text:
- Research Paper
- Open Access
Genetic associations with micronutrient levels identified in immune and gastrointestinal networks
© The Author(s) 2014
- Received: 1 February 2014
- Accepted: 12 May 2014
- Published: 31 May 2014
The discovery of vitamins and clarification of their role in preventing frank essential nutrient deficiencies occurred in the early 1900s. Much vitamin research has understandably focused on public health and the effects of single nutrients to alleviate acute conditions. The physiological processes for maintaining health, however, are complex systems that depend upon interactions between multiple nutrients, environmental factors, and genetic makeup. To analyze the relationship between these factors and nutritional health, data were obtained from an observational, community-based participatory research program of children and teens (age 6–14) enrolled in a summer day camp in the Delta region of Arkansas. Assessments of erythrocyte S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH), plasma homocysteine (Hcy) and 6 organic micronutrients (retinol, 25-hydroxy vitamin D3, pyridoxal, thiamin, riboflavin, and vitamin E), and 1,129 plasma proteins were performed at 3 time points in each of 2 years. Genetic makeup was analyzed with 1 M SNP genotyping arrays, and nutrient status was assessed with 24-h dietary intake questionnaires. A pattern of metabolites (met_PC1) that included the ratio of erythrocyte SAM/SAH, Hcy, and 5 vitamins were identified by principal component analysis. Met_PC1 levels were significantly associated with (1) single-nucleotide polymorphisms, (2) levels of plasma proteins, and (3) multilocus genotypes coding for gastrointestinal and immune functions, as identified in a global network of metabolic/protein–protein interactions. Subsequent mining of data from curated pathway, network, and genome-wide association studies identified genetic and functional relationships that may be explained by gene–nutrient interactions. The systems nutrition strategy described here has thus associated a multivariate metabolite pattern in blood with genes involved in immune and gastrointestinal functions.
- Systems nutrition
- Community-based participatory research
- Genetic analysis
- Network analysis
Systems thinking and methodologies hold greater promise in understanding the complex phenotypes of chronic disease or response to nutrients in foods than the focus on individual genetic variants or the identification of independent environmental factors (Patel et al. 2010, 2012a, b) that influence biological processes. An increasing number of reports employ systems designs and analysis of high-dimensional data from studies of obesity, cardiovascular, nutrition, diabetes, drug, toxicology, immunology, gut microbiota, medicine, health care, and health disparities (Slikker et al. 2007; Auffray et al. 2009; Gardy et al. 2009; Kalupahana and Moustaid-moussa 2011; Kleemann et al. 2011; Roux 2011; Karlsson et al. 2011; Afacan et al. 2012; Meng et al. 2013).
"…Since the components of such a [sic] system are likely to be interrelated in complex ways, and since the synthesis of the parts of individual genes are presumably dependent on the functioning of other genes, it would appear that there must exist orders of directness of gene control ranging from simple one-to-one relations to relations of great complexity" (Beadle and Tatum 1941). (Emphasis added)
With the exception of several publications that include dietary intake variables as a part of omics-based systems (Morine et al. 2010, 2011, 2012) or genomic analysis (Nettleton et al. 2010), many systems studies have implicitly analyzed biological processes as closed systems since environmental variables were not included in the analysis. Biological processes occur in open systems (Von Bertalanffy 1950), and ex vivo factors, which include nutrients and other naturally occurring chemicals in food, can alter biochemical processes and signaling networks occurring within the organism (Kaput and Rodriguez 2004). Excluding external factors that influence internal biological processes generates an incomplete system at best, likely an inaccurate understanding of the interactions between environment and genetic makeup, and from a practical standpoint, misses an opportunity to identify modifiable factors that influence health.
This report details the design and conduct of a discovery-based pilot study that accounts for (1) the known genetic uniqueness of individual humans (Olson 2012), (2) the intra-individual variability in homeostatic measurements (Williams 1956; Illig et al. 2010; Suhre et al. 2011), and (3) the challenge of characterizing complex phenotypes resulting from small contributions of many genetic and environmental factors (Goldstein 2009). The participants in the Delta Vitamin Obesity intervention study were children and teens (age 6–14) enrolled in a summer day camp that was a component of a community-based participatory research (CBPR) program. CBPR is a form of translational research that engages the participant, members of the community, and scientists in research, education, and health-promoting activities for improving personal and public health (McCabe-Sellers et al. 2008). A detailed description of the intervention and results obtained by aggregating data from individuals for population-level analysis such as metabolite and protein variation in relation to BMI, sex, and age has been reported (Monteiro et al. 2014).
In this report, analysis of the data from the Delta Vitamin Obesity (Monteiro et al. 2014) is extended to further characterize metabolite–metabolite interactions with discovery-based methods that identify systems-wide relationships between metabolites, proteins, nutrient intakes, and genetic makeup. Principal component analysis (PCA) was used to analyze plasma homocysteine (Hcy); vitamins A, D, E; riboflavin; thiamine; pyridoxal; and erythrocyte S-adenosylmethionine and S-adenosylhomocysteine (SAH) metabolites. A quantitative variable (met_PC1) from the PCA was defined and used for discovering metabolite–protein correlations as well as thousands of genotypes associated with met_PC1 values. Two recent studies have used inferences based on heritability and Bayesian approaches to identify thousands of SNPs associated with height and weight (Hemani et al. 2013) and rheumatoid arthritis (Stahl et al. 2012) to demonstrate that complex phenotypes are the result of thousands of SNPs. Subsequent data mining methods associated genes and proteins identified in this report to biological functional classes including predominantly immune and gastrointestinal function. Finally, the challenges of conducting case–control studies in light of genetic and cultural differences within and between populations are discussed.
Materials and methods
Participants and CBPR methods
A description of the summer day camp in the Marvell, AR (USA) school district, 24-h dietary intakes, body weight and height, blood sampling and processing, and proteomic and genomic analysis are provided in (Monteiro et al. 2014). In brief, assessments were conducted before the beginning of the camp (baseline), at the end of 5 weeks of the camp (end of camp), and 1 month after camp ended (post-camp). Metabolite and dietary intake data were averaged across the three assessments for the analysis in this study. Thirty-six participants were recruited in year 1, and 19 completed all three assessments. In the second year, 72 participants enrolled and 42 completed three assessments. None of the children or adolescents (age 6–14) was taking prescribed medicines, nor did they have overt malnutrition, active infection, or known genetic disease that could alter metabolism. All participants were healthy African American children and adolescents. Results for the three assessments are reported. The biomedical research protocol was approved by the FDA's Research Involving Human Subjects Committee (RIHSC) and the University of Arkansas for Medical Sciences (UAMS) Institutional Review Board (IRB).
Total Hcy was analyzed in plasma using a Hcy HPLC Kit (ALPCO Immunoassays, Salem, NH) and a UPLC Waters Acquity HSS T3 column (2.1 × 50 mm, 1.8 µm) coupled with an Acquity HSS T3 1.8 µm VanGuard pre-column at 40 °C.
Lipid-soluble vitamins
Vitamins were determined using LC/MS/MS (NCTR-FDA-USA): 250 µL of plasma, in a 1.5-mL Eppendorf microcentrifuge tube, was spiked with stable isotope-labeled standards and mixed with 740 µL of MeOH. Samples were held at 4 °C for 30 min. About 500 µL of hexane was added, and samples were centrifuged at 13,000×g for 12 min (4 °C). The (top) hexane layer was transferred into a total recovery autosampler vial, and the sample was subsequently extracted with two additional 500 µL hexane portions, each time transferring the hexane layer into the autosampler vial. The combined hexane extracts were placed under a stream of nitrogen gas, dried, and reconstituted in 50 µL of 50:50 MeOH/ACN. Ten microlitre sample was injected on an Acquity UPLC equipped with a 2.1 mm × 50 mm (1.7 µm particle) BEH C18 column held at 35 °C. The mobile phase A was 90:10 water/ACN, and the mobile phase B was 50:50 MeOH/can with a flow rate of 0.5 mL/min. Metabolites were analyzed on a Xevo TQ operated in positive APCI ionization mode using the following parameters: source temperature was 145 °C, corona was 15 uA, probe temperature was 575 °C, and desolvation gas flow rate was 600 L/h. Multiple reaction monitoring (MRM) was optimized by direct infusion of standards. The transitions monitored for vitamin A were m/z 269 → 109 (cone E = 35 V, collision E = 15 V) and m/z 269 → 93 (cone E = 26 V, collision E = 14 V). The transition monitored for vitamin E was m/z 431 → 165 and for (d3) vitamin E was m/z 434 → 165 (cone E = 35 V, collision E = 15 V). The transition monitored for 25-hydroxy vitamin D3 was m/z 401 → 159 and m/z 407 → 159 for the (d6) 25 hydroxy vitamin D3 with cone and collision energies of 24 and 28 V, respectively.
