Characterization of transgenic mice expressing cancer-associated variants of human NOTCH1

The Notch1 receptor plays a critical role in cell fate decisions during development. Activation of Notch signaling has been implicated in several types of cancer, particularly T-cell acute lymphoblastic leukemia (T-ALL). Consequently, several transgenic mouse strains have been made to study the role of Notch1 in T-ALL. However, the existing Notch1 transgenic lines mimic a translocation event found in only ～1% of T-ALL cases. Here we describe three novel NOTCH1 transgenic mouse strains that have Cre-inducible expression of the entire human NOTCH1 locus, each possessing a common mutation found in T-ALL. Unlike existing Notch1 transgenic strains, these NOTCH1 transgenic strains express full-length receptors from an endogenous human promoter that should be susceptible to a number of Notch antagonists that have recently been developed. These strains will allow researchers to modulate Notch signaling to study both normal development and cancer biology.     

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The blood-brain barrier (BBB) consisting of EC, astrocyte end-feet, pericytes and the basal membrane is responsible for the protection and homeostasis of the brain parenchyma. In vitro BBB models are common tools to study the structure and function of the BBB at the cellular level. A considerable number of different in vitro BBB models have been established for research in different laboratories to date. Usually, the cells are obtained from bovine, porcine, rat or mouse brain tissue (discussed in detail in the review by Wilhelm et al. ¹). Human tissue samples are available only in a restricted number of laboratories or companies ^2,3. While primary cell preparations are time consuming and the EC cultures can differ from batch to batch, the establishment of immortalized EC lines is the focus of scientific interest.Here, we present a method for establishing an immortalized brain microvascular EC line from neonatal mouse brain. We describe the procedure step-by-step listing the reagents and solutions used. The method established by our lab allows the isolation of a homogenous immortalized endothelial cell line within four to five weeks. The brain microvascular endothelial cell lines termed cEND ⁴ (from cerebral cortex) and cerebEND ⁵ (from cerebellar cortex), were isolated according to this procedure in the Förster laboratory and have been effectively used for explanation of different physiological and pathological processes at the BBB. Using cEND and cerebEND we have demonstrated that these cells respond to glucocorticoid- ^4,6-9 and estrogen-treatment ¹⁰ as well as to pro-infammatory mediators, such as TNFalpha ^5,8. Moreover, we have studied the pathology of multiple sclerosis ¹¹ and hypoxia ^12,13 on the EC-level. The cEND and cerebEND lines can be considered as a good tool for studying the structure and function of the BBB, cellular responses of ECs to different stimuli or interaction of the EC with lymphocytes or cancer cells.     

Slow development of ALS-like spinal cord pathology in mutant valosin-containing protein gene knock-in mice

Pathological features of amyotrophic lateral sclerosis (ALS) include, in addition to selective motor neuron (MN) degeneration, the occurrence of protein aggregates, mitochondrial dysfunction and astrogliosis. SOD1 mutations cause rare familial forms of ALS and have provided the most widely studied animal models. Relatively recent studies implicating another protein, TDP-43, in familial and sporadic forms of ALS have led to the development of new animal models. More recently, mutations in the valosin-containing protein (VCP) gene linked to the human genetic disease, Inclusion Body Myopathy associated with Paget''s disease of bone and frontotemporal dementia (IBMPFD), were found also to be associated with ALS in some patients. A heterozygous knock-in VCP mouse model of IBMPFD (VCP^R155H/+) exhibited muscle, bone and brain pathology characteristic of the human disease. We have undertaken studies of spinal cord pathology in VCP^R155H/+ mice and find age-dependent degeneration of ventral horn MNs, TDP-43-positive cytosolic inclusions, mitochondrial aggregation and progressive astrogliosis. Aged animals (∼24–27 months) show electromyography evidence of denervation consistent with the observed MN loss. Although these animals do not develop rapidly progressive fatal ALS-like disease during their lifespans, they recapitulate key pathological features of both human disease and other animal models of ALS, and may provide a valuable new model for studying events preceding onset of catastrophic disease.     

