Host-dependent genetic variation in freshwater pearl mussel (Margaritifera margaritifera L.)

Freshwater fishes are vulnerable to changes in water quality, physical habitat and connectivity resulting from drought, particularly in regulated rivers. When adequate river flows return, the recovery of populations might depend on the duration and consequences of drought. Rivers of the highly regulated Murray–Darling Basin in south-eastern Australia terminate at two large, shallow lakes that are separated from the estuary by tidal barrages. Over-abstraction of water and widespread prolonged drought (1997–2010) placed the lakes under severe environmental stress, culminating in critical water level recession from 2007 to 2010. Concurrently, most freshwater fish populations collapsed. We investigate shifts in fish assemblages resulting from habitat inundation in the lakes following the drought. The inundation and re-connection of the lakes and fringing habitats led to a substantial reduction of salinity throughout the region, and aquatic vegetation shifted from salt-tolerant to freshwater species. Fish assemblages became increasingly characterized by common freshwater taxa (ecological generalists), including high proportions of alien species. There were no indications of population recovery for three threatened species. The findings emphasize that short-lived fishes with specialized habitat requirements are vulnerable to severe population declines during prolonged drought in regulated rivers, which might restrict their recovery when adequate flows return.     

Role of mef2ca in developmental buffering of the zebrafish larval hyoid dermal skeleton

Phenotypic robustness requires a process of developmental buffering that is largely not understood, but which can be disrupted by mutations. Here we show that in mef2ca^b1086 loss of function mutant embryos and early larvae, development of craniofacial hyoid bones, the opercle (Op) and branchiostegal ray (BR), becomes remarkably unstable; the large magnitude of the instability serves as a positive attribute to learn about features of this developmental buffering. The OpBR mutant phenotype variably includes bone expansion and fusion, Op duplication, and BR homeosis. Formation of a novel bone strut, or a bone bridge connecting the Op and BR together occurs frequently. We find no evidence that the phenotypic stability in the wild type is provided by redundancy between mef2ca and its co-ortholog mef2cb, or that it is related to the selector (homeotic) gene function of mef2ca. Changes in dorsal–ventral patterning of the hyoid arch also might not contribute to phenotypic instability in mutants. However, subsequent development of the bone lineage itself, including osteoblast differentiation and morphogenetic outgrowth, shows marked variation. Hence, steps along the developmental trajectory appear differentially sensitive to the loss of buffering, providing focus for the future study.     

Reactive Oxygen Species-Mediated DJ-1 Monomerization Modulates Intracellular Trafficking Involving Karyopherin β2

Mutations in DJ-1 are a cause of recessive, early-onset Parkinson''s disease (PD). Although oxidative stress and mitochondrial integrity have been implicated in PD, it is largely unknown why neurons degenerate. DJ-1 is involved in oxidative stress-mediated responses and in mitochondrial maintenance; however, its specific function remains vague. Here we show that DJ-1 exhibits neuronal dynamic intracellular trafficking, with dimeric/monomeric cycling modulated by the oxidative environment. We demonstrate that oxidative stress enhances monomerization of wild-type cytosolic DJ-1, leading to nuclear recruitment. The pathogenic DJ-1/E163K variant is unable to homodimerize but is retained in the cytosol upon wild-type DJ-1 heterodimerization. We found that this wild-type/pathogenic heterodimer is disrupted by oxidative stress, leading to DJ-1/E163K mitochondrial translocation. We further demonstrated that endogenously expressed wild-type DJ-1 is imported into neuronal nuclei as a monomer and that nucleo-cytoplasmic transport is oxidative stress mediated. We identified a novel proline-tyrosine nuclear localization signal (PY-NLS) in DJ-1, and we found that nuclear monomeric DJ-1 import is mediated by an oxidative stress-dependent interaction with karyopherin β2. Our study provides evidence that oxidative stress-mediated intracellular trafficking of DJ-1, mediated by dynamic DJ-1 dimeric/monomeric cycling, is implicated in PD pathogenesis.     

