text:
- Methodology
- Open Access
MetaPro-IQ: a universal metaproteomic approach to studying human and mouse gut microbiota
© The Author(s). 2016
- Received: 3 February 2016
- Accepted: 2 June 2016
- Published: 24 June 2016
The gut microbiota has been shown to be closely associated with human health and disease. While next-generation sequencing can be readily used to profile the microbiota taxonomy and metabolic potential, metaproteomics is better suited for deciphering microbial biological activities. However, the application of gut metaproteomics has largely been limited due to the low efficiency of protein identification. Thus, a high-performance and easy-to-implement gut metaproteomic approach is required.
In this study, we developed a high-performance and universal workflow for gut metaproteome identification and quantification (named MetaPro-IQ) by using the close-to-complete human or mouse gut microbial gene catalog as database and an iterative database search strategy. An average of 38 and 33 % of the acquired tandem mass spectrometry (MS) spectra was confidently identified for the studied mouse stool and human mucosal-luminal interface samples, respectively. In total, we accurately quantified 30,749 protein groups for the mouse metaproteome and 19,011 protein groups for the human metaproteome. Moreover, the MetaPro-IQ approach enabled comparable identifications with the matched metagenome database search strategy that is widely used but needs prior metagenomic sequencing. The response of gut microbiota to high-fat diet in mice was then assessed, which showed distinct metaproteome patterns for high-fat-fed mice and identified 849 proteins as significant responders to high-fat feeding in comparison to low-fat feeding.
We present MetaPro-IQ, a metaproteomic approach for highly efficient intestinal microbial protein identification and quantification, which functions as a universal workflow for metaproteomic studies, and will thus facilitate the application of metaproteomics for better understanding the functions of gut microbiota in health and disease.
- Gene catalog
- Gut microbiota
- Metaproteomics
- Metagenomics
- Protein identification
- Quantification
Intestinal microorganisms, namely the gut microbiota, have been shown to be important in multiple aspects of physiological processes relating to health and disease, including nutrition, metabolism, and immunity [1]. Host genetics and nutrition have been shown to affect the composition of gut microbiota; conversely, the microbial metabolites or the microbes themselves can also regulate host metabolic processes [1–4]. Disruption of the homeostasis surrounding these host-microbe interactions has recently been shown to participate in the development of many diseases including obesity, diabetes, and inflammatory bowel disease (IBD) [1]. Therefore, the gut microbiota is emerging as an important topic for public health and scientific researchers.
Next-generation sequencing (NGS) has been widely applied in gut microbiota studies using various experimental approaches [5]. Classically, NGS studies have focused on characterizing the taxonomic profile of the gut microbiota through targeted amplicon sequencing (e.g., 16S hypervariable regions) or through shotgun metagenomics. More recently, NGS has been used to characterize the metabolic potential of the gut microbiota through metatranscriptomic analysis [6]. While these approaches are quite useful, they are unable to demonstrate that the predicted biological processes are actually present in the gastrointestinal tract. Metaproteomics, which examines all the expressed proteins in a microbial community, has been shown to provide invaluable functional information for the gut microbiota [7]. Moreover, the application of proteomics and metaproteomics to host-gut microbe interactions will help to provide meaningful information on the roles of microbiota [8]. Although the first shotgun metaproteomic study of the gut microbiota was reported in 2009 [7], only few follow-up large-scale gut metaproteomic studies have been published. The reasons for this lack of progress have been discussed in several reviews [8–12] and include (1) the inability to detect low abundant proteins with current mass spectrometers coupled to the high diversity of the gut microbiota and, more importantly, (2) the low efficiency in identifying gut microbial peptide or proteins from acquired mass spectrometry (MS) spectra. The latter is mainly due to the lack of a suitable database (in terms of database coverage and size) for peptide-spectra matching (PSM) which is the key step for current bottom-up proteomic studies.
The ideal database for proteomics should be composed of all potentially expressed proteins in samples being analyzed with no additional spurious sequences; however, this is challenging for gut metaproteomics due to the enormous diversity and individual variations of gut microbiome [12]. The NCBI nr database is often used in metaproteomics due to its high sequence coverage. Unfortunately, its enormous size (~80 million entries) makes the database searching extremely time consuming and also provides less sensitive peptide identifications when using target-decoy approach for false discovery rate (FDR) filtering [13]. To overcome these weaknesses, customized databases are often employed and are composed of a list of known gut microbial genomes that are manually chosen and combined to generate a synthetic metagenome database [14–16]. However, it is unclear how representative these synthetic metagenome databases are as compared to the actual metagenome since many intestinal microbes are un-cultivable and/or their genomes have not been sequenced. In addition, the researcher's choice of which bacteria taxa to include in the database is often arbitrary and may result in biases, which also makes the cross-study comparisons difficult. An alternative to manually curating a custom database is using a matched metagenome database. Briefly, either a "representative" subset or all the samples being investigated are subjected to metagenomic sequencing to compile a database of genes [14]. Since metagenomic sequencing introduces additional costs, it has not been generally performed in currently reported metaproteomic studies. These challenges have undoubtedly contributed to the fact that most metaproteomic publications only identify around 3000 proteins, which is far less than expected. Recently, Jagtap et al. [13] proposed a two-step database search strategy where a first search is performed against the target-only version of a database to generate a smaller refined database, which is then used for a second classical target-decoy database search [17]. This has been shown to increase the sensitivity of peptide identification and greatly increase the number of peptides and proteins identified. In a recent study by Tanca et al. [18] over 13,000 peptides corresponding to 9000 proteins were identified for mouse cecum samples by combining a matched metagenome database with the aforementioned two-step strategy. To the best of our knowledge, this is the highest protein identification for a single gut metaproteomic study to date. However, as mentioned above, the matched metagenome approach suffers from the need for metagenomic sequencing and the differences of databases used in different studies. Thus, a comprehensive gut microbial gene or protein database, which covers the expressed proteins for all gut microbial species, will make gut metaproteomics more affordable, comprehensive and comparable, which will largely promote wide application of metaproteomics in microbiome studies.
The Metagenomics of Human Intestinal Tract (MetaHIT) [19, 20] and the Human Microbiome Project (HMP) consortiums [21] have aimed to compile comprehensive gut microbial gene catalogs based on high-throughput sequencing to facilitate the analysis of multi-omic data. More recently, the close-to-complete human and mouse gut microbial gene catalog databases have been published and made freely available. Thus, in this study, we leveraged these gene catalogs as unified databases for protein identification in gut metaproteomics. We subsequently developed a high-performance and universal approach for gut MetaProteome Identification and Quantification (MetaPro-IQ), which led to the quantification of approximately 120,000 peptides corresponding to >30,000 protein groups (defined as the cluster of proteins identified by the same set or a subset of peptides) in a single experiment. More importantly, since the gene catalog databases are centrally curated and publicly accessible, the current approach can be widely applied for gut microbiota studies to generate comparable results from different researchers. Thus, the MetaPro-IQ shows great potential as a universal approach which would largely promote the wide application of gut metaproteomics.