Modeling climate change impacts on overwintering bald eagles

Bald eagles (Haliaeetus leucocephalus) are recovering from severe population declines, and are exerting pressure on food resources in some areas. Thousands of bald eagles overwinter near Puget Sound, primarily to feed on chum salmon (Oncorhynchus keta) carcasses. We used modeling techniques to examine how anticipated climate changes will affect energetic demands of overwintering bald eagles. We applied a regional downscaling method to two global climate change models to obtain hourly temperature, precipitation, wind, and longwave radiation estimates at the mouths of three Puget Sound tributaries (the Skagit, Hamma Hamma, and Nisqually rivers) in two decades, the 1970s and the 2050s. Climate data were used to drive bald eagle bioenergetics models from December to February for each river, year, and decade. Bald eagle bioenergetics were insensitive to climate change: despite warmer winters in the 2050s, particularly near the Nisqually River, bald eagle food requirements declined only slightly (<1%). However, the warming climate caused salmon carcasses to decompose more rapidly, resulting in 11% to 14% less annual carcass biomass available to eagles in the 2050s. That estimate is likely conservative, as it does not account for decreased availability of carcasses due to anticipated increases in winter stream flow. Future climate-driven declines in winter food availability, coupled with a growing bald eagle population, may force eagles to seek alternate prey in the Puget Sound area or in more remote ecosystems.     

The Saccharomyces cerevisiae W303-K6001 cross-platform genome sequence: insights into ancestry and physiology of a laboratory mutt

Saccharomyces cerevisiae strain W303 is a widely used model organism. However, little is known about its genetic origins, as it was created in the 1970s from crossing yeast strains of uncertain genealogy. To obtain insights into its ancestry and physiology, we sequenced the genome of its variant W303-K6001, a yeast model of ageing research. The combination of two next-generation sequencing (NGS) technologies (Illumina and Roche/454 sequencing) yielded an 11.8 Mb genome assembly at an N50 contig length of 262 kb. Although sequencing was substantially more precise and sensitive than whole-genome tiling arrays, both NGS platforms produced a number of false positives. At a 378× average coverage, only 74 per cent of called differences to the S288c reference genome were confirmed by both techniques. The consensus W303-K6001 genome differs in 8133 positions from S288c, predicting altered amino acid sequence in 799 proteins, including factors of ageing and stress resistance. The W303-K6001 (85.4%) genome is virtually identical (less than equal to 0.5 variations per kb) to S288c, and thus originates in the same ancestor. Non-S288c regions distribute unequally over the genome, with chromosome XVI the most (99.6%) and chromosome XI the least (54.5%) S288c-like. Several of these clusters are shared with Σ1278B, another widely used S288c-related model, indicating that these strains share a second ancestor. Thus, the W303-K6001 genome pictures details of complex genetic relationships between the model strains that date back to the early days of experimental yeast genetics. Moreover, this study underlines the necessity of combining multiple NGS and genome-assembling techniques for achieving accurate variant calling in genomic studies.     

Fire‐induced tree mortality in a neotropical forest: the roles of bark traits,tree size,wood density and fire behavior

Large‐scale wildfires are expected to accelerate forest dieback in Amazônia, but the fire vulnerability of tree species remains uncertain, in part due to the lack of studies relating fire‐induced mortality to both fire behavior and plant traits. To address this gap, we established two sets of experiments in southern Amazonia. First, we tested which bark traits best predict heat transfer rates (R) through bark during experimental bole heating. Second, using data from a large‐scale fire experiment, we tested the effects of tree wood density (WD), size, and estimated R (inverse of cambium insulation) on tree mortality after one to five fires. In the first experiment, bark thickness explained 82% of the variance in R, while the presence of water in the bark reduced the difference in temperature between the heat source and the vascular cambium, perhaps because of high latent heat of vaporization. This novel finding provides an important insight for improving mechanistic models of fire‐induced cambium damage from tropical to temperate regions. In the second experiment, tree mortality increased with increasing fire intensity (i.e. as indicated by bark char height on tree boles), which was higher along the forest edge, during the 2007 drought, and when the fire return interval was 3 years instead of one. Contrary to other tropical studies, the relationship between mortality and fire intensity was strongest in the year following the fires, but continued for 3 years afterwards. Tree mortality was low (≤20%) for thick‐barked individuals (≥18 mm) subjected to medium‐intensity fires, and significantly decreased as a function of increasing tree diameter, height and wood density. Hence, fire‐induced tree mortality was influenced not only by cambium insulation but also by other traits that reduce the indirect effects of fire. These results can be used to improve assessments of fire vulnerability of tropical forests.     

