Identifying Predictors of Activity Based Anorexia Susceptibility in Diverse Genetic Rodent Populations

Animal studies are very useful in detection of early disease indicators and in unravelling the pathophysiological processes underlying core psychiatric disorder phenotypes. Early indicators are critical for preventive and efficient treatment of progressive psychiatric disorders like anorexia nervosa. Comparable to physical hyperactivity observed in anorexia nervosa patients, in the activity-based anorexia rodent model, mice and rats express paradoxical high voluntary wheel running activity levels when food restricted. Eleven inbred mouse strains and outbred Wistar WU rats were exposed to the activity-based anorexia model in search of identifying susceptibility predictors. Body weight, food intake and wheel running activity levels of each individual mouse and rat were measured. Mouse strains and rats with high wheel running activity levels during food restriction exhibited accelerated body weight loss. Linear mixed models for repeated measures analysis showed that baseline wheel running activity levels preceding the scheduled food restriction phase strongly predicted activity-based anorexia susceptibility (mice: Beta  =  −0.0158 (±0.003 SE), P<0.0001; rats: Beta  =  −0.0242 (±0.004 SE), P<0.0001) compared to other baseline parameters. These results suggest that physical activity levels play an important role in activity-based anorexia susceptibility in different rodent species with genetically diverse background. These findings support previous retrospective studies on physical activity levels in anorexia nervosa patients and indicate that pre-morbid physical activity levels could reflect an early indicator for disease severity.     

The pelagic system in the photic zone of the tropical Atlantic

Summary A multidisciplinary study of the carbon budget in the upper 300 meter of permanently stratified waters was started by two NECTAR-expeditions (North Equatorial Current Trans Atlantic Research) with HMS TYDEMAN in 1977 and 1978 (BAARS, ZIJLSTRA and TIJSSEN, 1979). In 1979 additional measurements were performed during the Gulf of Guinea-expedition with MS TYRO.Primary production in the nutrient depleted mixed layer of the North Equatorial Current estimated from the diurnal cycle in the O₂ concentration (TIJSSEN, 1979) and in POC (POSTMA and ROMMETS, 1979) revealed values 4–10 times higher than the data from the¹⁴C method in the literature: 800–2000 against ca. 200 mg C/m²/day. Moreover,¹⁴C incubations performed in bottle volumes of 4 liter and over 2 hour periods, instead of the recommended 12 hours for oligotrophic waters, gave 5–15 times higher values as incubations in the commonly used 300 ml (or smaller) bottles (GIESKES, KRAAY and BAARS, 1979). In the latter bottles a dramatic decrease of chlorophyll concentrations was observed, suggesting either heavy damage to fragile microflagellates by glasswall contacts and/or insufficient nutrient recycling by the lack of zooplankton in small samples. This then could account for the phenomena of low production and decreasing algal stock in long incubations with small bottles. These results suggest that today's picture of the primary production in the world's oceans (DE VOOIJS, 1979) needs probably a thorough reexamination. On the other hand, experiments in the North Sea and in the Gulf of Guinea did not indicate an effect of bottle size on¹⁴C incorporation and the¹⁴C method gave comparable estimates of primary production as the high precision oxygen method (photometric endpoint detection in the Winkler titration,cf. TIJSSEN, 1979). However, in these waters nutrient concentrations are always clearly above detection levels so that all previous data from somewhat richer areas may have been correct. In relation to the large oligotrophic parts of the oceans a question remains about the fate of the probably high primary production. Is it consumed by the algae themselves by night (POSTMA, 1980), are bacteria and microzooplankton more important consumers than formerly thought, or is the daily ration of zooplankters much higher as expected from extrapolation of filtration experiments in more eutrophic waters? We hope to clarify this topic during the coming NECTAR'82 expedition.Another main objective of the programme concerns the character of the deep chlorophyll maximum in permanently stratified waters. This layer is in the North Equatorial Current located at the depth of the nitratocline and at ca. 1% of the incident light at the surface (SPITZER and WERNAND, 1981). Primary production in the layer seems to be negligible while detailed vertical profiles of zooplankton, obtained with a Longhurst-Hardy Plankton Recorder, revealed no obvious concentrations in the layer. Chlorophyll analysis by thin-layer-chromatography (GIESKES, KRAAY and TIJSSEN, 1978) demonstrated that more than half of the chlorophyll a in the layer consists of an isomer which bleaches rapidly when transferred to higher light levels. The hypothesis was formulated that the chlorophyll maximum layer, at least in this region of the Atlantic, is an accumulation of chlorophyll breakdown products with a quite long turnover time at low light levels. In contrast to these findings, chlorophyll maxima near West-Africa and in the Gulf of Guinea were located at 5–10% light and contained more living algal cells (most of them as small as 1–3 m) than the nutrientpoor mixed layer above it. Primary production profiles had therefore a bimodal shape, with peaks at 30–50% light and at the chlorophyll maximum. Chlorophyll isomers, now analysed with High Pressure Liquid Chromatography, were also in these waters present at nearly all stations and comprised 30–70% of total chlorophyll comparable to the situation in the North Equatorial Current (GIESKES and KRAAY, 1981). It could be shown that these isomers are not involved in primary production, so that well established relationships between chlorophyll, light and primary production, found in oceanographic literature, have to be reconsidered.     