Water-soluble vitamins
About 250 µL of plasma was mixed with 1 mL of (4 °C) acetonitrile in a 1.5-mL Eppendorf microcentrifuge tube. The sample was vortexed briefly and then centrifuged at 13,000×g for 10 min at 4 °C. The supernatant was transferred to a total recovery autosampler vial, and the solvent was evaporated. Samples were reconstituted in 250 µL of water (Optima grade), and 10 µL of sample was injected onto an Acquity UPLC equipped with an HHS T3 2.1 × 100 mm, (1.8 µm particle) UPLC column. Mass spectrometric detection was performed on a Xevo TQ (Waters) operated in ESI positive mode using the following parameters: source temperature was 150 °C, capillary voltage (kV) was 2.2, desolvation temperature was 400 °C, and desolvation gas flow rate was 800 L/h. MRMs for target analytes were optimized by direct infusion of standards. The transition monitored for pyridoxal was m/z 168 → 94 (cone E = 16 V, collision E = 22 V), for pyridoxine m/z 170 → 134 (cone E = 22 V, collision E = 22 V), for thiamine m/z 265 → 122 (cone E = 20 V, collision E = 12 V), for riboflavin m/z 377 → 243 (cone E = 40 V, collision E = 22 V), and for folic acid m/z 442 → 295 (cone E = 22 V, collision E = 12 V).
Red blood cell S-adenosyl-l-methionine (SAM) and S-adenosyl-l-homocysteine (SAH)
Red blood cell samples stored at −70 °C were randomly assayed in batches of 20. About 600 µL of red blood cells was added to tubes containing 150 µL of ice-cold trichloroacetic acid (40 % w/v), plus 330 µL 0.1 M sodium acetate trihydrate, and then vortexed. Samples were incubated at 4 °C for 30 min, followed by centrifugation at 15,000 rpm for 15 min. About 150 µL of supernatant was filtered using a 0.22-µm filter, spun at 5,000 rpm for 5 min, and transferred to vials for chromatographic analysis of SAM. The remainder of the supernatant was transferred to a clean tube for ether extraction. Samples were extracted twice with 300 µL, and any remaining ether was evaporated under argon before filtration and transferred to UPLC vials for the analysis of SAH. Standards for SAM and SAH were obtained from Sigma (St. Louis, MO). Chromatographic separation was achieved on an Acquity HSS T3 column (2.1 × 50 mm, 1.8 µm) coupled with an Acquity HSS T3 1.8 µm VanGuard pre-column at 40 °C. The peaks were separated isocratically with an elution time of 5.0 min for SAM and 2.0 min for SAH at 97 % A (buffer) and 3 % B (methanol). The buffer composition for SAM was 50 mM potassium phosphate and 10 mM heptane sulfonic salt adjusted to pH 4.38 with phosphoric acid. The composition for the SAH mobile phase was 50 mM potassium phosphate. Column equilibration time required for SAM was 90 min, while equilibration time for SAH was just 30 min at flow rates of 0.575 mL/min. Buffers and solvents are filtered using 0.22-µm filters prior to use. Samples were held at 4 °C for the duration of the analysis. The injection volume for samples and standard was 10 µL. Detection was performed with a photodiode array detector set to monitor wavelengths 210–400. Standard was prepared in a range from 0.78 to 25.00 pmol/µL for SAH and from 0.32 to 10.40 µL for SAM. A standard curve was generated to allow for automated calculation of results using the Waters Empower software.
The plasma proteome was quantified for 110 samples from 6 different time points (3 in year 1 and 3 in year 2) but data from 61 at time point 1 were used in these analyses due to missing samples. SomaLogic Inc. (Boulder, CO) performed all proteomic assessments and was blinded to the clinical characteristics of participants in this study. Samples were analyzed as previously described (Gold 1995; Brody and Gold 2000; Gold et al. 2010; Ostroff et al. 2010; Brody et al. 2012).
Genomic analysis
DNA preparation
About 1 mL of whole blood sample from each participant was used for DNA extraction. The genomic DNA samples were extracted and purified using the QIAamp DNA Blood Mini Kit (QIAGEN, Valencia, CA), following the protocol provided by the manufacturer. The quality and quantity of each DNA samples were measured using a NanoDrop 8000 (Thermo Scientific, Wilmington, DE). The Infinium Whole Genome Genotyping technology with the HumanOmni1-Quad version 1.0 kits (Illumina, San Diego, CA) was used for genotyping analyses following the manufacturer's protocol. The arrays were scanned on a high-resolution iScan (Illumina) and processed using the BeadStudio software version 3.1 (Illumina). The overall genotyping call rate on all samples was above 98 %. Data from 45 unique participants (15 participants attended both years) met these criteria.
Preprocessing of genotyping data
Raw SNP data were first preprocessed, removing SNPs with a GC score <0.7, and those that were not genotyped in all participants. SNPs with minor allele frequency <0.1 and those significantly diverging from Hardy–Weinberg equilibrium were also removed. The remaining SNPs were filtered to include only those present in the metabolic/protein–protein interaction (PPI) network used in the analysis, resulting in a final dataset of 125,959 SNPs.
Network analysis
A metabolic/PPI network was constructed based on the human interaction networks manually curated databases (Ma et al. 2007; Yu et al. 2012). The largest connected component of this network comprised 116,210 interactions between 13,705 genes, containing 125,959 SNPs present on the Illumina 1 M Quad Array. The network was partitioned into topological modules using the spinglass.community function in the igraph library in R (Csardi and Nepusz 2006) resulting in 58 topological modules (mean module size: 236 nodes; SD: 564 nodes).
SNP-, gene-, and network-level analyses
Significant correlations between genotype and met_PC1 levels were assessed in each SNP using generalized estimating equations (GEE), as implemented in the geepack library in R (Højsgaard et al. 2006). Met_PC1 was modeled as a function of genotype at each SNP locus, controlling for age, gender, average Healthy Eating Index, and sibling relationships among the participants (the latter being included as an independence correlation structure in the GEE models). Although some participants attended both years of the camp, only one genotype per participant was used in this analysis. Resulting p values were corrected for multiple testing using the procedure proposed by Benjamini and Hochberg (1995). Nominal p values were used as input for the VEGAS algorithm, which accounts for size, level of polymorphism, and linkage disequilibrium relationships within genes to determine genewise p values from SNP-level results (Liu et al. 2010). Genes reaching significance (q < 0.1) were used in hypergeometric tests (implemented using the HTSanalyzeR library in R) to determine significant enrichment of each of the 58 modules in the interaction network. Modules with q value <0.1 were considered as significantly enriched in genes related to micronutrients. In order to assess the biological processes that may be directly or indirectly implicated by genetic variation in our met_PC1 genes, the functional profile of each significant module was determined using the ClueGO (Bindea et al. 2009) plugin for Cytoscape. ClueGO functional profiles illustrated in Fig. 6 and Supplementary files include KEGG pathways that are significantly overrepresented among module nodes, using hypergeometric tests and correcting p values using the Benjamini and Hochberg method (see Bindea et al. 2009) for technical details on the generation of functional profile networks.
Significant genes were also analyzed in the context of the ArrayTrack QTL database (Harris et al. 2009; Xu et al. 2010) to determine significantly overrepresented QTL phenotypes. Gene sets were constructed by combining all genes within 1 Mbp of QTL mapping to each of the 36 phenotypes (containing at least one significant gene from our analysis) in the ArrayTrack database. Hypergeometric tests were then performed to identify which QTL phenotypes were significantly enriched in the significant genes from our analysis.
PCA of metabolite levels
Given the strong patterns of correlation among the plasma metabolites, PCA was used to identify latent metabolite variables. The first principal component (met_PC1) explained 41 % (Fig. 1b, c) of the variation in metabolite profile and stratified the participants primarily based on their levels of vitamin A, Hcy, SAM/SAH, thiamine, pyridoxal, and vitamin E. Vitamin D and riboflavin contributed to the second principal component and explained 5 % of the variation (Fig. 1b, c) in the dataset. To our knowledge, these nutrient–nutrient associations have not been previously reported and would not have been identified by standard single-variant analysis. Although met_PC1 is a continuous variable, the analysis and heat map indicate metabolic patterns that could be used to group individuals for different nutritional interventions.
Proteomic associations with metabolite patterns
Correlation analysis and hierarchical clustering produced two main branches differing in the percentage of plasma-soluble and membrane proteins versus cytosolic proteins (Fig. 2; see brackets at bottom). We previously observed two clusters of blood versus cytosolic proteins associated with erythrocyte SAM/SAH ratios (Monteiro et al. 2014). The cytosolic proteins in the blood were likely produced by apoptotic processes, although the current data cannot discriminate between normal and pathological cell death.