The utility of PacBio circular consensus sequencing for characterizing complex gene families in non-model organisms

Background

Molecular characterization of highly diverse gene families can be time consuming, expensive, and difficult, especially when considering the potential for relatively large numbers of paralogs and/or pseudogenes. Here we investigate the utility of Pacific Biosciences single molecule real-time (SMRT) circular consensus sequencing (CCS) as an alternative to traditional cloning and Sanger sequencing PCR amplicons for gene family characterization. We target vomeronasal gene receptors, one of the most diverse gene families in mammals, with the goal of better understanding intra-specific V1R diversity of the gray mouse lemur (Microcebus murinus). Our study compares intragenomic variation for two V1R subfamilies found in the mouse lemur. Specifically, we compare gene copy variation within and between two individuals of M. murinus as characterized by different methods for nucleotide sequencing. By including the same individual animal from which the M. murinus draft genome was derived, we are able to cross-validate gene copy estimates from Sanger sequencing versus CCS methods.

Results

We generated 34,088 high quality circular consensus sequences of two diverse V1R subfamilies (here referred to as V1RI and V1RIX) from two individuals of Microcebus murinus. Using a minimum threshold of 7× coverage, we recovered approximately 90% of V1RI sequences previously identified in the draft M. murinus genome (59% being identical at all nucleotide positions). When low coverage sequences were considered (i.e. < 7× coverage) 100% of V1RI sequences identified in the draft genome were recovered. At least 13 putatively novel V1R loci were also identified using CCS technology.

Conclusions

Recent upgrades to the Pacific Biosciences RS instrument have improved the CCS technology and offer an alternative to traditional sequencing approaches. Our results suggest that the Microcebus murinus V1R repertoire has been underestimated in the draft genome. In addition to providing an improved understanding of V1R diversity in the mouse lemur, this study demonstrates the utility of CCS technology for characterizing complex regions of the genome. We anticipate that long-read sequencing technologies such as PacBio SMRT will allow for the assembly of multigene family clusters and serve to more accurately characterize patterns of gene copy variation in large gene families, thus revealing novel micro-evolutionary patterns within non-model organisms.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-720) contains supplementary material, which is available to authorized users.     

Genome-wide association and biological pathway analysis for milk-fat composition in Danish Holstein and Danish Jersey cattle

Background

The milk fat profile of the Danish Holstein (DH) and Danish Jersey (DJ) show clear differences. Identification of the genomic regions, genes and biological pathways underlying the milk fat biosynthesis will improve the understanding of the biology underlying bovine milk fat production and may provide new possibilities to change the milk fat composition by selective breeding. In this study a genome wide association scan (GWAS) in the DH and DJ was performed for a detailed milk fatty acid (FA) profile using the HD bovine SNP array and subsequently a biological pathway analysis based on the SNP data was performed.

Results

The GWAS identified in total 1,233 SNPs (FDR < 0.10) spread over 18 chromosomes for nine different FA traits for the DH breed and 1,122 SNPs (FDR < 0.10) spread over 26 chromosomes for 13 different FA traits were detected for the DJ breed. Of these significant SNPs, 108 SNP markers were significant in both DH and DJ (C14-index, BTA26; C16, BTA14; fat percentage (FP), BTA14). This was supported by an enrichment test. The QTL on BTA14 and BTA26 represented the known candidate genes DGAT and SCD. In addition we suggest ACSS3 to be a good candidate gene for the QTL on BTA5 for C10:0 and C15:0. In addition, genetic correlations between the FA traits within breed showed large similarity across breeds. Furthermore, the biological pathway analysis revealed that fat digestion and absorption (KEGG04975) plays a role for the traits FP, C14:1, C16 index and C16:1.

Conclusion

There was a clear similarity between the underlying genetics of FA in the milk between DH and DJ. This was supported by the fact that there was substantial overlap between SNPs for FP, C14 index, C14:1, C16 index and C16:1. In addition genetic correlations between FA showed a similar pattern across DH and DJ. Furthermore the biological pathway analysis suggested that fat digestion and absorption KEGG04975 is important for the traits FP, C14:1, C16 index and C16:1.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1112) contains supplementary material, which is available to authorized users.     

