CCL5 gene variants, RANTES serum concentrations and protein levels in atherosclerotic plaques and risk for coronary events.The chemokine RANTES /CCL5 is involved in the pathogenesis of cardiovascular disease in mice, whereas less is known in humans. We hypothesised that its relevance for atherosclerosis should be reflected by associations between CCL5 single nucleotide polymorphisms and no common haplotype showed significant associations with coronary events. Also in the CARDIoGRAM study , none of these CCL5 SNPs was significantly associated with coronary artery disease. In the prospective Athero-Express biobank study, RANTES plaque levels were measured in 606 atherosclerotic lesions from patients who underwent carotid endarterectomy. RANTES content in atherosclerotic plaques was positively associated with macrophage infiltration and inversely associated with plaque calcification. However, there was no significant association between RANTES content in plaques and risk for coronary events (mean follow-up 2.8±0.8 years).We conducted a case-cohort study within the population-based MONICA/KORA Augsburg studies. Baseline RANTES serum levels were measured in 363 individuals with incident coronary events and 1,908 non-cases (mean follow-up: 10.2±4.8 years). Cox proportional hazard models adjusting for age, sex, body mass index, metabolic factors and lifestyle factors revealed no significant association between RANTES and incident coronary events . None of six CCL5 genotypes and (iii) RANTES content in carotid plaques and either coronary artery disease or incident coronary events in our cohorts suggests that RANTES may not be a novel coronary risk biomarker. However, the potential relevance of RANTES levels in platelet-poor plasma needs to be investigated in further studies.High RANTES plaque levels were associated with an unstable plaque phenotype. However, the absence of associations between (i) RANTES serum levels, (ii)  Inflammation is one of the hallmarks of atherosclerosis Chemokines are proinflammatory cytokines that recruit leukocytes to sites of tissue damage or infection Several lines of evidence indicate that RANTES plays a role in the pathogenesis of cardiovascular diseases. In mice, RANTES is expressed in atherosclerotic lesions and both RANTES antagonists and deletion of the gene encoding the RANTES receptor CCR5 can reduce the progression of atherosclerosis or early myocardial reperfusion CCL5 genotypes, systemic RANTES levels as well as RANTES levels in atherosclerotic plaques and risk for coronary events. We tested the first two parts of this hypothesis by assessing the relationship between CCL5 gene (encoding RANTES protein) variants and RANTES serum levels with cardiovascular risk in the German MONICA/KORA Augsburg case-cohort study. In addition, the association between CCL5 genotypes and CAD was analysed in the large CARDIoGRAM study We hypothesised that the relevance of RANTES in the development of atherosclerosis should be reflected by associations between This study is a prospective case-cohort study Response rates for the different surveys were 75.7% (S1), 74.4% (S2) and 73.1% (S3). The total number of study participants was 13,427  aged 25–64 (S1) or 25–74 years . All subjects were prospectively followed within the KORA framework until 2002.The outcome variable “coronary events” was defined as combined endpoint that consisted of incident non-fatal myocardial infarction (MI) and fatal coronary death or sudden cardiac death before the age of 75 years. Cases were identified through the MONICA/KORA Augsburg coronary event registry. In addition, follow-up questionnaires were sent to persons who had moved out of the study area. Until December 2000, a major non-fatal MI was diagnosed based on the MONICA algorithm . Since January 2001 myocardial infarction was diagnosed according to criteria defined by the European Society of Cardiology (ESC) and the American College of Cardiology (ACC) Due to the low incidence of coronary events in individuals below 35 years of age, we limited the present study to persons aged 35–74 years at baseline . Due to missing blood samples, 1187 subjects had to be excluded which led to a total source population of 9531 subjects . Participants with self-reported prevalent coronary heart disease were also excluded, leading to a final source population of 9300 subjects . For the case-cohort study, we selected a random sample of the source population  stratifying by sex and survey. The final stratum-specific sample sizes of this subcohort were used together with the stratum-specific sizes of the source population to compute sampling fractions, and the inverse of the sampling fractions yielded the survey and sex-specific sampling weights. All cross-sectional analyses were performed in the subcohort which included 2077 subjects  after exclusion of subjects with missing values for classical cardiovascular risk factors and essential inflammatory markers. Until December 31, 2002, 397 study participants had a coronary event according to our endpoint definition . After further exclusion of subjects with incomplete information on relevant variables, the present study was based on 2271 participants . The majority of cases  occurred before the end of 2000 when the diagnosis criteria were changed from the MONICA algorithm to the ESC/ACC criteria. Cases with incident coronary events included 175 participants with non-fatal MI and 188 cases with fatal coronary death or sudden cardiac death. The mean follow-up time (± standard deviation) was 10.2±4.8 years.Sociodemographic