Genetic analyses
Analysis of genotype–metabolite correlations within a global protein interaction network
Nominal p values were used as input for the VEGAS algorithm, which accounts for size, level of polymorphism, and linkage disequilibrium relationships within genes to determine genewise p values from SNP-level results (Liu et al. 2010). The result was 1,875 statistically significant genes associated with the met_PC1 variable, which were unevenly distributed among 46 of the 58 modules (Supplement 3).
Network modules significantly enriched in met_PC1 genes
Module ID
Module size
Expected hits
Observed hits
p value
Adjusted p value
1.33 × 10−10
3.86 × 10−9
2.23 × 10−3
3.23 × 10−2
6.23 × 10−3
6.02 × 10−2
1.22 × 10−2
8.86 × 10−2
Network modules significantly enriched in neighborhood micronutrient genes
Module ID
Module size
Expected hits
Observed hits
p value
Adjusted p value
8.23 × 10−35
2.39 × 10−33
9.07 × 10−21
1.32 × 10−19
1.74 × 10−22
1.68 × 10−19
Functional and genetic analyses of statistically significant genes and modules
In order to assess the biological processes that may be directly or indirectly implicated by genetic variation in our met_PC1 genes, the functional profile of each significant module was determined using data mining tools including the ClueGO plugin in Cytoscape (Bindea et al. 2009), the KEGG pathway database (http://www.genome.jp/kegg/pathway), ArrayTrack QTL (Xu et al. 2010) database, and literature mining. All pathways described in the ClueGO analysis results were significantly overrepresented in the given module (adjusted p value <0.05).
Module 18: Functional annotation
Module 2: Functional annotation
Module 2 is functionally enriched in immune function pathways and processes influenced by or involved in infectious diseases (Supplement 4, Barograph). Over 70 % of the genes involved in complement/coagulation pathways are found in this pathway. In addition, disease pathways affected by inflammation such as Alzheimer's, type 1 diabetes, and rheumatoid arthritis are also represented in this module. Proteomic analysis of blood proteins demonstrated the association between a combination of metabolites including micronutrients (met_PC1) and inflammatory processes (Fig. 2).
Module 52: Functional annotation
Module 52 is the largest in the network with over 2300 genes of which 422 had SNPs statistically associated with met_PC1. Genes and pathways involved in immune functions are enriched in module 52 (Supplement 4, BarGraph52) with cytokine signaling and other immune pathways overlapping with Module 2. The secretory and absorption pathways in Module 18 also have components in Module 52. About 75 % of the phosphatidylinositol and inositol phosphate pathways involved in proliferation, survival, migration, and differentiation in different cell types including the development and regulation of B-lymphocyte and T-lymphocyte functions (So and Fruman 2012) are found in Module 52. Functional analysis also highlights the known links between diabetes and immune function, since type 1 and type 2 diabetes genes and pathways and ~50 % of the insulin signaling pathway occur in Module 52.
Quantitative trait loci mapping and cofactor analyses
Lists of statistically significant genes alone, in modules, or mapped to QTLs identify potential candidate genes for a given phenotype. However, biological processes are necessarily controlled by gene–environment interactions. To associate the genes identified by data mining methods with nutrients, GeneCards and EBI's cofactor database were searched for each of the genes mapping to QTLs for plasma levels of leptin, adiponectin, glucose, and for type 2 diabetes mellitus (T2DM) loci. Many of the statistically significant met_PC1 genes that mapped to these loci had a metal cofactor, and only a few required organic cofactors (not shown). For example, CD320, the transcobalamin receptor, mapped to the GLUCO3_H QTL (glucose level) on chromosome 19. LRP2, which is involved in vitamin uptake, mapped to a chromosomal region (GLUCO15_H on chromosome 2) associated with hyperglycemia. Several met_PC1 genes (CHKA which is involved in choline metabolism; NOX4, TM7SF2, ALDH3B1, NDUFS8 are associated with NADPH) mapped to serum adiponectin level QTLs. Two genes (SHMT1, cofactors pyridoxal phosphate and folate; ALDH3A2–NADPH) mapped to serum leptin QTLs on chromosome 17. DNMT3B mapped to a serum cholesterol QTL and to a T2DM susceptibility locus on chromosome 20.
Met_PC1 correlated proteins mapping to quantitative trait loci (definitions)
Plasma cellular
Body weight
Serum levels
Health and disease processes result from a complex interaction between multiple genes and environmental factors. The systems nutrition analyses reported here used data from dietary intakes, plasma and erythrocyte metabolite levels, plasma proteins, and genetic makeup in a cohort of children/teens aged 6–14. Discussed below are (1) the main biological results, (2) strategy and methodological considerations, and finally, (3) implications for health and disease research.
Biological findings
Met_PC1 and SAM/SAH
Principal component analysis (PCA) identified a metabolite pattern, met_PC1, with positive and negative correlations between plasma micronutrients, plasma Hcy, and SAM/SAH ratio in erythrocytes. Plasma vitamin A and Hcy correlated positively with SAM/SAH, and vitamin E, thiamine, and pyridoxal correlated negatively in this population. While statistical associations do not prove causality, these correlations suggest that micronutrients and metabolites operate within a network that includes SAM/SAH metabolism. Altering the proportion of metabolites relative to each other may alter methylation potential and therefore epigenetic reactions. Others have shown that SAM/SAH correlated with differences in methylation at metastable epialleles based on season and food availability (Waterland et al. 2010). Changes in epigenetic programming at critical developmental windows such as in utero, early childhood, or during puberty have been associated with developmental plasticity, health, and susceptibility to chronic diseases in adults (Barker et al. 1993; Gluckman et al. 2009; Kussmann et al. 2010).
Met_PC1 and plasma proteins
Met_PC1 was also associated with levels of pro-inflammatory proteins. Individuals with high vitamin A, Hcy (but still below the clinical cutoff of 15 µmol/L), and SAM/SAH had lower levels of many of these inflammatory proteins. The correlation was modest for any single protein to met_PC1 value. However, certain proteins shared similar correlation coefficients and functional analysis based on gene ontologies, and some of these correlated proteins that participated in the same networks. Since met_PC1 is an empirically defined value specific to this study, the correlations among these plasma metabolites will necessarily require testing in other genetic makeups and environments.
Met_PC1 and global protein topological analysis
To discover whether the met_PC1 variable was associated with genetic variation in molecular interaction networks or subsystems, a metabolic/protein–protein interaction network was constructed based on two manually curated interaction databases (Ma et al. 2007; Yu et al. 2012). The network was partitioned into topological modules, each of which was assessed for significant enrichment with met_PC1-correlated genes using a hypergeometric test. Three modules were identified, 2 of which contained substantial numbers of immune and metabolic function genes and the third included genes in a range of secretory and gastrointestinal functions. Although the met_PC1 genes were not directly functionally annotated to every one of the identified processes/pathways in these modules, they may either be directly contained in these processes/pathways, or indirectly connected via a small number of degrees of separation. Variation in plasma micronutrient levels and Hcy, and erythrocyte SAM and SAH, and specifically the ratios of these metabolites relative to each other, was associated with genetic variations in immune and gastrointestinal functions. Chronic disturbance in gastrointestinal function (such as that seen in inflammatory bowel disease, Crohn's disease, and environmental enteropathy) may directly contribute to micronutrient deficiencies due to altered nutrient absorption (Valentini et al. 2008). Although the cohort in the present study did not present with diagnosed intestinal disorders, it may be the case that a range of SNPs contributes to subclinical variation in gastrointestinal function, which then relates to variation in micronutrient levels. Additional focused work on enterocytes and intestinal immune cells would be required to clarify the potential functional consequences of the SNPs identified in this study.
Metabolite principal component 1 (Met_PC1) was derived from the relationship of vitamins A, E, thiamine, pyridoxal, and the metabolites Hcy, SAM/SAH ratio. Correlations of met_PC1 to immunity were not unexpected since a rich literature exists for individual micronutrients and various aspects of immune function and regulation (Bhaskaram 2002; Maggini et al. 2007; Baeke et al. 2010; Ströhle et al. 2011; Ooi et al. 2012). The genetic analysis nevertheless revealed new insights into the many genes and their functions that may be associated with different plasma levels of metabolites and therefore with diet.
Strategy and methodological considerations
- 1.
The use of CBPR with biomedical and network biology analyses. Community-based research engages participants in the research study and provides opportunities for health- and nutrition-related exchanges between community members and researchers. Community-based research is done in "real" time with lifestyle and other environmental conditions that are not under the control of the researcher. These factors likely introduce noise into the study and analyses, but the measured biological "signals" include the contribution from those unmeasured influences. Our goal was to measure as many physiological and environmental variables as possible to associate signals with phenotype. In addition, community-based results from such studies are likely to be translated more rapidly to individuals and populations (McCabe-Sellers et al. 2008).
