutation. Prediction of Pah1 structure by AlphaFold shows that Trp-637 and the catalytic residues Asp-398 and Asp-400 in the haloacid dehalogenase-like domain almost lie in the same plane, suggesting that these residues are important to properly position the enzyme for substrate recognition at the membrane surface. These findings underscore the importance of Trp-637 in Pah1 regulation by phosphorylation, membrane association of the enzyme, and its function in lipid synthesis.     

Relationships of morphological and phototextural attributes of presumptive ovine zygotes and early embryos to their developmental competence in vitro: a preliminary assessment using time-lapse imaging

The assessment of morphology and digital image opacity may provide valuable information on the present embryo quality. Time-lapse imaging has been employed in research to establish a means of monitoring the dynamic nature of preimplantation embryo development. The aim of present study was to use time-lapse imaging for assessing various prospective morphometric and phototextural markers of the developmental potential of in vitro-derived ovine embryos. Oocytes were obtained by scarification of ovaries from nine Polish Longwool ewes. After in vitro maturation (IVM) and fertilization (IVF) of oocytes with fresh ram semen, the development of embryos to the blastocyst stage was monitored and evaluated using Primo Vision time-lapse imaging technology. Commercially available Image-Pro^® Plus software was used to measure zona pellucida thickness, embryo diameter, total area of the perivitelline space, cellular grey-scale pixel intensity and cellular pixel heterogeneity. Statistical assessment of all attributes was done at various time points during embryo development (i.e., presumptive zygote stage: t(0); first cleavage detected at t(2) or t(3); and second cleavage detected at t(4) or t(6)). Out of thirty-seven zygotes analyzed in this study, five did not divide, 26 arrested before and six developed to the blastocyst stage. Our present results indicate that most parameters analyzed did not differ among embryos varying in their developmental fate except for the perivitelline space area that was greater (P<0.05) for non-dividing zygotes than future blastocysts at the presumptive zygote stage (4040±1850 vs. 857±262 µm², respectively; means±SEM). Consequently, the measurement of perivitelline space at t(0) can potentially be used to prognosticate developmental potential of in vitro-produced ovine embryos albeit further confirmational studies are needed.