and lifestyle variables as well as data on parental history of myocardial infarction were collected through standardised interviews. Standardised medical examinations including collection of a non-fasting venous blood sample for the determination of biomarkers were performed. All procedures and routine laboratory methods have been described in detail elsewhere Serum levels of RANTES were determined using the Human CCL5/RANTES Quantikine ELISA kit from R&D Systems  as described http://www.hapmap.org) to obtain complete coverage of the CCL5 gene for a 9.2-kb locus of the CCL5 gene . All SNPs were in Hardy-Weinberg equilibrium (p>0.2). Genotyping was carried out with matrix-assisted laser desorption ionisation-time of flight analysis of allele-dependent primer extension products as reported Six single nucleotide polymorphisms (SNPs) were selected on the basis of data from the International HapMap project  and t tests on regression coefficients (SURVEYREG), respectively. In case of non-normality, t tests were carried out for log-transformed variables, and results were given as geometric means with antilogs of standard errors of the adjusted log-means.CCL5 genotypes and RANTES serum levels with incident coronary events were assessed by Cox proportional hazard models. The proportional hazards assumptions for the Cox models were tested for all models graphically before the analyses and demonstrated that proportional hazards can be assumed. The case-cohort design required correction of the variance estimation based on the sampling weights to give standard error estimates for the parameter estimates. Correction for standard errors was made using the method by Barlow CCL5 genotypes, the significance level was adjusted for multiple testing according to Bonferroni to 0.05/6 = 0.0083 for each SNP. All calculations were performed with SAS .The associations of CCL5 alleles with CAD were assessed by calculating unadjusted odds ratios (OR) with corresponding 95% CI and p values per specified allele. The nominal significance level was adjusted for multiple testing according to Bonferroni to 0.05/6 = 0.0083 for each SNP in this study. The level of genome-wide significance that corrected for all tests performed in the GWAS was set at 5×10−8.The transatlantic CARDIoGRAM  Consortium combines data from 14 genome-wide association studies (GWAS) comprising >22,000 cases with CAD and >60,000 controls, all of European ancestry The Athero-Express study is an ongoing biobank study with a longitudinal study design, comprising atherosclerotic plaques obtained from patients who underwent carotid endarterectomy (CEA) as described The collected atherosclerotic plaques were processed according to a standardised protocol The segments adjacent to the culprit plaque lesions were used for protein isolation. Segments were dissolved in Tris buffer according to a standardised protocol and quantified for the total amount of protein Baseline RANTES levels in plaques were statistically analysed by the non-parametric Mann Whitney-U test. The association of RANTES expression with categorised histological plaque characteristics was assessed using Spearman's correlation test. The association between plaque haemorrhage and RANTES expression was determined by the Mann Whitney U test.We used unadjusted Cox regression analyses to determine the association of RANTES plaque levels at baseline with cardiac outcome during the follow-up. In order to exclude potential confounding with respect to the association of RANTES plaque levels and cardiac endpoint, we performed multivariate analyses including all determinants that were significantly associated with RANTES expression levels at baseline. The statistical analyses were performed using SPSS 15.0 .Individuals who experienced a coronary event during the follow-up (cases) differed in most baseline characteristics from individuals without incident coronary events (non-cases). Cases were older, had a higher BMI, higher waist-to-hip ratio, higher blood pressure, more frequently a history of hypertension or diabetes, higher total and LDL cholesterol levels, lower HDL cholesterol levels, higher ratio of total to HDL cholesterol, higher systemic concentrations of CRP and IL-6, and were less frequently physically active. These data have been reported before RANTES serum levels did not differ significantly between cases and non-cases . Cox proportional hazard models showed that increasing tertiles of RANTES serum concentrations were neither associated with increased risk of coronary events after adjustment for age, sex and survey , model 1trend = 0.90) and 1.0, 1.04 (0.67–1.61) and 1.06 (0.68–1.66) for a follow-up time of five years (ptrend = 0.88) and thus very similar to results obtained from the complete follow-up period.In order to assess whether RANTES serum levels may be more informative for a shorter follow-up period, we repeated the analysis for coronary events occurring in the first three years  or in the first five years . HRs (95% CIs) for increasing tertiles were 1.0, 1.10 (0.62–1.98) and 1.03 (0.57–1.86) for a follow-up time of three years , and additional adjustment for this variable in model 3 had no impact on HRs (data not shown).CCL5 SNPs rs2107538, rs2280789, rs47966120 and rs3817655 had significantly lower RANTES serum levels (padditive≤3.1×10−8) than carriers of the major alleles in the MONICA/KORA subcohort, whereas no differences were found for rs2280788 and rs1065341 as reported previously Carriers of the minor alleles of the CCL5 SNPs, haplotypes were constructed. None of the