- 2.
Data from this study were previously analyzed at the group level (such as between SAM/SAH groups) and at the population level (Monteiro et al. 2014). We have also extensively analyzed dietary intake patterns, metabolomics, proteomic, and genomic data for individual participants in this study. For example, dietary intake variables were compared to metabolite patterns in each participant to determine whether common patterns could be identified at the individual level, and DNA ancestry was analyzed for each individual for the possibility of using genetic admixture mapping methods (Cheng et al. 2010) (data not shown). Methods which identify groups of individuals with related metabolic features but still allow for n-of-1 analysis may extend the recent personal omics analysis for molecular and medical phenotypes (Chen et al. 2012). Reporting data and results from studies with more than one individual, however, may require development of novel publication strategies.
- 3.
Levels of metabolites in each participant were analyzed and shown in one figure as opposed to reporting results of the average metabolite level in separate graphs. Although such methods are common in transcriptomic and metabolomics literature, we identified patterns of metabolite levels that revealed unanticipated nutrient–nutrient statistical interactions. Standard PCA converted the graphic representation of metabolite levels to a statistic specific called metabolite principal component 1 (met_PC1). Met_PC1 represented 6 strongly and one weakly associated (vitamin D) measured metabolites and their interactions. Although the value of this statistic is specific for the study reported here, similar methods may allow for more comprehensive analyses of interacting metabolites.
- 4.
Analyzing genetic differences based on met_PC1 in a metabolic/PPI network partitioned into topological modules allowed for the identification of physiological functions (immune, metabolic, and secretory) associated with gene–metabolite relationships identified in our statistical analysis. Rather than seeking a small number of SNPs with large effect on our phenotype, our network-based approach inherently highlights multivariate groups of functionally related SNPs/genes that are statistically associated with a phenotype. Defined interventions can be developed from our results and, equally importantly, tested by measuring parameters of immune and GI function that were identified in this study.
Implications for reproducibility
The results described in this manuscript and recent publications on intra-individual variability in physiological status found in environments that have large changes in nutrient availability (Dominguez-Salas et al. 2013) demonstrate the difficulty in replicating biomedical research, particularly for genetic and gene–environment interaction associations. Hierarchical clustering of proteins (Fig. 2) and SNPs (Fig. 5) correlated with met_PC1 helps visualize the proteomic and genotypic differences as combinations of SNPs or proteins rather than as single markers (even though these were derived from univariate analysis and corrected for multiple comparisons). No single protein or SNP is always correlated with met_PC1. This is what we would expect to observe, in part because of gene–gene and gene–environment interactions, epigenetic regulation, and other interactions. This perspective better fits the biological reality of multiple genes and their products contributing to a complex phenotype (in this case met_PC1). Our working hypothesis is that testing these associations in other populations and also in others experimental designs would be needed to identify common patterns of variants within these genotype data sets that might explain the percentage of genetic contribution to a given phenotype. Subsets of genes will contribute or not contribute to a complex phenotype (e.g., obesity and diabetes) based on interactions with diet or other environmental factors. In addition, the adaptations to diverse environments over human evolution may have selected different collections of genes for similar environments in different environments. The most notable, and still controversial example, is the different functional adaptations to Tibetan and Andean high altitudes (Beall 2007). Nevertheless, we predict that gene–environment interactions producing the same phenotype will have overlapping genes (much like Venn diagrams). Some pathways and therefore genes will be shared, and others may contribute less significantly in different genetic subpopulations. Discovering these similarities and differences may lead to an understanding of targeting diet and lifestyles to optimize health.
This research was funded by the Division of Personalized Nutrition and Medicine at the US FDA National Center for Toxicological Research (Jefferson, AR), the USDA Agricultural Research Service Delta Obesity Prevention Research Unit (Little Rock, AR), and the Nestlé Institute of Health Sciences (Lausanne, Switzerland). The research team gratefully acknowledges the contributions of the Phillips County community members in the Marvell (AR) School District for their participation in this research. This research was funded by the Division of Personalized Nutrition and Medicine at the US FDA National Center for Toxicological Research (Jefferson AR), the USDA Agricultural Research Service Delta Obesity Prevention Research Unit (Little Rock, AR), and the Nestlé Institute of Health Sciences (Lausanne, Switzerland). We thank Donna Mendrick, Li-Rong Yu, and Ritchie Feurs for critically reading this manuscript.
Conflict of interest
J.K. is employed by the Nestle Institute of Health Sciences, a for-profit company. No other authors declared a conflict of interest.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.
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i finally got to see the movie, "the pursuit of happyness" this weekend and it was as touching as i expected it to be, but it was a hard one to watch.
my blogs are never too personal, but it seems that the more i write, the more comfortable i become...so here goes.
the movie talks about this amazing father and his amazing strength to keep his head above water for his child. it's crazy but my whole life i've kept how i grew up this big secret. it's not that i'm ashamed, but i didn't want to put my mother thru it all again...i brought it up once as a kid and she started crying because she hadn't known i'd remember. nothing big. we were just really poor. we would wash our clothes in bathtubs, hang them to dry in the oven, walk everywhere...even with chicken pox...sometimes sleep on floors. my mom was young but she seriously made those times fun for me. she'd blast music and we'd dance and do our thing...years later my sisters would tease me for hanging on to toys or taking care of things in an almost anal retentive kind of way...but it simply because i learned to appreciate the gifts that i knew not everyone could get. that movie hit really close to home. we walked out and for the first time in front of another person i cried about it all. my guy was really cool about the whole thing and sat and talked to me for a while. i'm so scared of going back to that place. i don't know if i could be as strong as my mom once was. i don't know if i have that kind of hustle...people that have never experienced that kind of life can watch will smith's movie and then just move on..."good flick, i'm glad he got rich, lets go home and play with our ipods and be merry" but there's so much more to the story. we get so hung up on our lives we forget that there are people with real struggles just trying to keep their heads above water. you'll see will smith deal with that in the movie...
i can't wait to go home this weekend and hug the crap out of my mom. when you watch that movie you'll get why...she's a lot like will smith was. a girl i work with was explaining how everyone thinks her mother is beautiful because she never leaves the house without makeup or her hair done...my mom may not be the makeup wearing girlie girl type (now you know where i get it from) but she's still the most beautiful woman i've ever met. it's hard to keep focus being out here among the cali folks, but at least it came back. people probably question why i can be so harsh towards people i feel are priveleged and bratty about it. i try not to be, but it's just so disgusting to watch someone be ungrateful.
so there ya go...just in time my mind became clear again-- just in time for the holidays.
p.s. i really am lucky to have a boyfriend like the one i do. as he sits beside me watching lord of the rings for the upteenth time...i don't really care. it's good to have a best friend that has your back...even thru the really hard stuff that you normally can't share. merry christmas some more people..i can't say it enough!
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subset: commoncrawl


text:
To download Miami Nights: Singles In The City free java game, we recommend you to select your phone model, and then our system will choose the most suitable game files. Downloading is very simple: select the desired file and click "Java Miami Nights: Singles In The City - free download", then select one of the ways you want to get the file. Just a few easy steps and will enjoy playing new game at your phone!
Game description: Miami Nights: Singles In The City:
This newest " life simulator" will help you to carry out the most secret dreams in one of the most beautiful cities of the world - Miami. Create the character, using set of possible options: sex, hairdresses and a hair colour, face types and the figures, the most fashionable clothes of every possible colouring etc. You can enter into game the exact copy or the opposite. But appearance is not the most important thing! Your character possesses more than 40 parametres which will change depending on your behaviour and dialogue in game: culture, sexuality, charisma, kindness and even criminality! You can raise one parametres and ignore others, creating unique character and the person of the character! The city of Miami will appear before you in all beauty in more than 30 various locations: streets, beaches, hotels, restaurants, bars, clubs, private residences, gardens, a museum, a bookshop, rooms for rest and even prison! You can arrange the house as you wish, and to buy new posh real estate! But the most important part of game is dialogue and interaction with other characters. Some of them will help you to achieve heights, to give work and to get acquainted with the necessary people. You can choose how to behave with that or other character: to discuss various themes, to speak seriously or to joke, give every possible gifts, to flirt or fight! Be bad guy or good girl, make friends or get enemies - do everything you want to subdue Miami and become successful! Features of game: - Long-awaited continuation of hit game "Nights of New York: the Way to success" - The new improved drawing and animation - A great lot of new possibilities and options - More than 30 locations of a city of Miami - More than 40 attributes of the character - Choose own style of life: glamour or trash!