Results and discussion
Implementation of the MetaPro-IQ workflow for gut microbiota study
A gene/protein database is typically needed for the purpose of peptide identification and is readily available for most model organisms. A typical proteome database contains 5000–20,000 entries which leads to a reasonable database search time and FDR filtering sensitivity [17]. However, the gut microbiota consists of thousands of microbial species and most of them are remain largely unknown or lack any available genome or proteome sequences [23]. This makes the gut metaproteomic studies much more challenging. Therefore, in this study, we used the newly generated, well-annotated gut microbial gene catalog databases (available from http://meta.genomics.cn/ and http://gigadb.org/) [20, 24], to improve the database coverage for the gut microbial proteins. The human gut microbial gene catalog contains 9.9 million genes generated from >1200 metagenomic sequencing samples [20], and the mouse gut microbial gene catalog contains 2.6 million genes generated from 184 sequenced mouse samples of diverse genetic and environmental backgrounds [24]. These databases are the most comprehensive for human and mouse gut microbial genes to date, and more importantly, the application of these gene catalog databases allows the generation of unified protein lists from different studies, thus enabling easy cross-study comparisons. The latter is also one of the important disadvantages for the current gut metaproteomic studies as aforementioned.
The sizes of the abovementioned gene catalog databases are large (>106 entries), which will greatly limit the database search sensitivity [13]. Iterative database search strategy has been previously shown to increase the sensitivity of peptide identification using huge databases, particularly for the metaproteomics and proteogenomics [13]. Thus, the MetaPro-IQ conducted a first-step database search against the whole gene catalog database, which generated a reduced database from the original gene catalog based on the acquired MS spectra in a sample. The reduced database was then used for a classical target-decoy database search to obtain confident peptide/protein identifications [13, 17].
In addition to the peptide/protein identification, quantification is another important aspect for proteomics. Spectra counting-based label-free quantification had been used for protein quantitation in many previous gut metaproteomic studies [7, 14, 15]. However, for data generated from high-resolution mass spectrometers, such as orbitraps, precursor-intensity-based algorithms have been shown to have superior accuracy, particularly when dynamic exclusion is enabled for spectra selection [22, 25]. MaxQuant is a widely used quantitative proteomics software implemented with the advanced MaxLFQ algorithm, which is based on precursor signal intensity and advanced delaying normalization across samples [22]. The MetaPro-IQ approach adopted MaxQuant for the final-step quantification analysis, which enabled accurate quantifications of the identified microbial proteins.
MetaPro-IQ reached comparable performance with matched metagenome strategy
To compare the abilities of the two approaches for extracting functional information, all the quantified proteins were annotated with Clusters of Orthologous Group (COG) categories. Twenty-three COG categories were observed with the matched metagenome approach, which were all found with the MetaPro-IQ approach. There is no obvious difference in the relative abundance of the high abundant COG categories between the two approaches (Fig. 3e). Several COG categories such as B, Z, and X were solely present or with obviously higher observed LFQ intensity using the MetaPro-IQ approach (Fig. 3e). This may result from the lack of low abundant genes in matched metagenome databases due to inadequate sequencing depth. The low abundant genes may have relatively high protein-expression levels which are detectable using mass spectrometers, and thereby were identified by MetaPro-IQ approach.
To examine whether the above observations are dataset dependent, the murine fecal metaproteome dataset (MFM; two replicates with two runs for each replicate) from the study of Tanca et al. [18] were re-analyzed with the MetaPro-IQ workflow. In total, we quantified 19,497 peptides and 4549 protein groups for replicate 1 and 19,972 peptides and 4630 protein groups for replicate 2. More than 92 % of the peptides were quantified for both replicates (Additional file 2: Figure S1A). Tanca et al.'s study, using a matched metagenome database search strategy, identified 14,085 peptides for replicate 1 and 15,669 peptides for replicate 2 with an overlap of 63 % [18]. Compared to the matched metagenome strategy, the MetaPro-IQ workflow identified more peptides with a better overlap between replicates for their dataset. In addition, a Pearson's correlation coefficient of 0.89 was obtained between the two replicates, and more than 0.86 between runs (two mass spectrometry runs were conducted for each replicate in Tanca et al.'s study, Additional file 2: Figure S1B–F), which is also in agreement with the findings in their study.
Taken together, the MetaPro-IQ metaproteomic workflow using the gut microbial gene catalog database showed better performance for identifying gut microbial proteins, when compared to the workflow using a matched metagenome database. MetaPro-IQ allows high efficient protein identification from MS spectra in metaproteomics without the need for prior metagenomic sequencing (greatly reduces the experimental cost) and is readily applicable for all researchers from various disciplines.
MetaPro-IQ approach revealed metaproteome response of gut microbiota to diet in mice
More than 94 % of the key microbial proteins have a greater than twofold change between HFD and LFD groups, while thirty eight of them have more than a 100-fold change, representing the major responders to HFD (Additional file 1: Table S5). Proteins S-Fe9_GL0178463 and S-Fe7_GL0107273 were the most markedly changed proteins with more than 1000-fold decrease in the HFD group, and both of them were annotated as hypothetical proteins from Eubacterium plexicaudatum (Additional file 1: Table S6). Further examination of peptide identifications revealed that protein S-Fe9_GL0178463 had high-quality PSMs (scores up to 323) in all samples in the LFD group, but no PSM was obtained for HFD groups (Additional file 2: Figure S4). The MaxQuant software has been designed to identify and quantify peptides from precursor ions without any MS/MS scan, by matching to the high-quality PSMs in paralleled samples based on retention time and mass-to-charge (m/z) ratio [22]. This resulted in an average of three peptides quantified for the samples in HFD group (Additional file 2: Figure S4). The lack of PSM for protein S-Fe9_GL0178463 in the HFD group might be the result of low protein levels in the samples, such that their precursor ions were not abundant enough to be selected by mass spectrometer for fragmentation. However, the precursor ions were still present allowing accurate quantification and thereby selection of key proteins with huge differences between the different groups. This scenario, where certain peptides/proteins are almost totally absent from a single group, is expected to be common in gut microbial studies, where some species may be substantially inhibited by other species or environmental compounds in one group as compared to another.
MetaPro-IQ approach provides a universal, easy, and high-performance metaproteomic workflow for studying human or mouse gut microbiota and dramatically improves microbial protein identification and quantification. The workflow was established based on the usage of the close-to-complete gut microbial gene catalog database and an iterative database search. These enabled high protein-database coverage and better sensitivity for peptide identification. More importantly, it allows easy comparison for the results obtained from different studies. The MetaPro-IQ approach also makes matched metagenome sequencing unnecessary for general gut metaproteomic studies. Moreover, the workflow generated quantitative results based on precursor intensity and advanced normalization method, which allows accurate comparison across samples.
The limitation of the MetaPro-IQ also happens to the gene catalog databases since they are mainly generated from stool samples and will not represent all types of intestinal microbiomes, for example, the biopsy microbiomes. For uncommon samples, the gene catalog database could be enhanced with additional matched sample metagenomic sequencings, and thus will improve the performance of MetaPro-IQ. In addition, the current gut microbial gene catalog databases represent well for the gut-related bacteria and archaea; the studies focusing on other taxa such as fungi and virus will need further addition of their protein sequences or alternative databases. However, the concept of MetaPro-IQ is applicable for any research as long as the gene catalog or reference protein database is available.
