A novel strategy for defining haplotypes by selective depletion using restriction enzymes

Various single nucleotide polymorphisms (SNPs) have been investigated regarding association with gene expression levels or human diseases. Although different SNPs within one gene are frequently analyzed individually, it is highly probable that in the majority of the cases, a precise combination of SNP alleles, i.e., haplotype, determines a functional trait. Methods commonly used for haplotype determination, involving studies in families, cloning, or somatic cell hybrids, are expensive and time-consuming. We herein suggest a novel and simple strategy for haplotype determination, involving selective haplotype depletion with a restriction enzyme, followed by sequencing. We studied 11 LTA gene polymorphisms in 102 Brazilian individuals, and we applied this novel methodology for haplotyping 67 out of 70 LTA heterozygous individuals. We concluded that the method is rapid and efficient, and, as it includes only simple and widespread-used techniques, it could be used in most of the laboratories without further investment in equipments. The wider usage of haplotyping could be important to clarify contradictory results frequently observed among studies that focus on a single SNP. Maria Gerbase-DeLima and Andrey Morgun are co-senior authors.     

Why do female Lesser Spotted Eagles ( Aquila pomarina ) visit strange nests remote from their own?

There is very little confirmed information on the social organisation of breeding Lesser Spotted Eagle populations, the turnover rate of adults, and their nest-site and partner fidelity. According to established knowledge, however, breeding individuals are territorial and defend at least the immediate vicinity of the nest site against their own species. It has further been thought that females rearing young, as with the females of other raptor species, remain within a radius of only a few kilometres of their eyrie. Using GPS satellite telemetry and DNA microsatellite analysis (DNA STR typing), we were able to disprove this prevailing hypothesis. A satellite-tracked female flew over 50 km away from her eyrie (D) in at least two different directions and visited at least one other occupied eyrie (T). It was also established that at least two strange females arrived at her eyrie, which contained young, from as far away as 57 km, and probably remained there for some considerable time. The pool of alleles represented at the different loci analysed, as well as the distribution of these alleles among the individuals, excludes the possibility that these females could be sisters or even half-sisters. Visits of strange eagles at this eyrie were also confirmed by direct observation. It can therefore be assumed that males only exhibit territorial behaviour towards their own sex and not towards strange females and that females do not exhibit territorial behaviour towards other females; but all these assumptions must be confirmed by further studies. For the first time it could be proved by means of microsatellite analysis that almost all females studied used the same breeding site for 2 consecutive years. The longest established period in which both partners of a pair remained at the same breeding site was 3 consecutive years.     

Isolation, purification and properties of new restriction endonucleases from Bacillus badius and Bacillus lentus

We tentatively named two enzymes as BbaI and BleI, which were isolated and purified from Gram-positive mesophilic bacteria Bacillus badius 1458 and Bacillus lentus 1689 respectively, by ammonium sulphate precipitation, phosphocellulose and heparin-sepharose column chromatography. SDS-PAGE protein profiles for BbaI and BleI showed denatured molecular weights of 52 and 48 kDa, respectively. BbaI hydrolyzed pUC18 DNA into 1900 and 700 bp, pBR322 DNA into two fragments of 2800 and 1500 bp and Φ×174 DNA into 3800 and 1600 bp. BleI hydrolyzed pUC18 DNA into 1800 and 800 bp, pBR322 DNA into two fragments of 2700 and 1600 bp and Φ×174 DNA into 3700 and 1700 bp. The effects of temperature, ionic strength, pH and Mg²⁺ ion concentrations were studied to demonstrate some biochemical properties of BbaI and BleI. Maximum activities of these enzymes were observed at 37 °C (pH 8.0) with 100 mM NaCl and 10 mM Mg²⁺ concentrations.     

What is the Value of a Good Map? An Example Using High Spatial Resolution Imagery to Aid Riparian Restoration

Riparian areas contain structurally diverse habitats that are challenging to monitor routinely and accurately over broad areas. As the structural variability within riparian areas is often indiscernible using moderate-scale satellite imagery, new mapping techniques are needed. We used high spatial resolution satellite imagery from the QuickBird satellite to map harvested and intact forests in coastal British Columbia, Canada. We distinguished forest structural classes used in riparian restoration planning, each with different restoration costs. To assess the accuracy of high spatial resolution imagery relative to coarser imagery, we coarsened the pixel resolution of the image, repeated the classifications, and compared results. Accuracy assessments produced individual class accuracies ranging from 70 to 90% for most classes; whilst accuracies obtained using coarser scale imagery were lower. We also examined the implications of map error on riparian restoration budgets derived from our classified maps. To do so, we modified the confusion matrix to create a cost error matrix quantifying costs associated with misclassification. High spatial resolution satellite imagery can be useful for riparian mapping; however, errors in restoration budgets attributable to misclassification error can be significant, even when using highly accurate maps. As the spatial resolution of imagery increases, it will be used more routinely in ecosystem ecology. Thus, our ability to evaluate map accuracy in practical, meaningful ways must develop further. The cost error matrix is one method that can be adapted for conservation and planning decisions in many ecosystems.     