Spatial variability of the plankton trophic interaction in the North Sea: a new feature after the early 1970s

Traditionally, marine ecosystem structure was thought to be bottom‐up controlled. In recent years, a number of studies have highlighted the importance of top‐down regulation. Evidence is accumulating that the type of trophic forcing varies temporally and spatially, and an integrated view – considering the interplay of both types of control – is emerging. Correlations between time series spanning several decades of the abundances of adjacent trophic levels are conventionally used to assess the type of control: bottom‐up if positive or top‐down if this is negative. This approach implies averaging periods which might show time‐varying dynamics and therefore can hide part of this temporal variability. Using spatially referenced plankton information extracted from the Continuous Plankton Recorder, this study addresses the potential dynamic character of the trophic structure at the planktonic level in the North Sea by assessing its variation over both temporal and spatial scales. Our results show that until the early‐1970s a bottom‐up control characterized the base of the food web across the whole North Sea, with diatoms having a positive and homogeneous effect on zooplankton filter‐feeders. Afterwards, different regional trophic dynamics were observed, in particular a negative relationship between total phytoplankton and zooplankton was detected off the west coast of Norway and the Skagerrak as opposed to a positive one in the southern reaches. Our results suggest that after the early 1970s diatoms remained the main food source for zooplankton filter‐feeders east of Orkney–Shetland and off Scotland, while in the east, from the Norwegian Trench to the German Bight, filter‐feeders were mainly sustained by dinoflagellates.     

Geographic range determinants of two commercially important marine molluscs

Aim We modelled the spatial abundance patterns of two abalone species (Haliotis rubra Donovan 1808 and H. laevigata Leach 1814) inhabiting inshore rocky reefs to better understand the importance of current sea surface temperature (SST) (among other predictors) and, ultimately, the effect of future climate change, on marine molluscs. Location Southern Australia. Methods We used an ensemble species distribution modelling approach that combined likelihood‐based generalized linear models and boosted regression trees. For each modelling technique, a two‐step procedure was used to predict: (1) the current probability of presence, followed by (2) current abundance conditional on presence. The resulting models were validated using an independent, spatially explicit dataset of abalone abundance patterns in Victoria. Results For both species, the presence of reef was the main driver of abalone occurrence, while SST was the main driver of spatial abundance patterns. Predictive maps at c. 1‐km resolution showed maximal abundance on shallow coastal reefs characterized by mild winter SSTs for both species. Main conclusions Sea surface temperature was a major driver of abundance patterns for both abalone species, and the resulting ensemble models were used to build fine‐resolution predictive range maps (c. 1 km) that incorporate measures of habitat suitability and quality in support of resource management. By integrating this output with structured spatial population models, a more robust understanding of the potential impacts of threatening human processes such as climate change can be established.     

Refugee species: which historic baseline should inform conservation planning?

Understanding species' historical ranges can provide important information for conservation planning in the face of environmental change. Cromsigt et al. (this issue) comment on our recent European bison (Bison bonasus) range reconstruction, suggesting that bison were already 8000 years ago a refugee species (i.e. restricted to marginal habitat due to past human pressure) and that species distribution models (SDM) are generally of limited use for refugee species conservation. While we welcome this discussion, we find no evidence for the claim that human pressure prior to 8000 BP determined where bison occurred. More importantly, as human pressure is generally high and increasing, attempts to restore species across their former range may fail where the factors that relegated species into refugee status are still at play or where their optimal habitat has vanished. Identifying areas where human pressure is low and where refugee species have persisted over the last millennia is crucial, and SDM based on historical data are important for doing so. Refugee species suffer from the shifting baseline syndrome, but careful reality checks are needed and all available data should be considered before determining the baseline that should inform conservation planning.