Structural abnormalities in the primary somatosensory cortex and a normal behavioral profile in Contactin-5 deficient mice

Contactin-5 (Cntn5) is an immunoglobulin cell adhesion molecule that is exclusively expressed in the central nervous system. In view of its association with neurodevelopmental disorders, particularly autism spectrum disorder (ASD), this study focused on Cntn5-positive areas in the forebrain and aimed to explore the morphological and behavioral phenotypes of the Cntn5 null mutant (Cntn5^?/?) mouse in relation to these areas and ASD symptomatology. A newly generated antibody enabled us to elaborately describe the spatial expression pattern of Cntn5 in P7 wild type (Cntn5^+/+) mice. The Cntn5 expression pattern included strong expression in the cerebral cortex, hippocampus and mammillary bodies in addition to described previously brain nuclei of the auditory pathway and the dorsal thalamus. Thinning of the primary somatosensory (S1) cortex was found in Cntn5^?/? mice and ascribed to a misplacement of Cntn5-ablated cells. This phenotype was accompanied by a reduction in the barrel/septa ratio of the S1 barrel field. The structure and morphology of the hippocampus was intact in Cntn5^?/? mice. A set of behavioral experiments including social, exploratory and repetitive behaviors showed that these were unaffected in Cntn5^?/? mice. Taken together, these data demonstrate a selective role of Cntn5 in development of the cerebral cortex without overt behavioral phenotypes.     

The development of a homologous transformation system for Aspergillus oryzae based on the nitrate assimilation pathway: A convenient and general selection system for filamentous fungal transformation

Summary The development of an efficient and homologous transformation system for Aspergillus oryzae is described. This is based on nitrate reductase (niaD) of the nitrate assimilation pathway. The niaD system offers a number of inherent advantages over many other systems and may be of general use for nitrate-utilising filamentous fungi. Transformation frequencies of up to 800 transformants per microgram DNA are observed with A. oryzae. The preponderance of integration events take place at the resident niaD locus either by gene conversion (41%), single integration (23%) or multiple tandem integration (36%). Heterologous expression of the A. oryzae niaD gene in the filamentous fungi A. nidulans, A. niger and Penicillum chrysogenum is observed. That heterologous putative niaD hybridisation signals are seen with other fungal DNAs affords the oppotunity to isolate the corresponding niaD from various fungi in order to develop homolgous transformation. Co-transformation with the introduction of the non-selected markers pyrG, tub-2, and uidA has been achieved.     

When are genetic methods useful for estimating contemporary abundance and detecting population trends?

The utility of microsatellite markers for inferring population size and trend has not been rigorously examined, even though these markers are commonly used to monitor the demography of natural populations. We assessed the ability of a linkage disequilibrium estimator of effective population size (N_e) and a simple capture-recapture estimator of abundance (N) to quantify the size and trend of stable or declining populations (true N = 100–10,000), using simulated Wright–Fisher populations. Neither method accurately or precisely estimated abundance at sample sizes of S = 30 individuals, regardless of true N. However, if larger samples of S = 60 or 120 individuals were collected, these methods provided useful insights into abundance and trends for populations of N = 100–500. At small population sizes (N = 100 or 250), precision of the N_e estimates was improved slightly more by a doubling of loci sampled than by a doubling of individuals sampled. In general, monitoring N_e proved a more robust means of identifying stable and declining populations than monitoring N over most of the parameter space we explored, and performance of the N_e estimator is further enhanced if the N_e/N ratio is low. However, at the largest population size (N = 10,000), N estimation outperformed N_e. Both methods generally required ≥ 5 generations to pass between sampling events to correctly identify population trend.     

