A Gly98Val mutation in the N-Myc downstream regulated gene 1 (NDRG1) in Alaskan Malamutes with polyneuropathy.

The first cases of early-onset progressive polyneuropathy appeared in the Alaskan Malamute population in Norway in the late 1970s. Affected dogs were of both sexes and were ambulatory paraparetic, progressing to non-ambulatory tetraparesis. On neurologic examination, affected dogs displayed predominantly laryngeal paresis, decreased postural reactions, decreased spinal reflexes and muscle atrophy. The disease was considered eradicated through breeding programmes but recently new cases have occurred in the Nordic countries and the USA. The N-myc downstream-regulated gene (NDRG1) is implicated in neuropathies with comparable symptoms or clinical signs both in humans and in Greyhound dogs. This gene was therefore considered a candidate gene for the polyneuropathy in Alaskan Malamutes. The coding sequence of the NDRG1 gene derived from one healthy and one affected Alaskan Malamute revealed a non-synonymous G>T mutation in exon 4 in the affected dog that causes a Gly98Val amino acid substitution. This substitution was categorized to be "probably damaging" to the protein function by PolyPhen2 (score: 1.000). Subsequently, 102 Alaskan Malamutes from the Nordic countries and the USA known to be either affected (n = 22), obligate carriers (n = 7) or healthy (n = 73) were genotyped for the SNP using TaqMan. All affected dogs had the T/T genotype, the obligate carriers had the G/T genotype and the healthy dogs had the G/G genotype except for 13 who had the G/T genotype. A protein alignment showed that residue 98 is conserved in mammals and also that the entire NDRG1 protein is highly conserved (94.7%) in mammals. We conclude that the G>T substitution is most likely the mutation that causes polyneuropathy in Alaskan Malamutes. Our characterization of a novel candidate causative mutation for polyneuropathy offers a new canine model that can provide further insight into pathobiology and therapy of human polyneuropathy. Furthermore, selection against this mutation can now be used to eliminate the disease in Alaskan Malamutes.

Phagocytic uptake of Encephalitozoon cuniculi by nonprofessional phagocytes

Encephalitozoon cuniculi is an obligate intracellular, spore-forming parasite belonging to the microsporidia that can cause disseminated infection in immunocompromised persons. E. cuniculi spores infect host cells by germination, i.e., by explosively everting the polar filament, through which the spore contents (sporoplasms) are subsequently injected into the cytoplasm. In addition, we observed intracellular, nongerminated spores in various nonprofessional phagocytes. In MRC5 cells, the number of internalized spores was approximately 10-fold higher than the number of injected sporoplasms. Compared to the rate of uptake by human monocyte-derived macrophages, internalization rates by A549 cells, MRC5 cells, and 293 cells were 0.6, 4.4, and 22.2%, respectively. The mechanism of uptake was studied in MRC5 cells. Killed spores were internalized at the same rate as live spores, indicating that nongerminated parasites do not actively participate in cell entry. Cytochalasin D inhibited uptake of spores by 95%, demonstrating an actin-dependent process. By electron and epifluorescence microscopy, intracellular spores were found in a tightly fitting membrane-bound compartment. The vacuole containing the spores was positive for the lysosomal membrane protein LAMP-1 and colocalized with the late endosomal-lysosomal content marker rhodamine dextran. Our results show that, in addition to the unique way in which microsporidia infect cells, E. cuniculi spores enter nonprofessional phagocytes by phagocytosis and traffic into a late endosomal-lysosomal compartment.

Retrospective analysis of a nonforecasted rain-on-snow flood in the Alps – a matter of model limitations or unpredictable nature?