Mouse stool sample collection and processing
The animal experiments were performed at the Ottawa Hospital Research Institute and conducted in strict accordance with the guidelines on the Care and Use of Experimental Animals of Canadian Council on Animal Care (CCAC). The animal use protocol (2009-012) was approved by the Animal Care Committee at the University of Ottawa. A total of eight male C57BL/6J mice (Charles River, Sherbrooke, QC) were housed individually in the same room at 25 °C with a strict 12-h light/dark cycle. Food and water were available ad libitum. Mice were acclimatized to the facility for 2 weeks and fed a normal chow diet (containing 18 % fat by energy; Harlan Laboratories, Inc., Madison, WI) and then randomly divided into two groups (n = 4/group). One group of mice was fed a high-fat diet (containing 39.7 % fat by energy; TestDiet, St. Louis, MO), and the other group a low-fat diet (containing 15.8 % fat by energy; TestDiet) for 43 days. Body weight for each mouse was monitored weekly. Stool samples were collected at days 0, 14, 29, and 43 and stored at −80 °C until analysis.
For proteomic analysis, bacterial proteins were extracted from the stool samples. Briefly, stool samples (~1 g) were suspended in 1.5 mL ice-cold PBS (pH 7.0) with thorough vortexing. The slurries were centrifuged at 300g, 4 °C for 5 min. Supernatants were carefully collected, and the pellets were subjected to the above procedure three times. All the supernatants for each sample were then combined followed by three more centrifugations at 300g, 4 °C for 5 min to remove debris. The supernatant was then centrifuged at 14,000g, 4 °C for 20 min to pellet bacterial cells. The pellet was then re-suspended in fresh PBS and washed another three times. The bacterial cells were then lysed with 4 % sodium dodecyl sulfate (SDS) and 6 M urea in 50 mM Tris-HCl buffer (pH 8.0) with sonication. SDS was removed by protein precipitation in acidified acetone/ethanol buffer at −20 °C overnight. The precipitated proteins were dissolved in 6 M urea in 50 mM ammonium bicarbonate (pH 8) for trypsin digestion.
Human MLI sample collection and processing
Eligible subjects were under 18 years of age and scheduled to undergo diagnostic colonoscopy (Additional file 1: Table S1). The protocol was approved by the Research Ethics Board of the Children's Hospital of Eastern Ontario (CHEO). Colonoscopy preparation was done as per standard protocol modified to 1 day [40]. During colonoscopy, once the proximal ascending colon was intubated, any fluid and loose debris was aspirated and discarded. Thereafter, sterile water was flushed onto the mucosa to dislodge adherent mucus from mucosal epithelial cells and the mixture was then aspirated into a sterile container through the colonoscope. The samples were immediately placed on ice and transported to the lab for processing.
To remove any debris in the aspirate sample, a first centrifugation at 700g, 4 °C for 5 min was performed and the supernatant was transferred into a new tube. The bacterial cells were then collected with a centrifugation at 14,000g, 4 °C for 20 min. The pellets were then used for protein extraction according to the procedures described above.
Liquid chromatography-tandem mass spectrometry
In-solution trypsin digestion for bacterial proteins recovered from both mouse stool and human MLI samples was conducted as described previously [41]. Briefly, the proteins were first reduced and alkylated with 10 mM dithiothreitol (DTT) and 20 mM iodoacetamide (IAA), respectively. The urea concentration was then diluted to <1 M with 50 mM ammonium bicarbonate. Trypsin (Worthington Biochemical Corp., Lakewood, NJ) was then added at a protein-trypsin ratio of 50: 1 (w/w) for digestion overnight with agitation at 37 °C. The tryptic digest was desalted with a 10-μm C18 column and eluted with 80 % acetonitrile/0.1 % formic acid. The eluent was then evaporated with a Speed-Vac concentrator and tryptic peptides dissolved in 0.1 % formic acid for mass spectrometry analysis.
Tryptic peptides equivalent to 4 μg of proteins were loaded for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis on a Q Exactive mass spectrometer (ThermoFisher Scientific Inc.). The separation of peptides was performed on an analytical column (75 μm × 50 cm) packed with reverse phase beads (1.9 μm; 120-Å pore size; Dr. Maisch GmbH, Ammerbuch, Germany). A 4-h gradient was performed from 5 to 35 % acetonitrile containing 0.1 % formic acid at a flow rate of 200 nL/min. The instrument method consisted of one full MS scan from 300 to 1800 m/z followed by data-dependent MS/MS scan of the 12 most intense ions, a dynamic exclusion repeat count of 2, and repeat exclusion duration of 30 s. All data were recorded with the Xcalibur software and exported as.raw format for further analysis.
Metagenomic DNA extraction, sequencing, and gene prediction
Total DNA was extracted from intestinal aspirate samples using the Fast DNA spin kit (MP Biomedicals, Santa Ana, CA) and using a FastPrep-24 (MP Biomedicals). Briefly, MLI samples were thawed and contents pelleted by centrifuging at 14,000g for 10 min in a bench top centrifuge. The pellets were re-suspended in 1 mL of cell lysing solution (CLS)-TC and subjected to two mechanical lysis cycles at speed 6.0 for 40 s. The extracted DNA was then used to construct sequencing libraries using an Illumina TruSeq DNA Sample Prep kit v3 according to the manufacturer's instructions. The sequencing was performed on an Illumina HiSeq 2000 (generating paired-end 100-bp reads) at the Génome Québec Innovation Centre, McGill University (Montreal, Canada).
Gene prediction was performed on each sample individually using the previously published MOCAT pipeline [27]. Briefly, raw reads were first filtered and trimmed to remove sequencing adapters and low quality reads. Reads with human origin were removed using SOAPAligner 2 against the human genome database (hg19) [42]. The remaining reads were then used for scaftig assembly and assembly revision to generate assembled sequences for gene prediction with the MetaGeneMark algorithm [43]. The resulting gene sequence lists for each of the sample were then compiled into FASTA files and used as a matched metagenome database for benchmarking MetaPro-IQ.
Bioinformatics for metaproteome data analysis
Implementation of MetaPro-IQ approach
The implementation of the MetaPro-IQ approach is illustrated in Fig. 1 and details are highlighted in the "Results and discussion" section. The human and mouse gut microbial gene catalog databases were downloaded from the IGC website (http://meta.genomics.cn/) and the GigaScience Database (http://gigadb.org/dataset/view/id/100114/token/mZlMYJIF04LshpgP), respectively [20, 24].
In MetaPro-IQ, the first- and second-step database searches were carried out with X! Tandem (release 2015.04.01) [44, 45], and the third step was carried out with MaxQuant software (version 18.104.22.168) [46]. For the X! Tandem database search, each raw file obtained in the current study or from Tanca et al.'s study (downloaded from PeptideAtlas Repository at http://www.peptideatlas.org/PASS/PASS00355) was converted into mgf format with an in-house software platform. The tandem search was performed with up to two miss-cleavages (trypsin/P), carbamidomethylation of cysteine as a fixed modification, and oxidation of methionine as a potential modification. A fragment ion tolerance of 20 ppm and a parent ion tolerance of 10 ppm were used. All matched protein sequences for the first-step search were extracted as the sample-specific database (sample_all-macth.fasta). The X! Tandem outputs of the target-decoy database search (step 2) were summarized with an in-house software to generate an identified protein list at a FDR cutoff of 0.01 and maximum expect value of 0.05. The resulting protein list for all samples was then combined, and duplicates were removed for generating a "combined non-redundant database" to use for protein quantification using MaxQuant. Similar peptide identification parameters with X! Tandem database searches were used for MaxQuant (parameter files were uploaded together with the raw data and result files to ProteomeXchange). For quantification, the LFQ algorithm was used for label-free quantification. Both razor and unique peptides were used for protein quantification, and the minimum ratio count was set as 1. An alignment retention time window of 20 min and match time window of 5 min were applied to match the same accurate masses between different runs. Proteins identified by the same set or a subset of peptides were grouped together as one protein group.