Comprehensive Quantitative Comparison of the Membrane Proteome,Phosphoproteome, and Sialiome of Human Embryonic and Neural Stem Cells

Human embryonic stem cells (hESCs) can differentiate into neural stem cells (NSCs), which can further be differentiated into neurons and glia cells. Therefore, these cells have huge potential as source for treatment of neurological diseases. Membrane-associated proteins are very important in cellular signaling and recognition, and their function and activity are frequently regulated by post-translational modifications such as phosphorylation and glycosylation. To obtain information about membrane-associated proteins and their modified amino acids potentially involved in changes of hESCs and NSCs as well as to investigate potential new markers for these two cell stages, we performed large-scale quantitative membrane-proteomic of hESCs and NSCs. This approach employed membrane purification followed by peptide dimethyl labeling and peptide enrichment to study the membrane subproteome as well as changes in phosphorylation and sialylation between hESCs and NSCs. Combining proteomics and modification specific proteomics we identified a total of 5105 proteins whereof 57% contained transmembrane domains or signal peptides. The enrichment strategy yielded a total of 10,087 phosphorylated peptides in which 78% of phosphopeptides were identified with ≥99% confidence in site assignment and 1810 unique formerly sialylated N-linked glycopeptides. Several proteins were identified as significantly regulated in hESCs and NSC, including proteins involved in the early embryonic and neural development. In the latter group of proteins, we could identify potential NSC markers as Crumbs 2 and several novel proteins. A motif analysis of the altered phosphosites showed a sequence consensus motif (R-X-XpS/T) significantly up-regulated in NSC. This motif is among other kinases recognized by the calmodulin-dependent protein kinase-2, emphasizing a possible importance of this kinase for this cell stage. Collectively, this data represent the most diverse set of post-translational modifications reported for hESCs and NSCs. This study revealed potential markers to distinguish NSCs from hESCs and will contribute to improve our understanding on the differentiation process.Pluripotent embryonic stem cell (ESC)¹-derived neural stem cells (NSCs) can differentiate into neurons and glia cells of the central nervous system (1), including specialized neuron types like dopaminergic, representing a potential source for treatment of neurological diseases, such as Parkinson′s disease. Therefore, a better understanding of the cellular processes behind the changes of hESCs into NSCs, including solid markers for each cell type, is fundamental to move forward with a successful regenerative cell therapy and to investigate the early human neurogenesis processes.Many markers have been reported for the two types of stem cells (2, 3), however several of these markers are also identified in other stem or progenitor cells such as CD133 (Prominin-1) (4). Discovery of cell surface specific markers for differentiated stem cells is highly relevant for future clinical applications. In particular being able to distinguish the developmental stages of the differentiation from parental stem cells to fully mature cells would allow a correct manipulation and isolation of the cell type of interest. Moreover, such study would increase our understanding on the whole process of differentiation from embryonic cells to neural cells. Because plasma membrane-associated proteins are the key interface between cell and the surrounding environment, and they frequently present large extracellular domains suitable for antibody detection, they represent a great marker candidate potential. In addition, these proteins are very important in the cellular signaling process and cell–cell interaction and communication, processes very important for cellular differentiation. Furthermore, most membrane bound proteins involved in the abovementioned process are frequently regulated or otherwise