common haplotypes with frequencies of ≥1% in the subcohort had a statistically significant impact on the risk of coronary events compared to the most frequent haplotype CCTATA  in the CARDIoGRAM study  and there was a trend toward higher RANTES levels in plaques with increased macrophage infiltration (p = 0.054), suggesting an association between high RANTES levels and an unstable plaque phenotype. We also found a trend for an association between RANTES levels and lipid content, but not with smooth muscle cell infiltration, collagen content, or presence of plaque haemorrhage .th percentile, 75th percentile] 44.86 [29.04–74.96] vs 57.77 [34.52–79.90] ng/µg; respectively; p = 0.12). When the event rates of individuals with high (above the median of 45.52 ng/µg) and low (<45.52 ng/µg) RANTES levels in plaques were compared, unadjusted Cox regression analysis revealed no association with higher event risk for higher RANTES levels  , 51 study participants (8.5%) reached a cardiac endpoint (cases). These included 31 individuals who died and 20 individuals who underwent a cardiac intervention due to coronary artery disease. RANTES plaque levels did not differ between individuals without and with events (median [25 = 0.16) , Table 8CCL5 SNPs were not associated with CAD in the large CARDIoGRAM study, and (iii) RANTES levels were associated with an unstable phenotype of carotid plaques, but no independent predictor of future coronary events in the Athero-Express study.Our study has three main findings: (i) RANTES serum levels were not associated with incident coronary events in the MONICA/KORA Augsburg case-cohort study, (ii) functional RANTES/reduced levels of RANTES plasma levels elevated RANTES levels predicted future cardiovascular events in a small sample of patients with unstable angina pectoris not predictive of future cardiovascular events Prospective studies on systemic RANTES levels and cardiovascular outcomes are very scarce and controversial. Whereas higher 2-year cardiac mortality in male patients undergoing coronary angiography has been found associated with CCL5 SNPs could serve as biomarkers for cardiovascular risk. Most data are available for the CCL5 SNP rs2107538 which is also known as −403GA. This SNP is likely to be functional as we showed in the MONICA/KORA cohort before that carriers of the minor allele had significantly lower RANTES serum levels In recent years, several candidate studies analysed whether CCL5 locus in recent GWAS that covered this genomic region Given the low statistical power of the aforementioned single studies it is of interest that associations with genome-wide significance have not been reported for SNPs in or near the CCL5 genotypes and incident coronary events from a population-based study. Due to limited statistical power we sought to substantiate our findings in the CARDIoGRAM consortium, currently the largest database on gene variants and CAD worldwide. As recently published, the CARDIoGRAM study is adequately powered to reveal associations at genome-wide significance level (p<5×10−8) between common SNPs and CAD even for allelic OR of <1.10 CCL5 genotypes and CAD were not nominally significant and far from genome-wide significance. It should be noted that in contrast to the prospective MONICA/KORA Augsburg case-cohort that evaluated risk factors for incident coronary events the CARDIoGRAM study investigated gene variants that are associated with prevalent CAD and thus does not represent a perfect replication sample. However, we believe that any major impact of RANTES genotypes on coronary risk should also have been reflected by an association with prevalent disease so that the CARDIoGRAM data add relevant information to the initial findings from the MONICA/KORA Augsburg case-cohort study.The data from the MONICA/KORA study represent the first report on associations between The MONICA/KORA Augsburg study also allowed for the evaluation of haplotypes as risk factors of coronary events. Our data indicate that rare haplotypes may indeed comprise variants that are associated with increased coronary risk. However, this study does not have the statistical power to analyse this aspect in more detail, and data on DNA sequences would be needed to identify defined haplotypes with potential relevance for pathogenesis and prediction. Therefore, this finding is hypothesis-generating at best and needs corroboration in further studies.CCL5 SNPs that we included in our study and the major haplotypes based on them may not be relevant biomarkers for the prediction of cardiovascular events.Taken together, our data strongly suggest that the six Data from mouse models demonstrate that RANTES and its receptor CCR5 are expressed in atheromas, that they play a causal role in atherogenesis in these models and that interference with leukocyte migration at this level may represent a promising therapeutic approach to prevent or delay atherosclerotic processes CCL5 gene expression has indeed been reported in atherosclerotic tissue from individuals with symptomatic carotid plaques  compared to CCL5 gene expression in iliac arteries from organ donors CCL5Higher Our findings from the Athero-Express study extend current knowledge on RANTES plaque levels as potential biomarker because it relates RANTES plaque levels and risk for coronary events in a prospective design. Although we observed a trend toward higher risk for higher RANTES plaque levels, this association was completely abolished when we accounted for several established cardiovascular risk factors in a multivariate