Loss of stem cell regenerative capacity within aged niches

This work uncovers novel mechanisms of aging within stem cell niches that are evolutionarily conserved between mice and humans and affect both embryonic and adult stem cells. Specifically, we have examined the effects of aged muscle and systemic niches on key molecular identifiers of regenerative potential of human embryonic stem cells (hESCs) and post-natal muscle stem cells (satellite cells). Our results reveal that aged differentiated niches dominantly inhibit the expression of Oct4 in hESCs and Myf-5 in activated satellite cells, and reduce proliferation and myogenic differentiation of both embryonic and tissue-specific adult stem cells (ASCs). Therefore, despite their general neoorganogenesis potential, the ability of hESCs, and the more differentiated myogenic ASCs to contribute to tissue repair in the old will be greatly restricted due to the conserved inhibitory influence of aged differentiated niches. Significantly, this work establishes that hESC-derived factors enhance the regenerative potential of both young and, importantly, aged muscle stem cells in vitro and in vivo; thus, suggesting that the regenerative outcome of stem cell-based replacement therapies will be determined by a balance between negative influences of aged tissues on transplanted cells and positive effects of embryonic cells on the endogenous regenerative capacity. Comprehensively, this work points toward novel venues for in situ restoration of tissue repair in the old and identifies critical determinants of successful cell-replacement therapies for aged degenerating organs.     

Movement patterns of the White-tailed Sea Eagle (Haliaeetus albicilla): post-fledging behaviour,natal dispersal onset and the role of the natal environment

Exploratory movements and natal dispersal form essential processes during early life history stages of raptors, but identifying the factors shaping individual movement decisions is challenging. Global positioning system (GPS) telemetry thereby provides a promising technique to study movement patterns on adequate spatio-temporal scales. We analysed data of juvenile White-tailed Sea Eagles Haliaeetus albicilla (WTSE) in north-east Germany (n = 24) derived from GPS tracking to extensively analyse movements between fledging and emigration from the natal territory. Our goal was to determine the time point of fledging, characterize pre-emigration movements and the onset of natal dispersal while investigating the influence of the natal environment. WTSE fledged at an average age of 72 days and showed strong excursive behaviour during the post-fledging period regarding the number, distance and duration of excursions, yet with high individual variability. Excursive behaviour did not differ between sexes. On average, WTSE left the parental territory 93 days after fledging. The quantity of excursive behaviour delayed the timing of emigration and WTSE tended to postpone their emigration when foraging water was accessible within the boundaries of their parental territory. The overall results suggest that young WTSE assess the quality of the natal environment via pre-emigration movements and stay in their territory of origin for as long as internal and external conditions allow for it. Our study is one of the first to characterize post-fledging and natal dispersal movements of young WTSE to such an extent and applies modern techniques to understand related movements in relation to the natal environment. The results emphasize the urgent necessity for the extension of currently existing nest protection periods and guaranteeing sustainable management of potential breeding and foraging grounds for WTSE. Ultimately, the results are relevant for all large raptor species sensitive to human-related disturbance, as they support the increasing importance of regulations with spatio-temporal specifications for breeding populations of large raptors in densely human-inhabited areas with increasing alteration of land.     

AMP-activated Protein Kinase Stimulates Warburg-like Glycolysis and Activation of Satellite Cells during Muscle Regeneration

Satellite cells are the major myogenic stem cells residing inside skeletal muscle and are indispensable for muscle regeneration. Satellite cells remain largely quiescent but are rapidly activated in response to muscle injury, and the derived myogenic cells then fuse to repair damaged muscle fibers or form new muscle fibers. However, mechanisms eliciting metabolic activation, an inseparable step for satellite cell activation following muscle injury, have not been defined. We found that a noncanonical Sonic Hedgehog (Shh) pathway is rapidly activated in response to muscle injury, which activates AMPK and induces a Warburg-like glycolysis in satellite cells. AMPKα1 is the dominant AMPKα isoform expressed in satellite cells, and AMPKα1 deficiency in satellite cells impairs their activation and myogenic differentiation during muscle regeneration. Drugs activating noncanonical Shh promote proliferation of satellite cells, which is abolished because of satellite cell-specific AMPKα1 knock-out. Taken together, AMPKα1 is a critical mediator linking noncanonical Shh pathway to Warburg-like glycolysis in satellite cells, which is required for satellite activation and muscle regeneration.     

A Disintegrin and Metalloprotease 10 (ADAM10) Is Indispensable for Maintenance of the Muscle Satellite Cell Pool

Satellite cells (SCs) are muscle-specific stem cells that are essential for the regeneration of damaged muscles. Although SCs have a robust capacity to regenerate myofibers, the number of SCs decreases with aging, leading to insufficient recovery after muscle injury. We herein show that ADAM10 (a disintegrin and metalloprotease 10), a membrane-bound proteolytic enzyme with a critical role in Notch processing (S2 cleavage), is essential for the maintenance of SC quiescence. We generated mutant mice in which ADAM10 in SCs can be conditionally abrogated by tamoxifen injection. Tamoxifen-treated mutant mice did not show any apparent defects and grew normally under unchallenged conditions. However, these mice showed a nearly complete loss of muscle regeneration after chemically induced muscle injury. In situ hybridization and flow cytometric analyses revealed that the mutant mice had significantly less SCs compared with wild type controls. Of note, we found that inactivation of ADAM10 in SCs severely compromised Notch signaling and led to dysregulated myogenic differentiation, ultimately resulting in deprivation of the SC pool in vivo. Taken together, the present findings underscore the role of ADAM10 as an indispensable component of Notch signaling in SCs and for maintaining the SC pool.