A rain-on-snow flood occurred in the Bernese Alps, Switzerland, on 10 October 2011, and caused significant damage. As the flood peak was unpredicted by the flood forecast system, questions were raised concerning the causes and the predictability of the event. Here, we aimed to reconstruct the anatomy of this rain-on-snow flood in the Lötschen Valley (160 km2) by analyzing meteorological data from the synoptic to the local scale and by reproducing the flood peak with the hydrological model WaSiM-ETH (Water Flow and Balance Simulation Model). This in order to gain process understanding and to evaluate the predictability. The atmospheric drivers of this rain-on-snow flood were (i) sustained snowfall followed by (ii) the passage of an atmospheric river bringing warm and moist air towards the Alps. As a result, intensive rainfall (average of 100 mm day-1) was accompanied by a temperature increase that shifted the 0° line from 1500 to 3200 m a.s.l. (meters above sea level) in 24 h with a maximum increase of 9 K in 9 h. The south-facing slope of the valley received significantly more precipitation than the north-facing slope, leading to flooding only in tributaries along the south-facing slope. We hypothesized that the reason for this very local rainfall distribution was a cavity circulation combined with a seeder-feeder-cloud system enhancing local rainfall and snowmelt along the south-facing slope. By applying and considerably recalibrating the standard hydrological model setup, we proved that both latent and sensible heat fluxes were needed to reconstruct the snow cover dynamic, and that locally high-precipitation sums (160 mm in 12 h) were required to produce the estimated flood peak. However, to reproduce the rapid runoff responses during the event, we conceptually represent likely lateral flow dynamics within the snow cover causing the model to react "oversensitively" to meltwater. Driving the optimized model with COSMO (Consortium for Small-scale Modeling)-2 forecast data, we still failed to simulate the flood because COSMO-2 forecast data underestimated both the local precipitation peak and the temperature increase. Thus we conclude that this rain-on-snow flood was, in general, predictable, but requires a special hydrological model setup and extensive and locally precise meteorological input data. Although, this data quality may not be achieved with forecast data, an additional model with a specific rain-on-snow configuration can provide useful information when rain-on-snow events are likely to occur.

Higher plant diversity promotes higher diversity of fungal pathogens, while it decreases pathogen infection per plant

Fungal plant pathogens are common in natural communities where they affect plant physiology, plant survival, and biomass production. Conversely, pathogen transmission and infection may be regulated by plant community characteristics such as plant species diversity and functional composition that favor pathogen diversity through increases in host diversity while simultaneously reducing pathogen infection via increased variability in host density and spatial heterogeneity. Therefore, a comprehensive understanding of multi-host multi-pathogen interactions is of high significance in the context of biodiversity-ecosystem functioning. We investigated the relationship between plant diversity and aboveground obligate parasitic fungal pathogen (''pathogens'' hereafter) diversity and infection in grasslands of a long-term, large-scale, biodiversity experiment with varying plant species (1-60 species) and plant functional group diversity (1-4 groups). To estimate pathogen infection of the plant communities, we visually assessed pathogen-group presence (i.e., rusts, powdery mildews, downy mildews, smuts, and leaf-spot diseases) and overall infection levels (combining incidence and severity of each pathogen group) in 82 experimental plots on all aboveground organs of all plant species per plot during four surveys in 2006. Pathogen diversity, assessed as the cumulative number of pathogen groups on all plant species per plot, increased log-linearly with plant species diversity. However, pathogen incidence and severity, and hence overall infection, decreased with increasing plant species diversity. In addition, co-infection of plant individuals by two or more pathogen groups was less likely with increasing plant community diversity. We conclude that plant community diversity promotes pathogen-community diversity while at the same time reducing pathogen infection levels of plant individuals.

Independent polled mutations leading to complex gene expression differences in cattle

The molecular regulation of horn growth in ruminants is still poorly understood. To investigate this process, we collected 1019 hornless (polled) animals from different cattle breeds. High-density SNP genotyping confirmed the presence of two different polled associated haplotypes in Simmental and Holstein cattle co-localized on BTA 1. We refined the critical region of the Simmental polled mutation to 212 kb and identified an overlapping region of 932 kb containing the Holstein polled mutation. Subsequently, whole genome sequencing of polled Simmental and Holstein cows was used to determine polled associated genomic variants. By genotyping larger cohorts of animals with known horn status we found a single perfectly associated insertion/deletion variant in Simmental and other beef cattle confirming the recently published possible Celtic polled mutation. We identified a total of 182 sequence variants as candidate mutations for polledness in Holstein cattle, including an 80 kb genomic duplication and three SNPs reported before. For the first time we showed that hornless cattle with scurs are obligate heterozygous for one of the polled mutations. This is in contrast to published complex inheritance models for the bovine scurs phenotype. Studying differential expression of the annotated genes and loci within the mapped region on BTA 1 revealed a locus (LOC100848215), known in cow and buffalo only, which is higher expressed in fetal tissue of wildtype horn buds compared to tissue of polled fetuses. This implicates that the presence of this long noncoding RNA is a prerequisite for horn bud formation. In addition, both transcripts associated with polledness in goat and sheep (FOXL2 and RXFP2), show an overexpression in horn buds confirming their importance during horn development in cattle.