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Thank heavens for relationship calculators on family tree software: he's the husband of my ex-husband's half-7th great-grand aunt, (8th, to our children) through the RICE family line, up through to Susanna WHITE Winslow's daughter, Elizabeth! I decided to write a little bit about him after reading through a book I stumbled on in our B.C. Genealogical Society's library while volunteering there.
The book I found is: Ancestral Roots of Certain American Colonists Who Came to America before 1700, by Frederick Lewis Weis, 7th Edition by Walter Lee Sheppard, Jr., published by Genealogical Publishing Co. Inc., 1992. This book is a compilation of ascendancy of early colonists back to Saxon and English Monarchs, French, Charlemagne, Early Kings of Scotland and Ireland, and much more. As I have quite a few early colonists, I checked the names with my database, and found George CURWEN. (I also found Thomas TROWBRIDGE, but that's another story!) Many specific sources are listed, one of them refers to a chart made "by Rev. George Curwen 1698, made during his father's lifetime". This book is not a primary source, as it is a compilation from various other sources, none of which I have seen or examined. Still, it is an interesting start.
George was born 3 Nov 1610 in Sibbertoft, Northampton, England. We know his father's name was John CURWEN; his mother's name is stated to be Marjorie, but I have not seen proof. The other children were: Thomas, Matthew, Elizabeth, Sarah, and John. His older brother, Matthias Curwen, b. abt 1602, immigrated with his three children abt 1634, settling first in Ipswich, MA.
George's first wife was Elizabeth Herbert, daughter of the Hon. John Herbert, Mayor of Northampton, England. They married in England about 1635, having at least one or two children in England. She died on 15 Sep 1668, and he married on 22 September 1669 to Elizabeth WINSLOW Brook, the only daughter of (Governor) Edward WINSLOW and Susanna White (widow), Mayflower immigrants. Elizabeth (2nd) was a widow of Robert Brook who had died earlier; she had one son, also named Robert Brook.
George & Elizabeth (1st) had seven known children:
1. Abigail, b. 1 Aug, 1637, died by 1 yr of age
2. John, b. 25 July 1638; d. 12 Jul 1683; m., 5 children
3. Jonathan, b. 14 Nov 1640; d. 25 Jul 1718; m., 10 children
4. Hannah, b. 1642, died by 1 yr of age
5. Abigail, b.? bap. 30 Nov 1643
6. Hannah, b. 1 Jan 1645/6; d.21 Nov 1692
7. Elizabeth, b.? bap. 2 Jul 1648; d. bef 1685
After Elizabeth died on 16 September 1668, in Salem, George married for a second time in 1669 to Elizabeth WINSLOW Brook (widow). Corwin Genealogy in the United States, [by E.T. Corwin, pub. 1872 by S.W. Green, New York], lists the following children on page 78:
George & Elizabeth (2nd) had 3 children, all of whom married, with children:
9. Penelope,
10. Susannah
11. George
George was a wealthy and influential man in Salem, living in the city, owning four houses, warehouses, and 2 wharves in Salem, plus another warehouse and wharf in Boston. He also owned about 15,000 acres of various land parcels around the area. His family lived in one side of his very large home, and the other side had several businesses/trade establishments leased. He was made a Freeman in 1665, and was involved as a Selectman for most of the years from 1666-1676.
When he died 5 Jan 1684, his will showed an inventory valued at £5,964. 10s. 7d., which was a large fortune at the time. Some details of his inventory (e.g., silver-topped cane) are still kept by descendants. His portrait was commissioned and is in the Peabody Essex Museum in Salem, MA; a copy is hanging in the "Witch House", house, again owned by a Corwin descendant, in Salem. He is described as having a "fine, round forehead, large nostrils, high cheek bones, and grey eyes," in the Corwin Genealogy in the US book mentioned previously.
Much has been written in various articles, letters, journals and books of history and genealogy, concerning George CORWIN/CURWEN, and searching on several genealogy websites will turn up a number of books and histories which may be directly searched. Interestingly, the original surname in the 1200s, was spelled CULWEN and the spelling shifted in about 1433 - see next paragraph.
That book that started me on this post lists the direct lineage (with more details as known in the book) as follows on pages starting at 40-41, 37, 1-4, following generation numbers:
Captain George CURWEN - John CURWEN - Henry CURWEN - Thomas CURWEN - Thomas CURWEN - Margaret CURWEN (m. cousin Wm CURWEN) - Sir Christopher CURWEN - Sir Thomas CURWEN - Sir Christopher CURWEN - William CURWEN - Gilbert CULWEN - Sir Gilbert CULWEN, Knt. - Gilbert CURWEN (m. cousin Edith HARINGTON) - Gilbert CULWEN - Patric DE CULWEN - Thomas OF WORKINGTON, son of Gospatric, Lord of Workington in Cumberland.
The last named ancestor, Thomas of Workington, is known to have died soon after 13 Nov 1200. Both the Harington and Culwen/Curwen lines can be traced back to Aethelred II (King, 979-1016). Aethelred II traces back to CERDIC, King of the West Saxons in the early 500s. Yes, once one finds a landed gentry ancestor, there are interesting lineages to find, piecing them from various writings of battles, of famous people's visits, and from various collections of documents. Interesting puzzles.
Of course, all of us trace back to a fairly small group of people. I find it fascinating to see how "mixed up" we all are: Vikings and other Scandinavians, Germans, French, English, Dutch, and others. One day I'll be able to pay for genetic testing and find out even more about my deep ancestry details.
If you have questions or comments, do contact me via calewis at telus dot net,or add them to the Comments section below. Thank you for stopping by and reading.


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One of the most quiet places in your home is your garden. For this reason, the furniture is very popular these days is. With the purchase and establishment of a set of garden furniture, you can enjoy the relaxing atmosphere of the garden to sit and enjoy. Moreover, a beautiful set of garden furniture to enhance the beauty of your garden.
If you are thinking of buying furniture for yourself, you have to put in mind that you should always choose the one that suits your style and your garden. Today there are many types of outdoor furniture that can choose the one that best suits
The different types of garden furniture
If a number of tables and chairs is selected, the first thing to think about is the size of the area which is to be placed. The table and chairs should not be too small or too big for them. Choosing the right size table and chairs are your garden a more appropriate look.
With regard to the color of the furniture, you should choose the perfect flower in your garden to your taste and, of course, that the color of the flowers is convenient. There are no specific rules in a variety of colors for your outdoor furniture because of it. A matter of personal choice
Furniture spilling one of the most common types of furniture used in the garden. If it comes to buying one, you should make sure you choose the attractions cushions in keeping with the style of your garden.
If you want to sit in your garden and relax, it is always on hand if you have an umbrella with you, especially in the summer. This provides shade and protection from the sun's heat. From the choice of tables and chairs, you also need to select the type of umbrella and designs that harmonize well with the overall look of the garden.
Once you have bought the furniture for your garden, you may still be to improve through accessories and decorations adds to it. Even ordinary furniture look great if they come with decorations and stylish accessories.
Some of the accessories that can be used are outdoor lamps, birdhouses, birdbaths, and ornamental statues. These decorations naturally mixed with adjusting your outdoor garden. If you want to improve, you can select a mini ponds or wells have been diverted to give a classic look.
The health benefits of a seat on the slope in front of an ergonomic computer chair
One of the pioneers in offering the benefits of a seat that tilts forward was Danish surgeon Dr. A. C. Mandal. He said that the only way I could sit without the participation was back, bow on the front legs of the chair forward. He also noted that many school-age children, what they did and tried to ease the pain again by poorly designed school furniture.
Mandal recommended that the seat should be tilted 15 ° forward. If we need to take this position our lumbar spine not to soften the sitting posture and hips and torso to take care of the muscles in the optimal equilibrium position they are.
By tilting the seat forward change of focus behind the sitting bones (ischial tuberosity) directly above. As a result, the muscles are not tense or contract to maintain an upright posture. At the same time the lumbar curve (or lordosis) is restored, and the pressure is reduced in the discs.
A variation of the forward tilt of the seat back was the shape of a saddle. This has the effect that the opening leg and give greater stability to the pelvis. It also provides some lumbar lordosis. There are versions of this type of seat on the market that are popular among people. A disadvantage of this type of seat is, however, that his office should be set much higher legs to maintain order "position of the saddle."
Some of the most common early versions of the sloping seat objections were: –
He leaned forward to slide.
Excess weight was placed on the feet.
Clothes rather "Ruck up" ..
Balans Chair:
The Balans was one of the first presidents of the front slope to incorporate suggestions from Mandal in mass production – with preventing forward sliding knee rest.
Most of the weight on his knees and everything is taken that reduces pressure on the discs, muscles have to work harder to maintain an upright posture, because there is no registration. If the muscles are tired, there is a natural tendency to collapse.
In addition to these problems, knees and shins can become very sick because there is no provision to change the position of the knee after a while. To enter and exit the chair is a bit difficult due to disorder of the central column.