Mapping the Naked Neck (NA) and Polydactyly (PO) mutants of the chicken with microsatellite molecular markers

The bulked segregant analysis methodology has been used to map, with microsatellite markers, two morphological mutations in the chicken: polydactyly (PO) and naked neck (NA). These autosomal mutations show partial dominance for NA, and dominance with incomplete penetrance for PO. They were mapped previously to different linkage groups of the classical map, PO to the linkage group IV and NA being linked to the erythrocyte antigen CPPP. An informative family of 70 offspring was produced by mating a sire, heterozygous for each of the mutations, to 7 dams homozygous recessive for each locus. Three DNA pools were prepared, pool PO included 20 chicks exhibiting at least one extra-toe, pool NA included 20 non-polydactyly chicks showing the typical phenotype associated with heterozygosity for the naked neck mutation, and pool NP included 20 chicks exhibiting neither of the mutant phenotypes. Typings were done on an ABI-373 automatic sequencer with 147 microsatellite markers covering most of the genome. An unbalanced distribution of sire marker alleles were detected between pool PO, and pools NA and NP, for two markers of chromosome 2p, MCW0082 and MCW0247. A linkage analysis taking into account the incomplete penetrance of polydactyly (80%) was performed with additional markers of this region and showed that the closest marker to the PO locus was MCW0071 (5 cM, lod score = 9). MCW0071 lies within the engrailed gene EN2 in the chicken. In the mouse, the homologous gene maps on chromosome 5, close to the hemimelic extra-toes mutation Hx. In the case of the NA locus, markers of chromosome 3 were selected because CPPP was mapped on this chromosome. Analysis of individual typings showed a linkage of 5.7 cM (lod score = 13) between the NA locus and ADL0237 in the distal region of chromosome 3q. These results contribute to connecting the former classical map to the molecular genetic map of the chicken, and open the way to the identification of the molecular nature of two developmental mutations of the chicken that are known to occur in many breeds of chickens.     

On the relationship between an Asian haplotype on chromosome 6 that reduces androstenone levels in boars and the differential expression of SULT2A1 in the testis

Background

Androstenone is one of the major compounds responsible for boar taint, a pronounced urine-like odor produced when cooking boar meat. Several studies have identified quantitative trait loci (QTL) for androstenone level on Sus scrofa chromosome (SSC) 6. For one of the candidate genes in the region SULT2A1, a difference in expression levels in the testis has been shown at the protein and RNA level.

Results

Haplotypes were predicted for the QTL region and their effects were estimated showing that haplotype 1 was consistently related with a lower level, and haplotype 2 with a higher level of androstenone. A recombinant haplotype allowed us to narrow down the QTL region from 3.75 Mbp to 1.94 Mbp. An RNA-seq analysis of the liver and testis revealed six genes that were differentially expressed between homozygotes of haplotypes 1 and 2. Genomic sequences of these differentially expressed genes were checked for variations within potential regulatory regions. We identified one variant located within a CpG island that could affect expression of SULT2A1 gene. An allele-specific expression analysis in the testis did not show differential expression between the alleles of SULT2A1 located on the different haplotypes in heterozygous animals. However a synonymous mutation C166T (SSC6: 49,117,861 bp in Sscrofa 10.2; C/T) was identified within the exon 2 of SULT2A1 for which the haplotype 2 only had the C allele which was higher expressed than the T allele, indicating haplotype-independent allelic-imbalanced expression between the two alleles. A phylogenetic analysis for the 1.94 Mbp region revealed that haplotype 1, associated with low androstenone level, originated from Asia.

Conclusions

Differential expression could be observed for six genes by RNA-seq analysis. No difference in the ratio of C:T expression of SULT2A1 for the haplotypes was found by the allele-specific expression analysis, however, a difference in expression between the C over T allele was found for a variation within SULT2A1, showing that the difference in androstenone levels between the haplotypes is not caused by the SNP in exon 2.     

White adipose tissue reference network: a knowledge resource for exploring health-relevant relations