Genome profiling of sterol synthesis shows convergent evolution in parasites and guides chemotherapeutic attack

Sterols are an essential class of lipids in eukaryotes, where they serve as structural components of membranes and play important roles as signaling molecules. Sterols are also of high pharmacological significance: cholesterol-lowering drugs are blockbusters in human health, and inhibitors of ergosterol biosynthesis are widely used as antifungals. Inhibitors of ergosterol synthesis are also being developed for Chagas's disease, caused by Trypanosoma cruzi. Here we develop an in silico pipeline to globally evaluate sterol metabolism and perform comparative genomics. We generate a library of hidden Markov model-based profiles for 42 sterol biosynthetic enzymes, which allows expressing the genomic makeup of a given species as a numerical vector. Hierarchical clustering of these vectors functionally groups eukaryote proteomes and reveals convergent evolution, in particular metabolic reduction in obligate endoparasites. We experimentally explore sterol metabolism by testing a set of sterol biosynthesis inhibitors against trypanosomatids, Plasmodium falciparum, Giardia, and mammalian cells, and by quantifying the expression levels of sterol biosynthetic genes during the different life stages of T. cruzi and Trypanosoma brucei. The phenotypic data correlate with genomic makeup for simvastatin, which showed activity against trypanosomatids. Other findings, such as the activity of terbinafine against Giardia, are not in agreement with the genotypic profile.

A RAB3GAP1 SINE Insertion in Alaskan Huskies with Polyneuropathy, Ocular Abnormalities and Neuronal Vacuolation (POANV) Resembling Human Warburg Micro Syndrome 1 (WARBM1)

We observed a hereditary phenotype in Alaskan Huskies, which was characterized by polyneuropathy with ocular abnormalities and neuronal vacuolation (POANV). The affected dogs developed a progressive severe ataxia, which led to euthanasia between 8 and 16 months of age. The pedigrees were consistent with a monogenic autosomal recessive inheritance. We localized the causative genetic defect to a 4 Mb interval on chromosome 19 by a combined linkage and homozygosity mapping approach. Whole genome sequencing of one affected dog, an obligate carrier and an unrelated control revealed a 218 bp SINE insertion into exon 7 of the RAB3GAP1 gene. The SINE insertion was perfectly associated with the disease phenotype in a cohort of 43 Alaskan Huskies and it was absent from 541 control dogs of diverse other breeds. The SINE insertion induced aberrant splicing and led to a transcript with a greatly altered exon 7. RAB3GAP1 loss-of-function variants in humans cause Warburg Micro Syndrome 1 (WARBM1), which is characterized by additional developmental defects compared to canine POANV, whereas Rab3gap1 deficient mice have a much milder phenotype than either humans or dogs. Thus the RAB3GAP1 mutant Alaskan Huskies provide an interesting intermediate phenotype that may help to better understand the function of RAB3GAP1 in development. Furthermore, the identification of the presumed causative genetic variant will enable genetic testing to avoid the non-intentional breeding of affected dogs.

Chemopreventive function of retinoid X receptors in human squamous cell carcinoma of the skin

Retinoid therapy has been successful for the treatment of skin squamous cell carcinoma (SCC). A suppression of the predominant retinoid X receptor expressed in skin, retinoid X receptor α (RXRα), has been reported in skin SCC. These observations have led to the hypothesis that retinoid receptor loss contributes to the tumorigenic phenotype of epithelial cancers. To test this hypothesis, the RXRα gene was mapped in order to generate a targeting construct. Additionally the transcriptional regulation of the human RXRα a gene in keratinocytes was characterized after identifying the transcription initiation sites, the promoter, and enhancer regions of this gene. The structure is highly conserved between human and mouse. A nontumorigenic human skin-derived cell line called near diploid immortalized keratinocytes (NIKS) has the advantage of growing as organotypic raft cultures, under physiological conditions closely resembling in-vivo squamous stratification. We have exploited the raft culture technique to develop an in-vitro model for skin SCC progression that includes the NIKS cells, HaCaT cells, a premalignant cell line, and SRB 12-p9 cells, a tumorigenic SCC skin cell line. The differentiation, proliferation and nuclear receptor ligand response characteristics of this system were studied and significant and novel results were obtained. RXRs are obligate heterodimerization partners with many of the nuclear hormone receptors, including retinoic acid receptors (RARs), vitamin D3 receptors (VDR), thyroid hormone receptors (T3 R) and peroxisome proliferator activate receptors (PPARs), which are all known to be active in skin. Treatment of the three cell lines in raft culture with the RXR specific ligand BMS649, BMS961 (RARγ-specific), vitamin D3 (VDR ligand), thryoid hormone (T3R ligand) and clofibrate (PPARa ligand), and the combination of BMS649 with each of the 4 receptor partner ligands, resulted in distinct effects on differentiation, proliferation and apoptosis. The effects of activation of RXRs in each of the four-receptor pathways; in the context of skin SCC progression, with an emphasis on the VDR/RXR pathway, are discussed. These studies will lead to a better understanding of RXRα action in human skin and will help determine its role in SCC tumorigenesis, as well as its potential as a target for the prevention, treatment, and control of skin cancer. ^