Despite the various drawbacks mentioned, however, several thousand of these chairs were sold units to relieve the pressure of the inclined seat forward of the spine, where due to the obvious relief.
Balance Kneelsit chair
If you allow the seat rotates forward forward of the pelvis to tilt, this attitude creates less stress on the muscles, tendons and discs, as the spine is aligned correctly.
Also pulls the chest, increasing lung capacity and makes it easier to breathe better functioning of the heart's circulation and blood; It also allows an open abdomen, which aids digestion.
If your pelvis is tilted his lumbar spine curves naturally to the front and the rest of the spine follows. This is the ideal posture at work are – just like on a computer. If you have a folder – as in the Kneelsit chair – this backward tilt the spine and pressure on the discs and muscles can be used to relax, for example, read or just chat.
The real secret, however, care for spine healthy it is to have a chair that allows movement back free, natural flow between the front and swings. This provides a "pumping action" so that feeds and nourishes the cells of the intervertebral discs, exercises the muscles of the back and allowed to sit for hours with very little discomfort.


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Publication - Statistics publication
Scottish cancer patient experience survey 2015-2016: exploring differences in patient experiences
- Directorate:
- Population Health Directorate
- Part of:
- Health and social care, Research
- 9781788512510
Additional survey analysis which examines the relationship between the self-reported experiences of service users and a range of factors.
Pre-Existing Conditions
Respondents were asked to select all the pre-existing health conditions they had from a list provided. Those who selected only one condition have been assigned to that sub-category and those who selected more than one condition have been categorised as having multiple conditions. Individuals who selected that they had none of the conditions listed were used as the reference group.
This factor affected the likelihood of responding positively to 20 out of the 47 questions analysed. Only one of these questions was answered more positively by people with a pre-existing condition – whether anyone had discussed taking part in cancer research with them since their diagnosis; those with a physical disability were more positive compared to those with no pre-existing conditions.
All of the other questions were more negative for people with at least one pre-existing condition. These questions are spread across the survey with no clear patterns related to particular areas of care.
Looking at the distribution of number of questions affected by pre-existing health conditions, as shown in Figure 3, those with multiple conditions are more negative for the most questions, followed by those with chronic pain lasting for more than 3 months. Those with mental health conditions do not show any significant difference compared to those with no pre-existing conditions for any questions.
Figure 3: No. of questions affected by pre-existing health conditions when compared to those with no pre-existing conditions
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Tips for going into the property market with a budget
If you want to get involved in the property market and your finances are limited, you may wonder if you can realistically compete, but in fact most property investors started small, and you'll be surprised by the deals you can get if you have a decent deposit. The trick is knowing where to look and being ready to put in some hard work to turn the place you buy into something you can rent out for a good amount or sell at a comfortable profit.
Take your time when you're buying to let, you have a big advantage over most other buyers – you're not operating on a tight schedule, needing to find somewhere to move to before your current home belongs to someone else. Don't rush into buying the first thing you see – take as long as you can get away with and wait until you spot a good deal. Great places at low prices do come up, and if you're ready to pounce when they do, you can get a lot more for your money.
Consider different mortgage deals – There are many different types of mortgage deal out there, including ones specially designed to help first time buy to let buyers. Make sure the deal you get doesn't restrict your ability to sell at the point where you may wish to do so. If you want to sell, you should be aiming to make back your original deposit plus the value of any mortgage payments made, plus the cost of any work you do, plus 20%. If you rent the property, you should have a margin of 20% on top of your mortgage payments.
Find a good estate agent – A good estate agent can help you a great deal by assisting you in finding appropriate properties and giving you advice about getting the right mortgage, home insurance deal, etc. Belvoir Lincoln, for instance, can help you all the way through from searching for a property to getting it ready for the rental market. They can also help you to manage your tenants and arrange safety inspections to ensure that the property stays in good condition.
Look for a fixer-upper – Most landlords find properties in one of two ways: by looking for sellers who need to dispose of them quickly or by picking up places that have already been foreclosed on. Many properties like this are in poor condition, so it's important to do your research and have an understanding of what it costs to fix different types of damage – everything from a simple paint job to fitting a new heating system. This will enable you to determine how good a deal really is. Once you've developed skills like this, you'll be able to put them to use in other ways as well.
The next time your family is ready to move, you'll be able to find a better deal at a lower price, and your business experience will help you to get a good financing deal. You'll also have the option of buying further investment properties in the long term and watching your property business grow.


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What do you think of when you hear about AI? Do you picture your favourite sci-fi movie or a book that you read when you were younger? In that favourite book or movie, were the robots smart? Could they learn?
Today's software robots can. In AI, we can find a subset of machine learning called "deep learning," which is defined as networks that can learn unsupervised from unstructured data.
Now the bigger question is: Are you ready to take advantage of deep learning in your business? The vast ocean of data grows exponentially every day. If you and your company can't keep up, you'll be left behind.
It's time to utilize intelligent automation to help your business grow, keep organized, and stay on top of the competition. Let̵7;s discuss three advantages of deep learning for your company.
- Cost and time benefits.
- Quality and accurate results.
- Job growth.
Utilizing deep learning for your business will save you money and time in more ways than one. You, your company, and your employees will benefit when you decide to take advantage of what AI has to offer. Will you make a move toward deep learning in your company?
Maybe they aren't like the ones in books or movies, but robots are here, in one form or another. What is the future of AI? In some ways, that is up to you.
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SAN FRANCISCO (CBS.MW) -- Swiss pharmaceutical giant Novartis NVS, +1.21% said Thursday in its quarterly report that it has accepted an overture from Franco-German drugmaker Aventis AVE, -24.00% to start merger talks that could create the world's second-largest drug company. Novartis said first-quarter profit rose 22 percent to $1.29 billion. Analysts polled by Reuters had expected on average a net profit of $1.3 billion.
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If you fall and fail to report the accident and no witnesses are around to verify the fall, you will have a very difficult time succeeding on a slip and fall claim. Failure to document your fall and/or report it, can be fatal to your claim for compensation.
If you fall, and you do not need immediate medical attention, or have not called 911, look around to anyone who saw the fall. You can never have too many witnesses. Scan the area and look for what caused you to fall. Try to understand exactly what happened and it is essential that you report the incident immediately to owner of the location. Request the police to file a report and/or file an incident report to document your fall. Be specific and concise in the report. Be sure to indicate that you will be seeking medical attention. So, be safe and be alert. We all have a personal and legal duty to be alert to the world around us. If you are hurt, seek legal assistance so at the very least, the costs of your medical care are covered.


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I had a very crazy year in 2015. My life, was completely flipped upside down, turned wrong side out, ran over, drowned, and then laid out flat to dry. It's okay though! It ended with very positive and promising things ahead. Me saying that it's okay, is also part of why my year ended the way it did.
There are a million things that went on in my life this year, and I won't get into details, because really the most important thing that went on, was the one thing that absolutely affected everything else in my life; my depression.
Depression is such a taboo topic in our society. I never use to talk about it, what will people think of me if they knew I'm on medication? If they knew there are days when I can't get out of bed? What if they knew that while on the outside I seem fine, "perfect" even, really on the inside I'm destroying myself? I feel like we are all trained from a very young age to never show weakness. We're trained to think of lobotomies, insane asylums, and people shuffling around in pajamas with blank looks on their faces whenever the words mental illness are uttered.
It's not even just society, it's our families, our friends, our peers. As an adolescent, a teenager, and a young adult I fought daily battles with myself, with my self esteem. I was a chubby kid, I had acne and frizzy hair, my family wasn't poor but we weren't rich either, so I never had the "cool" clothes. I was socially awkward, to the extreme. I grew up in a Mormon household; not only a Mormon household, but a shunned-by-our-church-community Mormon household. I was home school between the ages of 8 and 14; for the record, that's a very bad time to not be in school, learning the basics of human social interaction.
By the time it became just too much to handle, and I finally went on anti depressants while in my early 20's, I was so incredibly angry to learn that several of my family members had been on them for years. I felt so betrayed learning that, because I was always pretty much told to suck it up, that people had it worse, to stop complaining; I believe at one point I was even told to pray. This was the culture I was raised in; I was raised to believe that depression wasn't normal, it wasn't okay, it was something to hide.
I'd like to say that my battle ended there, and that I lived happily ever after. The placebo effect isn't something that only happens when someone isn't given actual medication. Sometimes, things have gotten so bad, that you don't even know what "normal" is anymore; maybe I never really did know "normal". So when I started taking the meds, yes, things "got better". What I didn't know though, was they could be even better. I made two giant mistakes: first, I thought that taking anti depressants would fix everything, all on its own; second, I thought after a couple of years that I was "cured", and no longer needed to take the meds.