Matched metagenome database search strategy
Matched metagenome database searches were performed with exactly the same parameters as the MetaPro-IQ approach, except that the metagenome database (gene sequences) was used for each sample instead of the whole gene catalog database. To ensure a fair comparison, the same multi-step database search strategy and MaxQuant quantification was performed. The outputs of MaxQuant (namely the results included in the txt folder) were then compared between the matched metagenome and MetaPro-IQ approaches.
Functional annotation into COGs
All the quantified protein sequences were aligned against the COG database (ftp://ftp.ncbi.nih.gov/pub/COG/COG2014/data) with DIAMOND using default parameters (e-value cutoff of 0.001) [47, 48]. The best hit for each query was selected for annotation, and the COG id, name, and category information for each of the matched sequences were extracted from the annotation file of the COG database. If the leading protein (defined as the top rank protein in a group; ranking is based on the number of peptide sequences, the number of PSMs, and the sequence coverage) in a protein group had no match for COG id, the others were checked for the existence of any match. The LFQ intensity of all the protein groups annotated with the same COG category were summed together to represent the COG category abundance for each sample.
Taxonomic analysis
Taxonomic analysis was performed based on taxon-specific unique peptides using Unipept (version 3.1) [31]. All of the quantified peptide sequences from MaxQuant were imported into the Unipept for analysis with the "Equal I and L" and "Advanced miss cleavage handling" options allowed. The results were then exported as a table format. The relative abundance of each taxon (namely phylum or species in this study) is represented as the sum of intensity for all the unique peptides found with Unipept.
Multivariate statistical analysis
The protein group results from MaxQuant were first imported into Perseus (version 184.108.40.206) to remove any contaminants, reverse sequences, and those identified only by site. The LFQ intensity was log10-transformed and used for statistical analysis. Only those protein groups with valid LFQ intensity values in at least 29 of the 32 samples (>90 %) were kept for subsequent statistical and multivariate analysis. This resulted in a stringently quantified dataset of 5299 protein groups for the bacteria from 32 stool samples. Hierarchical clustering analysis was also conducted in Perseus with default parameters. Significant proteins between HFD and LFD groups were identified by a two-sample t test with Benjamini-Hochberg FDR correction. Those proteins with a FDR corrected p value (q value) of <0.05 were considered as significant proteins.
PCA was performed in MATLAB (version 2010b, The MathWorks Inc.), and the first two PCs were used for generating the score plot. A prior missing value imputation was done with the nearest-neighbor method in MATLAB with the knnimpute function (http://www.mathworks.com/help/bioinfo/ref/knnimpute.html) [49].
COG, Clusters of Orthologous Groups; DTT, dithiothreitol; FDR, false discovery rate; HFD, high-fat diet; HMP, Human Microbiome Project; IAA, iodoacetamide; IBD, inflammatory bowel disease; LC-MS/MS, liquid chromatography-tandem mass spectrometry; LFD, low-fat diet; MetaHIT, Metagenomics of Human Intestinal Tract; MetaPro-IQ, metaproteome identification and quantification; MLI, mucosal-luminal interface; PCA, principal component analysis; PEP, posterior error probability; PSM, peptide-spectra matching; SDS, sodium dodecyl sulfate
We acknowledge the assistance of R. Singleton, A. Mack, and D. McCulloch in making this study possible.
This work was supported in part by the Genome Canada and the Ontario Genomics Institute (OGI-067), CIHR grant number GPH-129340, the Ontario Ministry of Economic Development and Innovation (REG1-4450), the Natural Sciences and Engineering Research Council of Canada (NSERC) council of Canada, and the Foundation Jean-Louis Lévesque. DF acknowledges a Canada Research Chair in Proteomics and Systems Biology. JB is the recipient of a CIHR/CAG Postdoctoral Award.
Availability of data and materials
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://www.proteomexchange.org) via the PRIDE [50] partner repository with the dataset identifiers PXD003527 and PXD003528, and all the raw reads of Illumina sequencing have been submitted to NCBI sequence read archive (SRA) under the accession of SRP068619.
Authors' contributions
DF, AS, DM, and XZ designed the study. XZ, JM, JIM, JL, JB, SAD, and CKC performed the experiments. XZ, ZN, RC, and MW analyzed the data. XZ and DF wrote the manuscript, and all the authors contributed to the editing and revision of the manuscript. All authors read and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Consent for publication
Consent to publish was obtained from parents of all the children.
Ethics approval and consent to participate
The animal use protocol (2009-012) was approved by the Animal Care Committee at the University of Ottawa, ON, Canada. The clinical sample collection protocol was approved by the Research Ethics Board of the Children's Hospital of Eastern Ontario (CHEO), Ottawa, ON, Canada. The consent to participate was obtained for all the participants.
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Aaron Shaffer began as a part-time move helper for JK Commercial 15 years ago, when the business unit only contained 20 employees. Now, he is JK Commercial's Training Manager, responsible for guiding new members of the team and helping existing employees achieve their goals through training and retraining. "Training helps you build your strengths, helps you look at your weaknesses, and helps you grow with the organization." Aaron is passionate about the Commercial Services JK provides, and hopes to continue rising through the ranks at JK Commercial. Watch Aaron's full story below. This video is part of JK's #TalentTuesday series, […]Read more
Month: July 2016
8 Moving Hacks that Cost You Nothing
Moving can be expensive. Moving is stressful. That is why you should use every trick at your disposal to make your move easier and less expensive. The following tips cost you nothing and will have you kicking yourself for not utilizing them before. 1. Use empty toilet paper rolls to organize cords and prevent tangling. The time spent untangling cords will make you tear your hair out. Utilizing this trick makes it quick and easy. 2. Wrap bottles of liquids such as shampoo in plastic wrap. The plastic wrap should contain the liquid and prevent leaking. 3. […]Read more
(NOW) Full and Part-Time Help Needed at JK
JK Moving Services is always looking to add full and part-time workers to help us manage the busyness of summer. If you are looking to work for a constantly growing company that promotes from within, apply to JK today! Our employee benefits extend beyond profit-sharing plans and health care — we routinely host a Farmer's Market at our office, invite ice cream trucks, and have a tuition-assistance program.Read more
Fire Breaks Out in a Moving Truck — Remember Your DO NOT PACK Items!
A few weeks ago in Phoenix, Arizona, a family nearly lost everything they owned. Around 1 a.m. on a Thursday, a fire broke out in a parked moving truck. Firefighters said they arrived to see an obvious working fire in the moving trailer, and quickly worked to put the fire out. (ABC15 Arizona's story can be read here) Though the firefighters were successful in putting out the fire, the household belongings inside the truck all needed to be removed from the trailer and were set in the parking lot outside before being loaded onto a new truck that arrived later. […]Read more
Nate Kost — From Part-Time Helper to Accounting Manager
Nate Kost began his career at JK Moving Services on the trucks as a moving helper. When JK found out he was looking for an accounting internship, the HR team reached out and let Nate know about an accounting internship available at JK. Now, Nate is JK's Accounting Manager, and earned his Master's degree through JK's tuition assistance program. It all started with a part-time job as a moving helper! We are glad that Nate took advantage of JK's employee benefits, and could not be more proud of his commitment to growth. Listen to Nate tell his story in the video below. This video is part of JK's #TalentTuesday […]Read more
Types of PCS Military Moves
If you are conducting a PCS military move (Permanent Change of Station), you may receive one of a few types of PCS orders. The government will pay for or reimburse military members for certain aspects of their PCS move, but each type has its own rules and regulations, so it is vital to plan your move according to the type of PCS order you receive. JK has moved thousands of service members over the years – including plenty of 3-star generals and admirals – so you can rest assured that we know all of the jargon used, and are experienced […]Read more

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Researching the Shady Family Tree
Dig into the past of the Shady Family Tree using the links below. We hope you find this information useful whilst you are unearthing your Shady Family Tree !