analysis. However, it should be noted that we found an association between RANTES and an unstable plaque phenotype which is in line with previous studies investigating the potential role of RANTES in the development of cardiovascular disease. It is noteworthy that some of the classical cardiovascular risk factors such as male sex, diabetes or systemic CRP were not significantly associated with the risk for coronary events in the multivariate model. We hypothesise that both the low number of events (compared to studies like the MONICA/KORA Augsburg case-cohort study) and the fact that the study sample represents a high-risk group that differs from the general population may have contributed to this finding.CCL5 SNPs, RANTES serum levels and risk for coronary events. Its strengths include the population-based design, a long follow-up and a large number of cases. The Athero-Express study also represents the first study to test whether RANTES plaque levels predict cardiovascular outcome. The fact that the same cohort and the same methods allowed us to identify other prognostic proteins in carotid plaques such as fatty-acid binding protein, matrix metalloproteinase-8 and osteopontin as predictors of cardiovascular events underlines that the study design is indeed suitable to address our study question The combination of three independent studies to investigate different biological levels  of RANTES in order to assess their use as potential biomarkers for the risk of coronary events represents the main strength of our study. Our findings are based on study cohorts that have been used before to identify novel associations between inflammation-related proteins , gene variants (CARDIoGRAM) and cardiovascular risk. The MONICA/KORA study is the first population-based study to examine the relationship between In the MONICA/KORA Augsburg case-cohort study we measured RANTES in serum samples which is important to note because RANTES is released by platelets during the clotting process. Therefore, we cannot exclude that the analysis of platelet-poor or platelet-free plasma would have led to different results. Platelet count is only available in MONICA/KORA survey 3, so that we could not adjust for this potentially relevant confounder in our analysis. A general limitation of current studies on RANTES and disease outcomes, including our own (MONICA/KORA and Athero-Express), is the fact that commercially available assays measure total RANTES protein, although variant forms including truncated proteins and proteins with different oxidation, glycation and glycosylation patterns exist. More research is needed to reveal whether these variants may be more informative in prospective studies than total RANTES levels CCL5 genotypes and (iii) RANTES content in carotid plaques with coronary artery disease or incident coronary events in our cohorts. These data indicate that RANTES/CCL5 gene variants and protein levels may not be novel biomarkers for the risk of coronary events in humans. However, our study could not address the questions whether measurement of RANTES in platelet-poor plasma or measurement of variant forms of RANTES proteins  may be more informative for cardiovascular risk prediction than the approaches we pursued. Moreover, we cannot exclude that the measurement of RANTES may be of prognostic benefit in specific patient groups.We found an association between high RANTES plaque levels and an unstable plaque phenotype, but no associations of (i) RANTES serum levels, (ii) Text S1Members and affiliations of the CARDIoGRAM Consortium are listed in the Supplementary Information.(DOC)Click here for additional data file.Text S2Funding information and competing interests of the CARDIoGRAM Consortium.(DOC)Click here for additional data file.Table S1Baseline demographic, lifestyle and clinical characteristics of the study participants without and with incident coronary event during follow-up (MONICA/KORA Augsburg case-cohort study).(DOC)Click here for additional data file.


A new approach is described here to predict ubiquitinated substrates of the E3 ubiquitin ligase, TRAF6, which takes into account its interaction with the scaffold protein SQSTM1/p62. A novel TRAF6 ubiquitination motif defined as [–(hydrophobic)–k–(hydrophobic)–x–x–(hydrophobic)– (polar)–(hydrophobic)–(polar)–(hydrophobic)] was identified and used to screen the TRAF6/p62 interactome composed of 155 proteins, that were either TRAF6 or p62 interactors, or a negative dataset, composed of 54 proteins with no known association to either TRAF6 or p62. NRIF (K19), TrkA (K485), TrkB (K811), TrkC (K602 and K815), NTRK2 (K828), NTRK3 (K829) and MBP (K169) were found to possess a perfect match for the amino acid consensus motif for TRAF6/p62 ubiquitination. Subsequent analyses revealed that this motif was biased to the C-terminal regions of the protein (nearly 50% the sites), and had preference for loops (~50%) and helices (~37%) over beta-strands (15% or less). In addition, the motif was observed to be in regions that were highly solvent accessible (nearly 90%). Our findings suggest that specific Lysines may be selected for ubiquitination based upon an embedded code defined by a specific amino acid motif with structural determinants. Collectively, our results reveal an unappreciated role for the scaffold protein in targeting ubiquitination. The findings described herein could be used to aid in identification of other E3/scaffold ubiquitination sites. The process of signal transduction is dependent upon specific protein-protein interactions, with a small number