Molecular genetic analyses of extracellular signaling pathways during murine retinal development

Extracellular signaling pathways initiated by secreted proteins are important in the co-ordination of tissue interactions in multi-cellular organisms, particularly during embryonic development. These signaling cascades direct diverse cellular events, including proliferation, differentiation and migration, in both autocrine and paracrine modes. In adult animals, abnormal function of these proteins often results in degenerative and tumourigenic syndromes. In this study, I have focused on elucidating the role of Bone Morphogenetic Protein (Bmp) signal transduction during neuronal specification and differentiation in the vertebrate embryo, using the mouse retina as a model. Using tissue-specific conditional knock-out approaches, the consequences of genetic loss-of-function of this signaling pathway on retinal physiology were examined. Mutant mice lacking Bmp type I receptor function displayed a range of retinal phenotypes, each of which appeared to be regulated at a different threshold of Bmp receptor activity. Novel essential functions for Bmp signaling were uncovered for retinal neurogenesis, cell survival, and axonal pathfinding at the optic disc. Further, BmprIa and BmprIa exhibited genetic interactions suggestive of functional redundancy. To further characterize the underlying molecular bases for the pleiotropic effects of Bmp receptors, retina-specific loss-of-function mutants of the obligate Bmp-activated transcriptional mediator Smad4 were generated. A comparison of the retina-specific Smad4 mutant phenotypes with those of the Bmp receptor mutant retina revealed that only a subset of retinal phenotypes, namely optic disc axon pathfinding and axial patterning were common for both classes of mutant animals. Thus, these results suggest that, contrary to the classic scheme of Bmp signal transduction, Smad4-independent pathways may be operative downstream of the type I receptors. Indeed, such alternative intracellular signaling cascades may constitute a molecular basis for the multiple cellular responses elicited by Bmp signaling. Finally, I tested whether the potential Bmp pathway targets, the extracellular ligands Fgf9 and Fgf15, mediate essential cellular processes in the retina. The analyses of Fgf9 −/−; Fgf15−/− mutant mice posit a novel shared role for these genes in intra-retinal axon pathfinding. Collectively, these studies have elucidated part of the molecular machinery directing mammalian neuro-retinal development, and provided useful in vivo models to study visual function. ^

Size-related stable isotope changes in Late Cretaceous and Recent planktic foraminifera from the western tropical Atlantic

The paleoecology of Cretaceous planktic foraminifera during the Late Cenomanian to Coniacian period (~95-86 Ma) remains controversial since much of the tropical marine record is preserved as chalk and limestone with uncertain geochemical overprints. Here we present delta13C and delta18O data from sieve size fractions of monospecific samples of exceptionally well preserved planktic foraminifera recovered during Ocean Drilling Program Leg 207 (Demerara Rise, western tropical Atlantic). Our results suggest that all species studied (Hedbergella delrioensis, Heterohelix globulosa, Marginotruncana sinuosa, Whiteinella baltica) grew primarily in surface waters and did not change their depth habitat substantially during their life cycle. Comparison of size-related ontogenetic trends in delta13C in Cretaceous and modern foraminifera further suggests that detection of dinoflagellate photosymbiosis using delta13C is confounded by physiological effects during the early stages of foraminifer growth, raising doubts about previous interpretations of photosymbiosis in small foraminifera species. We propose that obligate photosymbiosis involving dinoflagellates may not have evolved until the Campanian or Maastrichtian since our survey of Cenomanian-Coniacian species does not find the delta18O and delta13C size-related trends observed in modern foraminifer-dinoflagellate symbioses.

Bryoliths constructed by bryozoans in symbiotic associations with hermit crabs, Golfe d'Arguin, Mauritania

The Golfe d'Arguin offshore of northern Mauritania hosts a rare modern analogue for heterozoan carbonate production in a tropical marine setting. Dominated by ocean upwelling and with additional fertilisation by iron-rich aeolian dust, this naturally eutrophic marine environment lacks typical photozoan communities. A highly productive, tropical cosmopolitan biota dominated by molluscs and suspension-feeders such as bryozoans and balanids characterises the carbonate-rich surface sediments. Overall biodiversity is relatively low and the species present are tolerant against the eutrophic and low-light conditions, the strong hydrodynamic regime governed by ocean upwelling, and the unstable, soft-bottom seafloor with few hard substrata. Here, we describe an ectosymbiosis between the hermit crab Pseudopagurus granulimanus (Miers, 1881) and monospecific assemblages of the encrusting cheilostome bryozoan Acanthodesia commensale (Kirkpatrick and Metzelaar, 1922) that cohabits vacant gastropod shells. Nucleating on an empty gastropod shell, the bryozoan colonies form multilamellar skeletal crusts that produce spherical encrustations and extend the living chamber of the hermit crab through helicospiral tubular growth. This non-obligate mutualistic symbiosis illustrates the adaptive capabilities and benefits from a close partnership in a complex marine environment, driven by trophic conditions, high water energies and instable substratum. Sectioned bryoliths show that between 49 and 97 % of the solid volume of the specimens consists of bryozoan skeleton.