Optimal health is maintained by interaction of multiple intrinsic and environmental factors at different levels of complexity—from molecular, to physiological, to social. Understanding and quantification of these interactions will aid design of successful health interventions. We introduce the reference network concept as a platform for multi-level exploration of biological relations relevant for metabolic health, by integration and mining of biological interactions derived from public resources and context-specific experimental data. A White Adipose Tissue Health Reference Network (WATRefNet) was constructed as a resource for discovery and prioritization of mechanism-based biomarkers for white adipose tissue (WAT) health status and the effect of food and drug compounds on WAT health status. The WATRefNet (6,797 nodes and 32,171 edges) is based on (1) experimental data obtained from 10 studies addressing different adiposity states, (2) seven public knowledge bases of molecular interactions, (3) expert’s definitions of five physiologically relevant processes key to WAT health, namely WAT expandability, Oxidative capacity, Metabolic state, Oxidative stress and Tissue inflammation, and (4) a collection of relevant biomarkers of these processes identified by BIOCLAIMS (http://bioclaims.uib.es). The WATRefNet comprehends multiple layers of biological complexity as it contains various types of nodes and edges that represent different biological levels and interactions. We have validated the reference network by showing overrepresentation with anti-obesity drug targets, pathology-associated genes and differentially expressed genes from an external disease model dataset. The resulting network has been used to extract subnetworks specific to the above-mentioned expert-defined physiological processes. Each of these process-specific signatures represents a mechanistically supported composite biomarker for assessing and quantifying the effect of interventions on a physiological aspect that determines WAT health status. Following this principle, five anti-diabetic drug interventions and one diet intervention were scored for the match of their expression signature to the five biomarker signatures derived from the WATRefNet. This confirmed previous observations of successful intervention by dietary lifestyle and revealed WAT-specific effects of drug interventions. The WATRefNet represents a sustainable knowledge resource for extraction of relevant relationships such as mechanisms of action, nutrient intervention targets and biomarkers and for assessment of health effects for support of health claims made on food products.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-014-0439-x) contains supplementary material, which is available to authorized users.     

A worldwide perspective on the population structure and genetic diversity of bottlenose dolphins (Tursiops truncatus) in New Zealand

Bottlenose dolphins (Tursiops truncatus) occupy a wide range of coastal and pelagic habitats throughout tropical and temperate waters worldwide. In some regions, "inshore" and "offshore" forms or ecotypes differ genetically and morphologically, despite no obvious boundaries to interchange. Around New Zealand, bottlenose dolphins inhabit 3 coastal regions: Northland, Marlborough Sounds, and Fiordland. Previous demographic studies showed no interchange of individuals among these populations. Here, we describe the genetic structure and diversity of these populations using skin samples collected with a remote biopsy dart. Analysis of the molecular variance from mitochondrial DNA (mtDNA) control region sequences (n = 193) showed considerable differentiation among populations (F(ST) = 0.17, Phi(ST) = 0.21, P < 0.001) suggesting little or no female gene flow or interchange. All 3 populations showed higher mtDNA diversity than expected given their small population sizes and isolation. To explain the source of this variation, 22 control region haplotypes from New Zealand were compared with 108 haplotypes worldwide representing 586 individuals from 19 populations and including both inshore and offshore ecotypes as described in the Western North Atlantic. All haplotypes found in the Pacific, regardless of population habitat use (i.e., coastal or pelagic), are more divergent from populations described as inshore ecotype in the Western North Atlantic than from populations described as offshore ecotype. Analysis of gene flow indicated long-distance dispersal among coastal and pelagic populations worldwide (except for those haplotypes described as inshore ecotype in the Western North Atlantic), suggesting that these populations are interconnected on an evolutionary timescale. This finding suggests that habitat specialization has occurred independently in different ocean basins, perhaps with Tursiops aduncus filling the ecological niche of the inshore ecotype in some coastal regions of the Indian and Western Pacific Oceans.     

Design of a High Density SNP Genotyping Assay in the Pig Using SNPs Identified and Characterized by Next Generation Sequencing Technology

Background

The dissection of complex traits of economic importance to the pig industry requires the availability of a significant number of genetic markers, such as single nucleotide polymorphisms (SNPs). This study was conducted to discover several hundreds of thousands of porcine SNPs using next generation sequencing technologies and use these SNPs, as well as others from different public sources, to design a high-density SNP genotyping assay.

Methodology/Principal Findings

A total of 19 reduced representation libraries derived from four swine breeds (Duroc, Landrace, Large White, Pietrain) and a Wild Boar population and three restriction enzymes (AluI, HaeIII and MspI) were sequenced using Illumina''s Genome Analyzer (GA). The SNP discovery effort resulted in the de novo identification of over 372K SNPs. More than 549K SNPs were used to design the Illumina Porcine 60K+SNP iSelect Beadchip, now commercially available as the PorcineSNP60. A total of 64,232 SNPs were included on the Beadchip. Results from genotyping the 158 individuals used for sequencing showed a high overall SNP call rate (97.5%). Of the 62,621 loci that could be reliably scored, 58,994 were polymorphic yielding a SNP conversion success rate of 94%. The average minor allele frequency (MAF) for all scorable SNPs was 0.274.

Conclusions/Significance

Overall, the results of this study indicate the utility of using next generation sequencing technologies to identify large numbers of reliable SNPs. In addition, the validation of the PorcineSNP60 Beadchip demonstrated that the assay is an excellent tool that will likely be used in a variety of future studies in pigs.