Did you know that in the 1881 UK census there were 5 people with the Shady surname? In that year Shady was ranked number 3574 for popularity in the UK.
New to Family History? Check out our Getting Started page. Check out House Of Names to investigate the origins of the Shady surname or get a copy of the Shady family Crest (a great gift idea!).
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Seneca Falls, New York, is best known for its associations with the beginning of the women's rights movement in the United States - more than any other site in the country. Seneca Falls was the location of the First Women's Rights Convention, held over two days in July 1848. More than 300 women and men attended the convention which marked the beginning of the organized women's rights movement. The "Declaration of Sentiments," adopted during the convention and signed by 100 attendees, provided the agenda for subsequent women's rights activity.
Women's Rights National Historical Park was created by Congress in 1980 to preserve and interpret the nationally significant historical and cultural sites and structures associated with the struggle for equal rights for women.
The park consists of several buildings in Seneca Falls and nearby Waterloo, New York including the Hunt Home in Waterloo where the convention was planned; the M'Clintock Home in Waterloo where the Declaration of Sentiments was written; the Wesleyan Methodist Chapel in Seneca Falls where the convention was held; and the home of Elizabeth Cady Stanton in Seneca Falls.
For high resolution photographs of the park, see the photo album on this page or go to the photo gallery for more downloadable images. Photographs used from this web page must be credited to NPS.
Last updated: February 26, 2015


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Photos courtesy of Joanna McLendon.
Photos courtesy of Joanna McLendon.
On a crisp fall day in 1933, two black men stepped out of a modest middle-class home on Kansas City's Haskell street. Both were named John B. McLendon. Fifty-one-year-old railroad mail clerk John, Sr. got behind the wheel of his automobile, and 18-year-old John, Jr. slid into the passenger's seat. Father and son drove west past the meat-packing plants of the "Rattlebone Hollow" neighborhood, pointing the shiny, new DeSoto toward the town of Lawrence, where John the younger was beginning his studies at the University of Kansas. It was an easy enough forty-mile drive.
Decades later, John McLendon, Jr. told his friend and biographer Milton Katz what his father said as he was packing to leave. "Go up there and do what everybody else does, and try to do it in a way that won't have you getting hurt," he had told his son. "If you happen to get hurt, let me know. I'll be up there with my .44."
The man had reason to fear for the boy. John B. McLendon, Jr. was about to become the first black man to enroll in the physical education program at Kansas.
When they arrived, John, Sr. let his son out of the car, gave a final set of instructions, and drove away. John, Jr. entered Robinson Gymnasium and followed his father's parting advice to the letter. After a brief search, he found himself standing in an office doorway, looking in at the university's Director of Physical Education. Dr. James Naismith looked up and examined his unexpected guest. The ice was broken fairly easily, as Milton Katz recounted in Breaking Through, his biography of John McLendon:
[He announced] to this famous man that he was going to major in physical education, that he wanted to learn how to coach basketball, and that he understood that Dr. Naismith would be his advisor. "Who told you this?" Naismith inquired. "My father," answered McLendon. "Come on in, fathers are always right," Naismith declared.
Dr. Naismith had first nailed peach baskets to balconies at the Springfield, Massachusetts YMCA in 1891. In 1959, long after the great man had died, his name would grace the Basketball Hall of Fame in that same city. The nervous freshman in his office would eventually be called the "Father of Black Basketball" by Julius Erving. The two held many one-on-one talks about life and basketball, and it was during one such chat that Naismith expressed a life-changing theory to McLendon. "He said the ultimate game is to attack wherever the ball is and let your offense begin wherever you get the ball." At a time when slow-down basketball was a painful reality, Naismith's pupil took his mentor's words to heart, creating the fast break and the full-court press and using them to win a high school championship as a twenty-year-old student teacher.
"They really became friends," Katz told me during a phone conversation in 2010. "John actually said that he felt special under Naismith's tutelage. I don't think that was just bravado; he really felt that Naismith cared a lot about him. He thought McLendon was special."
Black athletes were playing for Clair Bee at Long Island University by 1933, but KU didn't put a black player on the court until LaVannes Squires joined the team in 1952. McLendon admitted to Katz that skin color wasn't the only obstacle standing between him and a spot on the Jayhawks—though quick and cerebral, McLendon had been cut from his high school basketball team three times. He was a former lifeguard and an excellent swimmer, though, and he expected to ace the mandatory swimming test required for his degree. But when McLendon showed up at the on-campus pool to work out, he found it half empty. He was told it was drained every Wednesday.
McLendon came back on Thursday, and found the pool empty again. Nobody had told him the pool was segregated, perhaps because no colored student had tried to swim there before. The school's passive-aggressive trick exposed a weakness he could exploit—a fear of direct confrontation. "You're going to have a pretty big water bill," McLendon told the attendant. "Because I'm going swimming every day."
Faced with McLendon's intransigence, university administration tried another indirect tack, offering to waive the swimming requirement based on his demonstrated proficiency as a former lifeguard. McLendon refused the poisoned fruit, adhering to his father's insistence that he earn his degree by doing what everyone else did, while simultaneously flaunting his injunction against risking bodily harm.
McLendon didn't ask anyone to share his fight for equal rights, but help came nonetheless. A white classmate circulated a petition and gathered over 1,000 signatures in support of "mixed swimming." Other physical education students arranged to meet John at the pool and work out with him in a show of solidarity. When things got testy, professor Naismith detailed a pair of intimidating football players to guard the Robinson pool during John's aquatic training. Tensions rose.
"Naismith was a very religious person, very liberal in his thinking, and a pure humanitarian, but for him to take McLendon under his wing I think was pretty extraordinary," Katz told me. "The fact that [McLendon] was the first African-American student in the PE program really meant a lot to Naismith. In that way, the color of his skin really did matter, and it was a positive thing."
Still the situation escalated. While walking across campus one day, John spotted a hand-lettered sign: "Do not swim with the nigger." John pulled that one down and collected several more that he found in the area and took them directly to Dr. Naismith. His mentor gathered up the offending propaganda and took it with him as he visited the university's chancellor and president in turn. His message to both men was simple and direct: if he ever saw another hateful sign on campus, he would resign immediately and seek employment elsewhere.
The threat worked. McLendon was allowed to meet with athletic director (and head basketball coach) Forrest "Phog" Allen to negotiate for the rights of black students who wanted to swim in the pool. Civil disobedience had brought his crusade to the attention of KU's most powerful men, and McLendon resolved to close the deal peacefully. When Allen stated—perhaps a bit disingenuously—that he hesitated to integrate the pool out of concern for McLendon's safety, the younger man had a ready answer: Open the pool to mixed swimming for two weeks, and if nothing untoward occurred during that time, the pool would be open to all. Allen agreed to the terms.