Benthic and fish community composition as well as water quality along the Thousand Islands north of the megacity Jakarta, Indonesia

Worldwide, coral reefs are challenged by multiple stressors due to growing urbanization, industrialization and coastal development. Coral reefs along the Thousand Islands off Jakarta, one of the largest megacities worldwide, have degraded dramatically over recent decades. The shift and decline in coral cover and composition has been extensively studied with a focus on large-scale gradients (i.e. regional drivers), however special focus on local drivers in shaping spatial community composition is still lacking. Here, the spatial impact of anthropogenic stressors on local and regional scales on coral reefs north of Jakarta was investigated. Results indicate that the direct impact of Jakarta is mainly restricted to inshore reefs, separating reefs in Jakarta Bay from reefs along the Thousand Islands further north. A spatial patchwork of differentially degraded reefs is present along the islands as a result of localized anthropogenic effects rather than regional gradients. Pollution is the main anthropogenic stressor, with over 80 % of variation in benthic community composition driven by sedimentation rate, NO2, PO4 and Chlorophyll a. Thus, the spatial structure of reefs is directly related to intense anthropogenic pressure from local as well as regional sources. Therefore, improved spatial management that accounts for both local and regional stressors is needed for effective marine conservation.

Biomass and substrate utilization of cold adapted bacteria in Fram Strait and the Western Greenlad Sea

During the 'Polarstern' expedition ARK-IV/2 in June 1987, water samples from 8 stations were taken to study biomass and substrate utilization of cold adapted bacteria. Bacterial biomasses determined from acridine orange direct counts (AODC) were between 0.4 and 31.4 µ/g C/l, and ATP concentrations amounted from <0.1 to 40 ng/l. Colony counts on seawater agar reached only 0.1% of AODC, but with the MPN-method 1 to 10% of AODC were recorded. With 14C-glutamic acid or 14C-glucose as tracer substrate in oligotrophic broth containing 0.5 mg trypticase and 0.05 mg yeast extract per liter of seawater, obligately oligotrophic bacteria could be detected in one water sample. Although incubation was at 2 °C, only psychrotrophic bacteria showing growth temperatures between 1 and 30 °C were obtained. Organic substrate utilizations by 106 isolates were tested at 4 and 20 °C. Most carbohydrates, organic acids, alcohols, and alanine were assimilated at both temperatures, but arginine, aspartate and ornithine were utilized only at 20 °C by almost all strains.

Seawater carbonate chemistry and calcium carbonate of Padina spp., photosynthesis of Padina pavonica in nature CO2 gradients experiment

Predicting the impacts of ocean acidification on coastal ecosystems requires an understanding of the effects on macroalgae and their grazers, as these underpin the ecology of rocky shores. Whilst calcified coralline algae (Rhodophyta) appear to be especially vulnerable to ocean acidification, there is a lack of information concerning calcified brown algae (Phaeophyta), which are not obligate calcifiers but are still important producers of calcium carbonate and organic matter in shallow coastal waters. Here, we compare ecological shifts in subtidal rocky shore systems along CO2 gradients created by volcanic seeps in the Mediterranean and Papua New Guinea, focussing on abundant macroalgae and grazing sea urchins. In both the temperate and tropical systems the abundances of grazing sea urchins declined dramatically along CO2 gradients. Temperate and tropical species of the calcifying macroalgal genus Padina (Dictyoaceae, Phaeophyta) showed reductions in CaCO3 content with CO2 enrichment. In contrast to other studies of calcified macroalgae, however, we observed an increase in the abundance of Padina spp. in acidified conditions. Reduced sea urchin grazing pressure and significant increases in photosynthetic rates may explain the unexpected success of decalcified Padina spp. at elevated levels of CO2. This is the first study to provide a comparison of ecological changes along CO2 gradients between temperate and tropical rocky shores. The similarities we found in the responses of Padina spp. and sea urchin abundance at several vent systems increases confidence in predictions of the ecological impacts of ocean acidification over a large geographical range.