of proteins – called 'hubs' possessing the ability to interact with many different partners to form multimeric signaling complexes referred to as signalsomes. These hubs mediate interactions by their modular protein domains that confer specific binding activity to their interacting partners. Protein p62 contains several structural motifs that allow it to function as a hub for protein-protein interactions. These motifs include an acidic interaction domain (AID/ORCA/PC/PB1) that binds the aPKC, a ZZ finger, a binding site for the RING finger protein TRAF6, two PEST sequences, and the UBA domain .Two TRAF6/p62 substrates, tyrosine receptor kinase A (TrkA)  and neurThere are approximately one thousand reported E3 ubiquitin ligases in eukaryotes. The preferred substrates for most of these ligases remain largely unknown. Moreover, it remains unclear how among the many Lysines (K) found in an ubiquitinated protein only a few are selected for post-translational modification. Like all signal transduction processes, ubiquitination is a result of specific protein-protein interactions and biochemical reactions occurring at the sub-cellular locales, mediated by modular protein domains. Furthermore, E3 ligases and scaffold proteins interact with numerous binding partners through their multi-domain structures. SQSTM1/p62 is one such scaffold that is known to be a crucial for bridging the E3 ligase, TRAF6 and its substrates while at the same time providing specificity for the enzyme-substrate reactions. We propose that the scaffold plays an integral component in activation of the E3 and may target the specificity of the ubiquitination reaction . Therefoin vivo and in vitro methods have been employed to identify ubiquitinated substrates and their sites, including proteome-scale analyses of the substrates . This program searches pre-defined SLiMs (Short Linear Motifs) in a protein sequence database and allows individual residues to vary as defined by the user. We utilized this program to search for perfect (10 amino acid) matches which we then compared to the results provided by our MotifFinder algorithm.To verify the accuracy of our search results both the experimental and negative datasets were submitted to SLiMSearch  were created for the sets of 7 (perfect fit) and 6 (near fit) amino acid motifs identified. This program generates a display of the amino acid distribution surrounding the core motif Lysine. The height of each letter in a stack is proportional to its frequency at that position in the motif set. Letters were further sorted with the most frequent amino acid on top.PSIPRED ,24 was uThe 155 proteins in the experimental dataset ranged in length from 103 to 4,572 amino acids with a median length of 565 amino acids (mean = 755.9 ± 609.5). A total of 7,592 Lysine residues (range = 1 – 260 per sequence) were found within the 155 sequences. This produced a median count of 33 Lysines per protein with one Lysine residues occurring on average every 19.7 residues (median = 17). The 54 protein negative dataset contained fewer total Lysines  but median values were similar between the two datasets. The negative dataset proteins ranged from 147 to 2,115 amino acids in length . Lysines ranged from 4 to 151 per protein with a median count of 29 and an average of one Lysine per 18.7 residues. Of the 7,592 total Lysines in the experimental dataset, 7,494 (98.7%) were located at positions within the sequence that could allow for a full 10 amino acid motif. In the negative dataset, 2,209 of 2,220 total Lysines (99.5%) were similarly available.P >0.3 for all tests).As expected given the large number of motif combinations analyzed , results from the motif searches yielded a wide distribution of matches in the regions surrounding individual Lysines Figure . Based oBecause low accuracy hits dominated all statistical comparisons and potentially masked meaningful differences, we next evaluated a subset of the distributions corresponding to match values > 4 Figure  were onlWhile informative, direct comparisons between the experimental and negative data sets did suffer from limitations. There existed an obvious bias within the experimental dataset because it was purposely assembled with proteins known or suspected to contain some form of our putative motif. This is not an unusual situation as many motif discovery studies use as input, sets of sequences hypothesized to contain a biologically important sequence pattern. These sequences are then searched for patterns that are unlikely to occur by chance. We attempted to estimate the probability of encountering perfect or near perfect matches within our datasets by chance using a randomized data approach. For this the frequency of amino acids found in the experimental dataset was used to randomly generate 999 test “proteins”. These 999 artificial proteins were then screened using the MotifFinder program. Despite its substantially larger size, all summary statistics for the randomized data were consistent with both the experimental and negative results. As examples, skewness (0.31) and kurtosis (-1.8) were effectively identical to the empirical datasets. In addition, the overall match frequencies again exhibited a strong fit to a discrete uniform distribution model Figure  and the -7 . A similar pattern was found for the near-perfect matches (6 variable amino acids) in the experimental dataset where 26 matches were observed compared to 19.8 predicted. As mentioned, it is obvious that bias did exist within the experimental dataset relate to protein selection, however even if