McLendon met with the other sixty-odd black students at KU and explained his plan: Stay away from the pool for two more weeks, and we can have it forever. When the two weeks passed quietly, Allen called McLendon a "smart-aleck" but lived up to his promise. The pool was open to all students from that day forward.
John McLendon wasn't done side-slipping racism by any means. He crashed the segregated spring dance with his future wife so they could dance to the music of black bandleader Andy Kirk and His Twelve Clouds of Joy. McLendon's popularity on campus grew to the point that he became the first black enrollee elected to student council in 1936.
After graduation, he took his first coaching job at North Carolina College for Negroes (now North Carolina Central) in Durham. He arranged the now legendary "secret game" in 1944, pitting his CIAA-Champion Eagles against an all-white Duke Medical School team that claimed to be the best in the state. The Eagles used McLendon's pioneering press and fast break to destroy the challengers 88-44. "The final score in the NCAA championship game that year was 44-42," Katz noted. McLendon then split the sides into integrated teams and had them work together. NCC player George Parks would later recall it fondly as "Just God's children, horsing around with a basketball."
McLendon went on to coach a three-peat NAIA national champion at Tennessee State, broke the color barrier as a head coach with the George Steinbrenner-owned Cleveland Pipers in the short-lived American Basketball League, and served a short but historically relevant stint at the helm of the ABA's Denver Rockets in 1969. McLendon claimed that Rockets management had a secret quota—six white players and six black players—for the team makeup, a charge the team's GM later denied. When McLendon was fired after just 28 games, however, two black players he had signed were cut the very same day.
Naismith's desire to promote social and spiritual advancement through sporting achievement was a life-long avocation. He took delight in the knowledge that his game had diversified and spread to exotic locales like Cuba, Turkey, Jamaica and China. He actively encouraged women to play, claiming that a group of teachers who watched his first YMCA classes subsequently sought, and obtained, his blessing to start the first women's team. He met his wife Maude at an 1892 women's tournament in which she was a star player. He wrote about how much he enjoyed watching Native American students play at Lawrence's Haskell Institute. According to Rob Rains' biography James Naismith, The Man Who Invented Basketball, Naismith sent a letter to his wife while stationed in France during World War I, in which he wrote "The human heart in a black or white skin wants to see right prevail." He wasn't talking specifically about basketball, but the sentiment—exceedingly rare in the early twentieth century—reveals something about the man who wrote it.
McLendon eventually coached an NCAA team at Cleveland State University, where the arena bears his name, and represented his country as an assistant coach of the 1972 Olympic basketball team. His long-overdue induction into the Naismith Memorial Basketball Hall of Fame came in 1979, just twenty years before his death. That same year, he finally got a call from Kansas, and the 54-year-old returned to his old stomping grounds to receive the school's Alumni Citation of Merit. It read, with maddening understatement: "This man, more than anyone else, is responsible for the integration of swimming and swimming pool programs at the University of Kansas."
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First off, we're not in the business of trying to figure out if an elephant never forgets anything. One can presume that even an elephant has left its cell phone at a restaurant once, or come in late with a rent check. But there's plenty of evidence that elephants have terrific memories in general, and they're not just good for counting cards or winning a part in the school play. Elephants' memories contribute to their remarkable intelligence in general.
Let's take an example. Between 1958 and 1961, Tanzania's Tarangire National Park experienced a terrible drought. In 1993, a second drought came to the area. Just like any other animal or person, elephants had to decide whether to stay in their area and hope the drought let up or take their chances migrating. Scientists found that elephant groups with mothers who had lived through the earlier drought left the park, while younger mothers stayed put [source: Foley]. Groups that left the park ended up having much higher survival rates.
Well, you think, what's so great about that? The smart elephants left the drought zone --seems commonsense. But keep in mind that the group of elephants that stayed behind had no matriarch old enough to have been a part of the first drought; there was a clear selective advantage in the memories of females who had experienced previous droughts [source: Goudarzi].
But long-term memory isn't the only bank that elephants use. They also demonstrate a strong working memory, which allows them to keep track of and even organize their social groups [source: Ritchie]. Elephants travel in large groups over wide ranges, and they keep track of family members within the group. Sounds easy enough -- until you realize there's 20 or 30 relatives to keep track of. But the elephants appear to use their sense of smell and memory to know the location of each member. When scientists surreptitiously deposited a urine sample of a trailing elephant in front of a leading elephant, the animals registered surprise. They seemed to remember where each elephant was in relation to them [source: Bates].
Elephants have also been shown to remember individual language differences. Researchers conducted a study where elephants were played recordings of men from the Masai -- who kill elephants to protect grazing land -- and the less-threatening Kamba tribe. Though both repeated the same phrase, a majority of elephants acted much more defensively to the Masai recording [source: AP]. So with evidence such as this, Elephants' memories have proven to be not just good, but incredibly useful.
Related Articles
- Associated Press. "Wild Elephants Can Recognize Different Human Languages, Study Finds." The Washington Post. March 12, 2014. (Oct. 16, 2014) http://www.washingtonpost.com/lifestyle/kidspost/wild-elephants-can-recognize-different-human-languages-study-finds/2014/03/12/17cf5c5e-a966-11e3-8d62-419db477a0e6_story.html
- Bates, Lucy A. et al. "African Elephants Have Expectations About the Locations Of Out-of-Sight Family Members." Biology Letters. 4, 1. Feb. 23, 2008. (Oct. 16, 2014) http://rsbl.royalsocietypublishing.org/content/4/1/34.full
- Briggs, Helen. "How Elephants Keep Tabs on Family." BBC News. Dec. 5, 2007. (Oct. 16, 2014) http://news.bbc.co.uk/2/hi/7127276.stm
- Foley, Charles et al. "Severe Drought and Calf Survival in Elephants." Biology Letters. Oct. 23, 2008. (Oct. 16, 2014) http://rsbl.royalsocietypublishing.org/content/4/5/541.full
- Goudarzi, Sara. "'Never Forgetting' Helps Elephants Survive, Study Says." National Geographic News. Aug. 19, 2008. (Oct. 16, 2014) http://news.nationalgeographic.com/news/2008/08/080819-elephants.html
- Ritchie, James. "Fact or Fiction?: Elephants Never Forget." Scientific American. Jan. 12, 2009. (Oct. 16, 2014) http://www.scientificamerican.com/article/elephants-never-forget/


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Special Education > Technical Assistance > Placement/Least Restrictive Environment (LRE)
Technical Assistance
Placement/Least Restrictive Environment (LRE)
Location, location, location. Yes, it does matter. The research overwhelmingly supports serving students in their least restrictive environment – which is why the following mandate is included in the federal legislation:
34 CFR 300.114 (a)(2) – Each public agency must ensure that – (i) To the maximum extent appropriate, children with disabilities, including children in public or private institutions or other care facilities, are educated with children who are nondisabled; and (ii) Special classes, separate schooling, or other removal of children with disabilities from the regular educational environment occurs only if the nature or severity of the disability is such that education in regular classes with the use of supplementary aids and services cannot be achieved satisfactorily.
Technical assistance resources on this page include research, articles, tools, templates, technical assistance centers, and Web sites. (Note: Use of the resources included on this site does not guarantee that the district's performance or determination status under section 616(d), will improve for the next reporting period.)
Least Restrictive Environment (LRE) – Ages 6 through 21 (Indicator 5)
Preschool Least Restrictive Environment (LRE) (Indicator 6)
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[Beware–this post contains a few spoilers for Season 4 of Vikings.]