manipulated to alter interaction partners and function by post-translational modifications (PTMs) such as phosphorylation and glycosylation.Protein phosphorylation and glycosylation are the most common PTMs in nature and they play an important role in many protein regulatory functions and cellular and biological processes. Protein phosphorylation is a dynamic PTM involved in many different cell signaling events like cell cycle, protein synthesis, protein degradation, differentiation, as well cellular proliferation and apoptosis (5). On the other hand, protein glycosylation has several roles in cell–cell interaction, cell-matrix interaction, communication and cellular signaling (6–8). A nine carbon sugar unit termed sialic acid (SA) can be bound to the nonreducing ends of glycans attached to certain membrane proteins, secreted proteins and lipids (9). SA is involved in many physiological processes such as cell–cell interaction and molecular recognition, and is important in different pathophysiological process, including brain development and cancer metastasis (10–14).The mass spectrometry-based proteomic approach is a powerful tool for characterization of proteins and their PTMs. Because of the low abundance of protein PTMs in comparison to their nonmodified counterpart in the cell or tissue, their detection and characterization are almost only possible by using advanced enrichment strategies. Although many studies have reported the proteome and phosphoproteome of hESCs and early differentiated stages (15–20), there are a limited number of studies available comparing the individual stages between hESCs and NSC (21, 22) especially at the PTM level. For example, Chaerkady and collaborators have reported the quantitative temporal proteomic analysis of hESC differentiation into neural cell types, including motor neurons and astrocytes (21). The authors identified a total of 1251 proteins including proteins differentially regulated during neural differentiation such as the solute carrier protein 3 member 2 (SLC3A2), a cell surface protein highly expressed only in the hESCs stage. However, the focus of this study was not on membrane bound proteins and the authors did not analyze any PTM profile during differentiation, which may also elicit important and more selective information regarding the molecular events underlying the different stages.To obtain more information about membrane proteins involved in the changes of hESCs to NSCs and also to investigate potential markers for the two distinct cellular stages, we performed a comprehensive quantitative mass-spectrometry-based proteome and PTM-ome study of membrane fractions isolated from hESCs and NSCs. We focused the PTM study on phosphorylation and SA N-linked glycosylation. This study allowed us to identify several significantly regulated proteins in hESCs and NSCs, including proteins involved in the early embryonic development as well as in the neural development. In the latter group of proteins we could identify Crumbs homolog 2 (CR2) as a potential novel NSC marker. By using selective reaction monitoring (SRM) we were able to verify a number of potential markers including CRB2 at protein and PTM level as well as CMP-N-acetylneuraminate-poly-alpha-2,8-sialyltransferase (SIA8D) at the PTM level across different cell lines beside the one used in this study. In addition, calmodulin-dependent protein kinase-2 (CaMKII) could be an important kinase for the NSC stage, because we identified the sequence recognition motif (R-X-X-pS/T) highly up-regulated in these cells. This is the consensus site for several kinases including CaMKII. Moreover, the analysis of the regulated dataset revealed an over-representation of the extracellular matrix (ECM)-receptor pathways, which are involved in diverse processes such as differentiation and proliferation (23). To our knowledge this comparative proteome study of the membrane-associated proteins that include quantitative analysis of protein phosphorylation and SA N-linked glycosylation is the most diverse set of PTMs reported for hESCs and NSCs.     