the probability estimates are inflated by 1 or more orders of magnitude the net conclusion remains; the total numbers of perfect and near-perfect motif matches observed were well beyond random expectations.Using the rate of occurrence of perfect matches (7 variable amino acids) in the randomized dataset as a crude estimator, we made a first approximation of the probability of encountering 8 motifs within 155 proteins. Based an initial expectation of 1 match per 999 proteins, the joint probability of finding 8 perfect motifs at random within 155 proteins was estimated to be extremely small at ~ 4.0 x 10Collectively, the results provided by comparisons of the experimental, negative and randomized datasets provided evidence in support of our hypothesis that a Lysine-centered ubiquitination motif might exist among TRAF6 / p62 interactors. The overall similarity between the negative and experimental match distributions argues that the selection of potential interacting proteins for the experimental group did not unduly bias the overall distribution pattern. Conversely, the presence of probabilistically unlikely large numbers of perfect and near perfect motif matches within the related experimental group proteins suggested that the motif is by itself or in association with an amino acid sequence of conserved biological importance.Next, we sought to identify amino acids that might play a critical role in ubiquitination site selection and investigated whether there were preferences for certain amino acids near the target ubiquitinated Lysines. This analysis focused on the well-defined proteins from Group I of the experimental dataset. Notably, when we examined the surrounding residues of the validated ubiquitinated Lysine with amino acids conserved at 7 variable positions in our hypothesized motif (perfect hit), we observed an enrichment of small residues (G/A) on the either side of the target site and high frequency of Valine at position 4, Leucine at position 6, and Aspartic Acid at position 7 Figure . A closeBecause post-translational modifications tend to be concentrated within specific structural regions of a protein, we further investigated structural constraints of the predicted Lysines. Only predicted Lysines from highly positive (conserved at 6 or 7 variable sites) motif sites were included in this analysis. These Lysines were classified as a high probability group. There was a total of 30 proteins in this category, 25 from the experimental dataset and 5 from the negative dataset. Eight of those 30 proteins had more than one predicted TRAF6/p62 ubiquitination site, such as one 7 match plus one 6 match motif. NRIF K19), TrkA K485), TrkB (K811), TrkC (K602 and K815), NTRK2 (K828), NTRK3 (K829) and MBP (K169) were found to possess a perfect match for the amino acid consensus motif for TRAF6/p62 ubiquitination . The second most common secondary structure was an alpha-helix . This was in agreement with previously reported findings on preferred in vivo ubiquitination sites in yeast proteins [We further sought to incorporate sequence information as well as information from sequence derived structural features of these proteins into the validation process. To do so, four potential structural features of the predicted high probability sites were evaluated: secondary structure, relative distribution within the protein, solvent accessibility, and the intrinsic disorder within the protein domain. Structural analysis was conducted using secondary structure, protein domain, and disorder prediction algorithms . Our resx Figure . Alpha hproteins . As an eproteins .in vitro and in vivo with the three C-terminal Lysines  essential for activation of IRF7 [The highest possible resolution for investigating structure–function relationships is that of individual residues and their corresponding microenvironments . To prov of IRF7 ,32. Simi of IRF7 . There w of IRF7 .Recent studies of all post-translationally modified proteins documented in Swiss-Prot have shown that most reversible modifications are found on protein surfaces . UbiquitTo study the subcellular distribution of the predicted TRAF6/p62 substrates, compartmentalization of the proteins in both the datasets was examined  was slightly higher than the total percentage of nuclear proteins (25%) found in the dataset . This unusually high conservation suggests that the ubiquitination of these sites may potentially be conserved across a wide spectrum of life forms, although this remains untested. A high degree of conservation among proteins that are ubiquitinated also suggests that they may have arisen early in the course of evolution. However, a significant number of ubiquitination sites differ in the ubiquitome and the extent of homology is not uniform because of the high diversity among the proteins. Nevertheless, evidence of conservation does suggest that ubiquitination is in each case indispensable for protein function, which is in turn essential for regulating cellular function. These highly conserved essential ubiquitination events may reflect how early forms of life used protein ubiquitination in specific housekeeping cellular functions. Interestingly, our results indicated that although the surrounding sequence regions may diverge, the critical residues remain conserved. Similar whole genome-scale studies have shown that 2,683 potential SUMO substrates are conserved between human and mouse based on pattern recognition and phylogenetic conservation [We sought to further validate the biological relevance of our hypothetical ubiquitination motif by