I've been thinking a lot about bears recently. Part of this is due to the theatrical release of the live-action Jungle Book ("Look for the bare necessities") as well as an ongoing project on Neil Gaiman's Odd and the Frost Giants (2008) in which Thor is transformed into a bear. I've heard that there's a bear in the 2015 film The Revenant, but to be honest, I have no desire to see it. Since I've moved to Colorado, I've seen at least seven bears, including a mother bear and her two cubs near the aptly named Bear Lake in Rocky Mountain National Park.
But most recently, an episode of the History Channel's The Vikings has brought human/bear relationships to my attention once again.
What I've seen of Season 4 (I'm a little behind) has been great so far. I've enjoyed watching Floki's storyline evoke that of his alleged ancestor Loki in significant ways, and the show has touched on the art of illuminated manuscripts. Poor Rollo is trying his best to please his French wife. Travis Fimmel as Ragnar continues to be mesmerizing. I've been particularly interested in the storyline of Björn Ironside, Ragnar's eldest son, who has decided to spend the harsh winter in the wilderness in an attempt to prove himself to his father.
But whennext encounters the bear, he does so fully armed and the two engage in battle.
Following the death of the bear,skins the bear and treats the wound he received from the beast. Later we see emerging from the ice in a scene reminiscent of baptism.
On one hand, the death of the bear will provide warmth (the bear skin), food, and much-needed confidence to
In fact, just before here and the poem in Old Norse here), Odin disguises himself as the ferryman Harbard who subsequently refuses passage to Thor. Ragnar tells the story in part to shame his wife Aslaug (she had an affair with a man named Harbard in the previous season), but given the larger frame of theencounters the bear for the final time, Ragnar has been telling the story of Hárbarðsljóð to his younger sons. In this flyting poem (a translation is available
While bears do not often show up in the Middle English romances with which I typically work, they do appear in the Old English and Norse material. The titular hero of Beowulf, for example, has linguistic connections to bears through his name ("Bee + Wolf"). Grettir, in Grettis saga Ásmundarsonar, spends most of his time fighting other outlaws and the occasional troll, but a few bears show up as well. But then there's the Saga of King Hrolf Kraki.
Even before Bodvar Bjarki appears, the motif of the Bear's Son appears when Queen Hvit, after unsuccessfully propositioning the king's son–aptly named Bjorn, curses Bjorn to live out the days of his life as a bear:
She then struck him with her wolfskin gloves, telling him to become a cave bear, grim and savage: 'You will eat no food other than your own father's livestock, and, in feeding yourself, you will kill more than has ever been observed before. You will never be released from the spell, and your awareness of this disgrace will be more dreadful to you than no remembrance at all.' (Chapter 19 from Jesse Byock's translation)
Bera, his beloved, follows after him and they live together for some time–even having children together (one of whom is Bodvar Bjarki, whose name means "Warlike Little-Bear"). Like Merida in Disney/Pixar's Brave, Bera recognizes the humanity in Bjorn's eyes. When Bodvar grows up, he seeks the court of Hrólfr Kraki and garners much glory. His connection to bears is more subtle than that of his father, as revealed in a battle scene:
Then Hjorvard and his men see a huge bear going before the King Hrolf's men, always nearest to where the king was. He kills more men with his paw than any five of the king's other champions. Blows and missiles glance off him. But he bursts under him both men and horses of King Hjorvard's army; and everything that comes in his way, he crushes in his teeth, so that panic sweeps King Hjorvard's army. (Chapter 50 from Peter Tunstall's translation)
Bodvar's companion, Hjalti, wonders where Bodvar is and leaves the battle to look for him. Upon finding Bodvar sitting motionless in a trance in the king's hall, Hjalti rebukes him–but alas, the spell has been broken. Bodvar was present at the battle–he had sent his spirit into the fierce bear–but now the tide will turn and Bodvar, the king, and their noble companions will fall.
The fourth episode of Vikings (the most recent episode that I've seen) did nothing further with's relationship to the bear. He defeats the sent to kill him and returns to his father's home (although now with a female companion), but I've not seen any evidence of change in him. Perhaps time will tell.


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November 02, 2011
Alaska has nearly 40 percent of the entire coastline of the United States, but is the only state without a program to coordinate management of these important areas. Thousands of Alaskans live near the coast, but because Alaska doesn't have a coastal management program, they have no voice in important decisions affecting coastal areas.
North Slope Borough Mayor Edward Itta supported the resolution. "The big resource development projects are in rural Alaska, and rural communities are generally strong supporters of these projects," Mayor Itta said. "If we work together early in the process, the chances of a timely and successful start are much better than if local people are just left out in the cold. Coastal management never stopped a project in the past. It just gave communities a stake in the project. What's not to like about that?"
State coastal management programs and local coastal management plans guarantee state and local participation in coastal development and a seat at the table during federal decision making. After operating successfully for 34 years, Alaska's Coastal Zone Management Program died when the Alaska Legislature failed in 2011 to renew it during both the regular session and two special sessions called in part to restore the program. Without a program in place, federal agencies need not consult state or local officials on questions of coastal development.
If the petition is signed by some 26,000 Alaska voters, and if the Alaska Legislature fails to enact substantially similar legislation during the 2012 regular session, "An Act establishing the Alaska Coastal Management Program" will appear on either the August 2012 Primary or the November 2012 Alaska General Election ballot. If voters approve the measure, the Act will create a coastal management program to replace the one dismantled in 2011.
The initiative is sponsored by the Alaska Sea Party, a group of municipal officials, local leaders, and interested voters committed to Alaska involvement in coastal development decisions. City and Borough of Juneau Mayor Bruce Botelho chairs the Alaska Sea Party's steering committee. He filed the initiative along with Kodiak Island Borough Mayor Jerome Selby and Kenai Peninsula Borough Assemblyman Mako Haggerty.
"When the Alaska Coastal Management Program was eliminated earlier this year, Alaska became the only state to weaken its oil spill prevention and response requirements," Botelho said. "Alaskans don't want another Exxon Valdez disaster, and they're tired of the political bickering that killed the very program that could prevent a similar tragedy."
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So that's how she does it! Bikini babe Naomi Campbell fits in some aerobics during relaxing holiday
13:53 GMT, 28 May 2012
Few supermodels achieve their figures through simple good genes.
And Naomi Campbell proved that she works hard for her amazing appearance when she was spotted getting in a little exercise during her holiday in Ibiza this week.
At 42, Naomi looked incredible in her skimpy black two-piece that displayed her long toned legs and flat stomach.
Fabulous after 40! Naomi Campbell shows off an impressive toned figure as she enjoys the sand, sun and sea in Ibiza
Miss Campbell fit some exercise into her otherwise relaxing day, doing some light water aerobics in the sea.
Her boyfriend Vladislav Doronin looked on as his beautiful girlfriend worked out.
Naomi showed off as much skin as possible in her brief bikini, but the Russian billionaire covered up in a T-shirt even in the water.
Splashing in the surf: Naomi is holidaying with boyfriend Vladislav Doronin
Got to keep that bikini body: Naomi got in some water aerobics as her Russian billionaire beau looked on
The couple are enjoying a break after flying from Austria where they attended the 20th Life Ball in Vienna.
Naomi, a longtime supporter, participated in a runway show to raise money for HIV and AIDS research.
Campbell appeared alongside Milla Jovovich, Karolina Kurkova and Keira Chaplin at the event.