Interaction between Genetic Predisposition to Adiposity and Dietary Protein in Relation to Subsequent Change in Body Weight and Waist Circumference

Background

Genetic predisposition to adiposity may interact with dietary protein in relation to changes of anthropometry.

Objective

To investigate the interaction between genetic predisposition to higher body mass index (BMI), waist circumference (WC) or waist-hip ratio adjusted for BMI (WHR_BMI) and dietary protein in relation to subsequent change in body weight (ΔBW) or change in WC (ΔWC).

Design

Three different Danish cohorts were used. In total 7,054 individuals constituted the study population with information on diet, 50 single-nucleotide polymorphisms (SNPs) associated with BMI, WC or WHR_BMI, as well as potential confounders. Mean follow-up time was ∼5 years. Four genetic predisposition-scores were based on the SNPs; a complete-score including all selected adiposity- associated SNPs, and three scores including BMI, WC or WHR_BMI associated polymorphisms, respectively. The association between protein intake and ΔBW or ΔWC were examined and interactions between SNP-score and protein were investigated. Analyses were based on linear regressions using macronutrient substitution models and meta-analyses.

Results

When protein replaced carbohydrate, meta-analyses showed no associations with ΔBW (41.0 gram/y/5 energy% protein, [95% CI: −32.3; 114.3]) or ΔWC (<−0.1 mm/y/5 energy % protein, [−1.1; 1.1]). Similarly, there were no interactions for any SNP-scores and protein for either ΔBW (complete SNP-score: 1.8 gram/y/5 energy% protein/risk allele, [−7.0; 10.6]) or ΔWC (complete SNP-score: <0.1 mm/y/5 energy% protein/risk allele, [−0.1; 0.1]). Similar results were seen when protein replaced fat.

Conclusion

This study indicates that the genetic predisposition to general and abdominal adiposity, assessed by gene-scores, does not seem to modulate the influence of dietary protein on ΔBW or ΔWC.     

Lifestyle Factors Associated with Type 2 Diabetes and Use of Different Glucose-Lowering Drugs: Cross-Sectional Study

Aims

To examine the lifestyle profile among persons with and without Type 2 diabetes mellitus (DM) and among users of different glucose-lowering drugs.

Methods

We used questionnaire data from a Danish health survey and identified presence of Type 2 DM and use of medications through medical databases. We calculated age- and gender-standardized prevalence ratios (PRs) of lifestyle factors according to Type 2 DM and different glucose-lowering drugs.

Results

Of 21,637 survey participants aged 25–79 years, 680 (3%) had Type 2 DM (median age 63 years) with a median diabetes duration of 5 years. Participants with Type 2 DM had a substantially higher prevalence of obesity (36% vs. 13%, PR: 3.1, 95% confidence interval (CI): 2.8–3.6), yet more reported to eat a very healthy diet (25% vs. 21%, PR: 1.2, 95% CI: 1.0–1.4) and to exercise regularly (67% vs. 53%, PR: 1.3, 95% CI: 1.2–1.4). Also, fewer were current smokers or had high alcohol intake. When compared with metformin users, obesity was substantially less prevalent in users of sulfonylurea (PR: 0.5, 95% CI: 0.4–0-8), and insulin and analogues (PR: 0.4, 95% CI: 0.3–0.7). Tobacco smoking was more prevalent in sulfonylurea users (PR: 1.4, 95% CI: 0.9–2.1) compared with metformin users. We found no material differences in physical exercise, diet or alcohol intake according to type of glucose-lowering drug.

Conclusions

Type 2 DM patients are substantially more obese than other individuals, but otherwise report to have a healthier lifestyle. Metformin use is strongly associated with obesity, whereas sulfonylurea use tends to be associated with tobacco smoking.     

A Biodiversity Indicators Dashboard: Addressing Challenges to Monitoring Progress towards the Aichi Biodiversity Targets Using Disaggregated Global Data

Recognizing the imperiled status of biodiversity and its benefit to human well-being, the world''s governments committed in 2010 to take effective and urgent action to halt biodiversity loss through the Convention on Biological Diversity''s “Aichi Targets”. These targets, and many conservation programs, require monitoring to assess progress toward specific goals. However, comprehensive and easily understood information on biodiversity trends at appropriate spatial scales is often not available to the policy makers, managers, and scientists who require it. We surveyed conservation stakeholders in three geographically diverse regions of critical biodiversity concern (the Tropical Andes, the African Great Lakes, and the Greater Mekong) and found high demand for biodiversity indicator information but uneven availability. To begin to address this need, we present a biodiversity “dashboard” – a visualization of biodiversity indicators designed to enable tracking of biodiversity and conservation performance data in a clear, user-friendly format. This builds on previous, more conceptual, indicator work to create an operationalized online interface communicating multiple indicators at multiple spatial scales. We structured this dashboard around the Pressure-State-Response-Benefit framework, selecting four indicators to measure pressure on biodiversity (deforestation rate), state of species (Red List Index), conservation response (protection of key biodiversity areas), and benefits to human populations (freshwater provision). Disaggregating global data, we present dashboard maps and graphics for the three regions surveyed and their component countries. These visualizations provide charts showing regional and national trends and lay the foundation for a web-enabled, interactive biodiversity indicators dashboard. This new tool can help track progress toward the Aichi Targets, support national monitoring and reporting, and inform outcome-based policy-making for the protection of natural resources.