I wasn't better. I thought I was, but I wasn't. I had just learned to hide it better, and instead of being sad all of the time, I had this overwhelming need to be perfect, to control everything in my life. Subconsciously, mind you; I really had no idea that this what was going on in my life. I was always stressed. If things didn't go how I had planned, if something unexpected happened, if my routine was disrupted in any way, I had a meltdown. It took a lot of shitty things happening, it took me hurting people I loved, it took people I loved hurting me, it took being alone for the first time in almost a decade, for me to see all of this for what it really was.
The first time I saw a therapist I was 28 years old (I'm now just 29). I didn't go for me, I went for my marriage, but it turned into being just me. I don't know how to describe therapy, I don't know how to tell someone why it works; I just know that it worked for me. I went back on my meds; in fact my doctor doubled my dosage. I didn't even know that was an option, I didn't even know that I truly did need a higher dose in order to get the full benefits of the medication. I think the most important thing I get out of therapy, is I learned how to love myself. I learned how to better my life, how to see things from a different perspective, how to handle stress, how to not blame anyone (including myself) for anything. I learned how to manage my depression. This was the key component that I was missing the first time I started taking anti depressants.
With the new year, I thought a lot about everything I have learned this year. It was so nice, actually, to look back and realize that even if in theory I had a shit year, I learned so much from it. I came across this post on Buzzfeed, and so much of it made so much sense to me. Before, I probably would've rolled my eyes, and made some sarcastic remark about someone not getting hugged enough as a child. Oh but no, it's true.
23 Things To Do To Improve Your Mental Health in 2016
Some of the ones that stuck out to me the most, the ones that I have decided will be my theme for 2016. Yes, my theme, I no longer will have New Years Resolutions, or set goals. Not to say a person shouldn't have goals; it's just that the word goal implies that if you don't meet it, that you've failed in some way.
- Learn to say no, without explaining yourself
This is so important! No, I can't do this thing for you. No, I can't make it to that event. No, I don't need to deal with this for any reason. I don't need to explain why I can't help, or why I can't go. It's not selfish, it's self care.
- Pick up a hobby that's only purpose is to make you happy
As I got older I got so wrapped up in "adulting". In work, in keeping my house clean, in managing my budget, in keeping my family happy. What about keeping me happy? What about doing something just because I like doing it? It's important.
- Budget for little indulgences that make you feel good every month
Buy yourself that book you've been wanting, go get a mani/pedi, go splurge on things for that new hobby you've picked up. You work hard, so you need to reward yourself.
- Cut should from your vocabulary
I should've worked out, I should've eaten better, I should've done that project-NOPE. I would like to work out, I would like to eat better, I would like to do this project. Cut the negative talk, don't beat yourself up for not doing something.
- Complain less
Oh this is a big one for me. Prime example of me putting this into action: I took Monday off work, so that I could take my car in to get repairs that it pretty much desperately needs at this point. However, Portland got a surprise snow and freezing rain storm on Sunday! Which meant that come Monday, my car was iced in, and the roads were a mess. I couldn't take my car in as planned. I could've been upset, I mean I was upset, but I could've let it ruin my day. I could've complained about it, and been grumpy. Instead I said it is what it is, I have a couple more PTO days coming up, I'll just take care of it then.
- Cut back on social media
I deactivated my Facebook on January 2nd. I did this for many reasons. It's been less than a week, and there are moments when I wonder what is going on in the world of Facebook, and others when I really don't care, because Facebook isn't the real world.
When I thought about how I wanted 2016 to be, I thought about what I really wanted and needed to be happy. I would like to workout more, I would like to drink less, I would like to have more adventures, I would like to spend more quality time with my husband (read: put down the iPhone dammnit!), I would like to find things that make me happy, I would like to make self care a priority.
I hope that if you are battling depression, that you realize that it is okay to have depression. I hope you realize that there are ways, that may sound cheesy, but there are ways to better your life. Cheers, to not a better year, but to a great year.


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2018 Nissan Titan
N/A • New
Asking Price
Ext. Color
Stock Number
8 Cylinder
Int. Color
Installed Options
Audio - SiriusXM Satellite Radio, Audio - Internet Radio: Pandora, NissanConnect - Satellite Communications, Phone Voice Activated, Electronic Messaging Assistance With Read Function, Phone Wireless Data Link Bluetooth, Electronic Messaging Assistance With Voice Recognition, Driver Information System, Multi-Function Display, Security Anti-Theft Alarm System, Crumple Zones Front, Crumple Zones Rear, Steering Wheel Mounted Controls Voice Recognition Controls, Stability Control, Power Windows: With Safety Reverse, Windows Front Wipers: Variable Intermittent, Pickup Truck Sliding Rear Window Manual Horizontal, Windows Rear Defogger, Power Windows: Front And Rear, Audio Streaming: Bluetooth, Audio - Antenna: Mast, Audio - Antenna: Diversity, Audio Voice Recognition, Audio MP3 Player: CD MP3 Playback, Audio Auxiliary Input: USB, Audio Auxiliary Input: Jack, Audio In-Dash CD: Single Disc, Audio Auxiliary Input: IPod/IPhone Integration, Audio - Radio Data System, Audio - Radio: AM/FM, Audio System 6 Speakers, Audio - Speed Sensitive Volume Control, Audio - Internet Radio: iHeartRadio, External Temperature Display, Fuel Economy Display Range, Trip Odometer 2, Fuel Economy Display MPG, Warnings And Reminders Lamp Failure Low Fuel Engine Oil Coolant, Trip Computer, Digital Odometer, Electroluminescent Instrumentation, Tachometer, Warnings And Reminders Maintenance Reminder, Rear Bumper Color Chrome, Mirror Color Black, Cargo Bed Light: LED, Exterior Entry Lights Puddle Lamps, Headlights Halogen, Tail And Brake Lights Rear Center Brakelight, Exterior Mirrors Power, Exterior Mirrors Integrated Turn Signals, Exterior Mirrors Manual Folding, Exterior Mirrors Heated, Air Conditioning - Front - Single Zone, Air Conditioning - Front, Air Conditioning - Air Filtration, Airbags - Front - Dual, Airbags - Passenger - Occupant Sensing Deactivation, Airbags - Front - Side, Airbags - Front - Side Curtain, Airbags - Side-curtain Rollover Sensor, Airbags - Rear - Side Curtain, Power Brakes, Braking Assist, ABS Brakes (4-Wheel), Electronic Brakeforce Distribution, Security Engine Immobilizer, Power Door Locks Anti-Lockout Feature, Security Perimeter Alarm, Child Seat Anchors LATCH System, Child Safety Locks, Energy Absorbing Steering Column, Body Side Reinforcements Side Impact Door Beams, Reading Lights Rear, Assist Handle Front, Reading Lights Front, Cruise Control, Power Steering Speed-Proportional, Multi-function Remote Keyless Entry With Panic Alarm, Steering Wheel Mounted Controls Cruise Controls, Multi-function Remote Keyless Entry, Power Outlet(s) 12V, Engine Push-Button Start, Overhead Console, Inside Rearview Mirror Manual Day/Night, Vanity Mirrors Dual Illuminating, One-Touch Windows: 2, Footwell Lights, Windows Illuminated Power Window Switches, Windows Lockout Button, Rear Seats Center Armrest: With Cupholders, Driver Seat Manual Adjustments: 4, Driver Seat Manual Adjustments: Recline, Passenger Seat Manual Adjustments: 4, Rear Seats Split Folding, Seats Front Seat Type: 40-20-40 Split Bench, Passenger Seat Manual Adjustments: Recline, Seats Cloth Upholstery, Rear Seats 60-40 Split Bench, Fixed Rear Headrests, Rear Seats Folds Up, Front Headrests Adjustable, Seatbelts Seatbelt Force Limiters: Front, Seatbelts Seatbelt Warning Sensor: Driver And Passenger, Front Seatbelts: 3-Point, Seatbelts Second Row 3-Point, Seatbelts Front Center Seatbelt: Lap Belt, Seatbelts Emergency Locking Retractors: Front, Seatbelts Emergency Locking Retractors: Front And Rear, Seatbelts Seatbelt Pretensioners: Front, Braking assist hill start assist, Traction Control With Trailer Stability Assist, Traction Control, Towing And Hauling Cargo Tie Downs, Front Suspension Type: Double Wishbones, Front Suspension Classification: Independent, Front Suspension Type: Strut, Suspension Rear Leaf Springs, Suspension Rear Twin-Tube Gas Shock Absorbers, Suspension Front Spring Type: Coil Springs, Suspension Front Shock Type: Twin-Tube Gas Shock Absorbers
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Indestructible Dog Toys: Our Top 5 Picks
Do you ever find yourself spending large amounts of money on dog toys that seem to only last, what appears to be, a few minutes against the mighty jaws of your pet? As good dog owners, we all want to provide the best chewing entertainment for our pups―we don't want them to be bored around the house after all.