Holiday mode: The supermodel carried her towel to her spot on the sand
An old pro: Naomi walked the runway at the 20th Life Ball in a stunning floor length gown at the Life Ball a few days earlier in Vienna
Before that, Naomi had travelled to Bethlehem where she marked her 42nd birthday.
'As much traveling as I've done, I've never come here,' Campbell told a Palestinian television station during her low-key visit.
'I'm happy to be here. Weapons and war, greed and oil … I hope it all stops. I care about health, about good vibrations, not destruction,' she said.
For charity: Naomi took part in a runway show alongside other models including Karolina Kurkova to raise money


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The Evolving Butterfly Effect
The Power of a Butterfly
It has been said, "A butterfly flapping its wings in Peru can start a change in the weather that leads to a typhoon in China." The important point of this idea is the power that is inside each and every one of us can be a powerful life changing experiences for ourselves and with others.
Throughout your life, there will be someone giving you insights and guidance that can help or give you something to think about. A message is shared you receive or reject it which has cause a reaction. The reaction is change and its effect on you and your life. This is the beauty of the evolving butterfly effect because it is about the power of being able to share insights and guidance that will help others.
Take a moment and think about how many times has your direction changed because you interacted with another person? Sometimes an observation from someone with more experience can change your destiny. Our connection with others helps us in reaffirming that which we know and feel. It helps us to stay focus and on point in taking continuous action in the direction of our goals, overcoming resistance, adversity, and temporary failures.
This is the true universal law of success and living life to its fullest one mind connecting with another mind interacting and sharing an inner power, as you spread your wings to make a difference in the world.
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Mon & Wed 9am to 6:30pm, Tues, Thurs & Fri 9am to 5pm Sat 12pm to 6pm
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Recently built, The EXO offers studio, one, two, and three bedroom layouts with modern conveniences and stunning skyline, bridge and river views. Most of our apartments include private balconies, and all apartments feature solid oak flooring, recessed lighting, and Bosch washer/dryers. Amenities include onsite covered parking, a resident lounge with free WiFi access, a fully-equipped fitness center, and a landscaped sky lounge overlooking Manhattan. We also offer a concierge service to fit your hectic schedule.
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The EXO is located at the corner of 21st Street and Astoria Boulevard. By public transit, The EXO is an 8 minute walk from the 30th Avenue N & W Train station. There is also a Q69 & Q100 bus stop adjacent to the property which services the Queensbridge F Train station in 9 minutes.


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In addition to the social "dangers" of snoring there are many studies that directly link patients with loud frequent snoring to (OSA) Obstructive Sleep Apnea. At night when snoring and sleep apnea coexist, the body no longer absorbs enough oxygen. Once this obstruction occurs, snoring becomes serious enough to have profound negative effects on a person's health. There have also been studies conducted that showed snoring to be a singular risk factor for high blood pressure (hypertension) and other cardiac diseases. Without a doubt, a snorer will experience some degree of daytime dysfunction due to their fragmented sleep. Snoring can cause morning headaches, concentration difficulties, reduced work performance and fatigue.
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According To His Will
Most Relevant Verses
And we know that God causes all things to work together for good to those who love God, to those who are called according to His purpose.
This is the confidence which we have before Him, that, if we ask anything according to His will, He hears us.
"All the inhabitants of the earth are accounted as nothing, But He does according to His will in the host of heaven And among the inhabitants of earth; And no one can ward off His hand Or say to Him, 'What have You done?'
I saw the ram butting westward, northward, and southward, and no other beasts could stand before him nor was there anyone to rescue from his power, but he did as he pleased and magnified himself.
"And a mighty king will arise, and he will rule with great authority and do as he pleases.
"And that slave who knew his master's will and did not get ready or act in accord with his will, will receive many lashes,
Then the word of the LORD came to me saying, "Can I not, O house of Israel, deal with you as this potter does?" declares the LORD. "Behold, like the clay in the potter's hand, so are you in My hand, O house of Israel. "At one moment I might speak concerning a nation or concerning a kingdom to uproot, to pull down, or to destroy it;read more.
if that nation against which I have spoken turns from its evil, I will relent concerning the calamity I planned to bring on it. "Or at another moment I might speak concerning a nation or concerning a kingdom to build up or to plant it; if it does evil in My sight by not obeying My voice, then I will think better of the good with which I had promised to bless it.
He made known to us the mystery of His will, according to His kind intention which He purposed in Him with a view to an administration suitable to the fullness of the times, that is, the summing up of all things in Christ, things in the heavens and things on the earth. In Him
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Sora Kasugano has a pensive air around her. She would be just another character from an eroge, except for the softly contrasted monochromatic colors of her skin, clothing, and hair caught my eye. I find all of the Yosuganosora characters illustrated by Hashimoto Takashi attractive, mainly for how well the line work and color palette match so well.
Houjun Otoyama sculpted Sora based on this illustration. The adaptation of that illustration to the figure, particularly in her body language is well done. Otoyama caught the pensive body language as well as the physical shape and tone. Her hips and subtle torso twist look far more solid in the figure. I'm a fan of tummies, so I enjoy the extra tone that Otoyama included on Sora. All that's missing is some water glistens, but I don't imagine there's currently a way to include that detail without it looking eww-chunky. Last thing you want to do with an already questionable desk ornament is to have to explain that stuff on her is supposed to be water.
Her towel is rendered surprisingly well for being made of plastic. As far as the base goes it does the job and is unobtrusive. The black rabbit doll acts as a counterweight to Sora's lightness. Keeping it on the base grounds the figure well. Check out one of the thumbnails at the end that show the figure without the rabbit doll and towel to see how the composition of the figure is a bit unsettled without these accessories.
MegaHouse's Sora Kasugano isn't completely chocolate and unicorns. Sora's head tilt looks better in the illustration, conveying a bit more personality than the more straight ahead positioning on the figure sculpt. Her figure face is a little more generic than resembling the illustration. As far as the detailing, shading in places is a bit over dark. In particular around her knees and ankles, it looks more like dirt than the natural darkening of these areas on an idealized body.
The over-shading isn't enough to detract from her legs, which are a bit more realistically proportioned than in a good number of figures, where the tendency is to really elongate the leg area. Sora's head and body are about the same combined length as her legs. Often times this proportion can make a drawing or figure appear stubby, rather than long and elegant. Sora appears to be anything but stubby.
I hope to see a Brilliant Stage treatment of some of the other Yosuganosora characters. Sora turned out better than I expected and a little bit more interesting than I thought she would be. Foo-bar-baz gave the figure 5-stars and I think its a good figure as well. I guess, if I had to score points against the sculpt itself, it would be her butt. The shape around her lower back, the tailbone on up seems off.


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|Daffodils II
oil on oil paper
8" x 10"
Thursday, 14 January 2016
Daffodils II
It snowed for all of five minutes and then melted. I won't be getting my outdoor easel out of mothballs just yet. Lol
2 comments:
Hi you :)ReplyDelete
Happy New Year. It's been an odd start to it over here but I'm glad to see you are taking the time to paint. I especially like your Salvation and what your thoughts. Keep painting for all of us! Cheers.
Just setting up my own daffodils and found this in my inbox - yours are so vigorous and cheering on this chilly morning. I'm enjoying the daily gift. These have nice depth and a sense of their physical presence.ReplyDelete
Thankyou so much for taking the time to comment, its always a pleasure receiving each and every one.
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