examining it in an evolutionary context. For this analysis a high-confidence set of TRAF6/P62 substrates, those with perfect matches to our putative motif, were selected for alignment. These proteins were TrkA, TrkB, TrkC, NRIF, NTRK2, NTRK3 and MBP Table . To checervation . In anotervation . This reervation , thus poervation .Most proteins in cells undergo post-translational modifications giving them structural and functional diversity for diverse roles in biological processes. Experimental identification and validation of posttranslational modifications (PTMs) is labor-intensive task and can be expensive in the absence of prior knowledge concerning PTMs. Analyzing the ‘ubiquitome’ is one of the most exciting and challenging tasks in current proteomics research. A lack of curated datasets of ubiquitinated proteins presents the ultimate limiting factor in studying substrate selection mechanism in ubiquitination making it difficult to evaluate, and compare target sites. As more and more ligases are identified there exists an urgent need to rapidly and precisely identify enzyme-specific substrates to decode their selectivity and specificity . Computain silico could yield more defined results. These prediction tools should be closely integrated into the interpretation of proteomic experiments.In this study, a computational tool was developed to predict Lysine ubiquitination sites from sequences using MATLAB programs and online prediction software. As more validated ubiquitinated sites from experimental data become available, and appropriate changes are made based on the available site data, further predictions can be made. The inclusion of structural information to improve the prediction tools could be another way to enhance the prediction performance as ubiquitination is an enzymatic process, and the interactions between target sites and enzymes concerned should be structurally satisfied. The model that we propose herein could be applied to other E3 Ub ligases that are known to employ scaffold proteins to aid in their substrate selection process. One such example is DYRK2–EDVP E3 ligase complex where DYRK2 not only is it serves as adaptor for assembly of the active Ub ligase complex, but it also phosphorylates its substrate and primes the substrate for degradation . Thus, uin vivo ubiquitination sites, prediction algorithms can be fine tuned and improved. A conserved motif that serves as a recognition determinant for TRAF6/p62 enzyme complex was identified. Studies have shown some structural preferences for ubiquitination of targeted proteins such as preferred choice of Lysines in loops and/or for easily accessible Lysines within α–helical regions [Here we identified the interactome of the active enzyme complex and studied the verified substrates for characterization of target sites to predict substrates. As proteomics methods identify additional  regions . Our finA total of 42 high probability TRAF6/p62 ubiquitination sites in 30 proteins were identified by our prediction approach. Structural analysis of these predicted TRAF6/P62 substrates showed that the predicted ubiquitination sites were biased towards the C-terminal domain of the protein ,32. SecoFindings from this study indicate that the ubiquitination site prediction is closely correlated with the amino acid property around the ubiquitination site. Our approach makes it possible to find putative novel ubiquitination sites that have not (yet) been experimentally identified. Thus, in the absence of experimental data, the prediction of novel ubiquitination sites can be taken as the first method of an experimental design for uncovering functionality of any protein of interest and elucidating its involvement in certain signaling cascades. Methods for computational prediction of peptide specificities and identification of substrates could be enhanced by combining different approaches and integrating various types of information. In addition, the prediction approach taken here combined with experimental verification will propel our understanding of ubiquitination mechanisms. Thus, a combination of both a computational and experimental approach could propel our understanding of ubiquitination dynamics into a new phase.bona fide in vivo ubiquitination site has been verified by mutagenesis along with verification of K63-ubiquitin linked chains for sites observed in TrkA, B, C, and NRIF [In conclusion, NRIF (K19), TrkA K485), TrkB (K811), TrkC (K602 and K815), NTRK2 (K828), NTRK3 (K829) and MBP (K169) were found to possess a perfect match for the amino acid consensus motif for TRAF6/p62 ubiquitination. The ability of a given Lysine residue to serve as a 85, TrkB AID: Atypical PKC-interaction domain; aPKC: Atypical protein kinase C; ELM: Eukaryotic linear motifs; FA: Formic acid; K: Lysine; KO: Knock-out; MBP: Myelin basic protein; NEMO: NF-kappa-B essential modulator; NRIF: Neurotrophin receptor interacting factor; NTRK2: Neurotrophic tyrosine receptor kinase 2; NTRK3: Neurotrophic tyrosine receptor kinase 3; PB1: Phox and Bem1; PEST: Proline Glutamate Serine Threonine; PTM: Post translational modification; RING: Really Interesting New Gene; SUMO: Small ubiquitin-related modifier; TAB2: TGF-beta activated kinase 1/MAP3K7 binding protein 2; TAB3: TGF-beta activated kinase 1/MAP3K7 binding protein 3; TRAF6: Tumor necrosis factor receptor associated factor 6; TrkA: Tropomyosin receptor kinase A; TrkB: Tropomyosin receptor kinase B; TrkC: Tropomyosin receptor kinase C; Ub: Ubiquitin; UBA: Ubiquitin-associated domain; ZIP: Zeta protein kinase C interacting protein; ZZ: ZZ-type Zinc finger domainThe authors declare that they have no competing interests.TSJ identified the consensus motif, assembled the protein datasets, compiled the search results, conducted the secondary structure analyses, conducted the SLiMSearch analysis, generated the randomized protein dataset, conducted parts of the statistical analyses and drafted the manuscript. MCW developed and programmed the MotifMaker and MotifFinder algorithms, conducted the motif searches using these programs, developed the probability estimates and participated in drafting of the manuscript. MWW conceived the study, led the project design, provided technical direction for the research and participated in drafting of the manuscript. All authors read and approved the final manuscript.