But having said that, it's no good if they're able to destroy and chew apart said toys in less than ten minutes!
Indestructible Dog Toys―Do They Exist?
Sure, dog toys can be patched up once or twice after falling victim to an attack but wouldn't it be better if the toys were sturdier and more durable in the first place? Are there such things as indestructible dog toys?
Considering the fact that dogs are able to chew through almost anything given the right motivation, the answer to that is probably no; however that doesn't mean that some of the dog toys aren't designed to be much higher quality compared to others.
As one who likes to spoil their pet rotten, we've bought dozens of toys over the years for our dog―from soft plush chews to light-up bouncy balls. To make your dog toy shopping a little easier, we've compiled a list of some of the best ones which we've encountered so far, here on our page!
Indestructible Dog Toys – Our Personal Favourites
#1: The Kong Knot Bear
The Kong Knot Bear was amongst one of the very first toys that we ever got for our pup. While we initially bought it for its cute looks, it has surprised us on many levels with its durability!
Inside its soft, furry exterior, the bear has a robust "rope skeleton" that enables it to hold up against the teeth of your pet. Did we also mention that it squeaks?
Besides its internal knotted structure, the toy also contains minimal stuffing so there are little to no worries about your pet being able to tear out, and ingest fluff. Our rambunctious guy ended up loving his bear so much that we ended up buying him a second in a different color! After three years, the original is still holding up, albeit the fact that it no longer has ears.
Kong Knotted Bear Toy
|The Kong Knotted Bear Toy features an internal knotted rope skeleton that makes it resilient, even against your pet's jaws. Soft yet durable, it's perfect for dogs of all sizes!
#2: Outward Hound Hedgehogz
We like to consider the Outward Hound Hedgehogz toy to be a staple in our home―we always have more than one at any given time! Don't let its gentle outer appearance fool you, this hedgehog is super well designed to last against extreme play.
Featuring limited seams, the toy is much more solidly built compared to many of the other plush dog toys out there. Not only does it squeak, but it also rattles and grunts when tossed and shaken!
Available in a variety of different sizes, this hedgehog is perfectly suitable for dogs of all sizes! In fact, there's even a "female version" of the toy, which comes complete with pink eyes shadow and toes!
Outward Hound Hedgehogz Toy
|This huggable Hedgehog Toy comes equipped with irresistible squeakers and noise-makers that are sure to keep your pets entertained!
#3: Jolly Pets Bounce-N-Play Ball Toy
If you're looking for a durable, ball toy for your pet―this is it. The Jolly Pets Bounce-N-Play Ball Toy is unique in that its made from a soft, rubbery-plastic material yet it's extremely resistant against wear.
Designed for sturdiness as well as with the safety of your pet in mind, the toy bounces and floats and doesn't require air to inflate. Even if your dog sinks his teeth into the ball and punctures a hole, it'll still be as good as new―it'll just gain a hardly noticeable battle scar in the form of a teeny mark.
We originally got ours at a clearance sale a couple years ago, and even after endless chomping sessions, it's still going strong. The manufacture, Jolly Pets is so confident in the quality of the product, they even offer a guarantee for their items!
Jolly Pets Bounce-n-Play Ball Toy
|This fruity scented ball toy is built for long-lasting fun! The Bounce-n-Play Ball Toy won't deflate even if your pet sinks his teeth into it!
#4: ZippyPaws Donutz Squeaky Plush Toy
Known for their cute and unique dog toys, ZippyPaws has created a resilient toy in the form of a sprinkled donut.
The first thing that you'd notice about the toy is its rugged surface―it's so sturdy that it actually requires more force than usual to get its two crescent-shaped squeakers to squeak!
Available in a bundle of different "flavours," the ZippyPaws Donutz Toy has no stuffing, making it a an appropriate and safe choice for your pet. Due to its donut-y design, it's also great for throwing and even rolling―it has definitely lasted longer than many of the other similar toys which we've purchased for our dog in the past.
ZippyPaws Donutz Squeaky Toy
|Not only does this no-stuffing donut toy come complete with sprinkles, but it also squeaks and rattles for maximum doggy fun!
#5: Goughnuts
Designed with aggressive chewers in mind, the Goughnut is a virtually indestructible chew toy that is in the shape of, you guessed it―a donut!
Made in the USA with the help of polymer and mechanical engineers, manufactures are so confident in its quality that it even comes complete with a product guarantee.
If your dog manages to chew his way through the outer layer into the red filling (which acts as a safety indicator), Goughnuts will be happy to replace the toy for you and your pet, free of charge!
Depending on how merciful your dog is with his chew toys, you can either get the ones designed for moderate chewers (e.g. orange, green, orange) or the black one if your pet is an extreme chewer.
Goughnut Chew Toy
|Designed with durability and safety in mind, the Goughnut is virtually indestructible with its specially engineered material!
What are some of your pup's favourite toys? Share with us in the comments below!


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Last Saturday, we teamed up with the Iceball 3-on-3 ("Are the Skills Still Ill in the Wind Chill?") crew, to host the fourth edition of this unique winter-tournament. The action went down at West 4th Street (W. 4th & 6th Ave) also known as "The Cage". "This is the only outdoor basketball tournament, which is held in the coldest month of the year, yet each year, we are able to get some of the toughest streetballers to come and compete" (founder Eric Henderson). "We are always looking to underline our "Play Hard" philosophy – and this bare-knuckle basketball bout illustrates what it's all about" (Benji Kesselbach, K1X NYC). Players included streetball legends like Tim "Headache" Gittens (who's team once again took home the victory) and Jay "Boogie" Brantley as well as Bronx-native Andre Martin, Jerrome Jones (Mt. Vernon), Jamaica-Queens resident Phillipe Gregoire, and Roudy "Movez Galore" St. Fleur (Brooklyn). The weather forecast for Saturday's games was windy and cloudy weather with a high of 45° F and low of 28° F. Only in New York!
Video: King Zulu
Organisation: Eric J. Henderson & Benji Kesselbach


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Two Exhibits in One - Horizons and Fibers of the Soul
Venue: Hallberg Center for the Arts | City: Wyoming, MN
Two Show Openings in One Night!
Opening Ceremonies take place at the Hallberg Center for the Arts from 4pm to 8pm. Free and open to the public come and meet and witness the artistic genius of three amazing artists. Complimentary refreshments will be served. The Hallberg Center for the Arts is located at 5521 East Viking Blvd, in Wyoming, MN, just minutes north of the Twin Cities on I35.
A Duo Art Exhibit by
MaryAnn Cleary and Andy Tinkham in the Main Gallery
Living in the Twin Cities metro, I don't always get the chance to see the full wonders of the night sky. While I cherish my opportunities to go up north to see things like the Milky Way and the Northern Lights (both favorite subjects of mine), I find that the bright lights of the city, rather than diminishing the experience, change it. They bring new challenges and convey new emotions.
- Andy Tinkham
To me, art is a way of expressing something in two dimensions that normally would be taken for granted by others in life. I love capturing the emotional essence of that moment in time. As I pursue my passion, I hope to capture those precious moments with the magic of a paintbrush or a stick of charcoal by utilizing light, contrast, value and color onto the paper or canvas. My ultimate goal is to take something ordinary and make it something magical and emotional to the viewer.
- MaryAnn Cleary
Fibers of the Soul
A Solo Feature in the Cube Gallery by Heather Tinkham
At its best, art is a way to bring more beauty, joy, honesty, and authenticity into a world that is increasingly busy and chaotic. My work extends the idea of art destined for gallery walls in to our everyday lives, as Artwear for both our bodies and our homes, as I see a life integrated with art as richer and more grounded overall.
- Heather Tinkham
Sponsored by
All Dates
- From Thursday, March 22, 2018 to Saturday, April 14, 2018
↳ Tuesday, Wednesday, Thursday, Friday & Saturday


text: I have been falling behind on blog posts, so I just pulled together a few of my most recent favoirte Pinterest pins. I hope to do #3 this weekend in my spare time between painting my sisters room and watching Drop Dead Diva. :) I will defintly tell you my end result. The new trend of reusing books/music sheets for pretty banners, I am really liking. It so simple and every sheet/page tells a story (maybe even literally; ha). The quote (#1) I plan to print out and put up in my room. The quote is so perfect for me because I often have a hard time "living in the moment". Now number three I was really upset when I clicked through and found out it was only $26 but it was out of stock in my size, of course. Maybe, I will just have to find a different dress on "CiChic".There is not much to tell about number four except that I just love it. It is so unique with the lights in the background and the bicycle in the front. That is it for this lovely Friday, enjoy.