Angiogenesis is the generation of mature vascular networks from pre-existing vessels. Angiogenesis is crucial during the organism' development, for wound healing and for the female reproductive cycle. Several murine experimental systems are well suited for studying developmental and pathological angiogenesis. They include the embryonic hindbrain, the post-natal retina and allantois explants. In these systems vascular networks are visualised by appropriate staining procedures followed by microscopical analysis. Nevertheless, quantitative assessment of angiogenesis is hampered by the lack of readily available, standardized metrics and software analysis tools. Non-automated protocols are being used widely and they are, in general, time - and labour intensive, prone to human error and do not permit computation of complex spatial metrics. We have developed a light-weight, user friendly software, AngioTool, which allows for quick, hands-off and reproducible quantification of vascular networks in microscopic images. AngioTool computes several morphological and spatial parameters including the area covered by a vascular network, the number of vessels, vessel length, vascular density and lacunarity. In addition, AngioTool calculates the so-called “branching index” (branch points / unit area), providing a measurement of the sprouting activity of a specimen of interest. We have validated AngioTool using images of embryonic murine hindbrains, post-natal retinas and allantois explants. AngioTool is open source and can be downloaded free of charge. The formation of new blood vessels from a pre-existing vascular plexus is called angiogenesis. This is a complex process that depends on tight co-ordination of several important cellular activities, including proliferation, differentiation and migration in vivo analysis of angiogenesis to earlier embryonic stages, or demands lengthy breeding of conditional knock-out systems to allow inducible deletion at later stages.Recent years have seen the identification of many important regulators of angiogenesis. Due to the vital role angiogenesis plays during embryonic development, knocking out such regulators often leads to embryonic lethality, typically from mid-gestation. This restricts In vitro assays rely on cultured endothelial cells and assay a particular aspect of endothelial cell biology such as cell motility in a transwell or tube formation in a three-dimensional matrix. In vivo assays give a wealth of information on many aspects of endothelial cell biology and are particularly useful when genetically altered model organisms are being examined. A well-established model system in the mouse, which allows characterization of developmental sprouting angiogenesis in embryos from E10, is the embryonic hindbrain in vivo experimental system today. An advantage of retinal angiogenesis is that the effects of activators or inhibitors of proteins of interest on angiogenesis can be analysed after intravitreal or systemic administration of such substances Many experimental systems allow studying different aspects of angiogenesis. These can be broadly divided into two groups. ex vivo analysis of developmental angiogenesis even in the majority of embryonically lethal mutants, given they are taken before the onset of embryonic wasting . Growing in a tissue culture incubator under defined conditions, explant cultures avoid potentially deleterious external influences, such as placental defects, heart defects or hypoxia. Two types of explants are commonly used. Para-aortic splanchnopleural explants grow over a period of two weeks on a layer of OP9 feeder cells and allow distinguishing between vasculogenesis and angiogenesis defects in vivo assays described above, allantois explants are useful for the analysis of several endothelial cell parameters, including cell proliferation, cell migration and sprouting.Embryonic explants, taken at an early developmental stage  allow for immediate Quantitative analysis of the vascular networks in the above systems is not standardized and tends to be done in a non-automated fashion, making the analysis process labour intensive and prone to human error. There is a lack of easily accessible and user-friendly software tools designed to perform comprehensive quantitative analysis of vascular networks. To remedy this, we designed AngioTool, a software for the quantitative analysis of angiogenesis with user-friendly interface and analysis flow. AngioTool computes several morphological and spatial parameters including the overall size of the vascular network, total and average vessel length, vascular density as well as lacunarity, which characterizes vessel non-uniformity by assessing the variation in foreground and background pixel mass densities across an image. Lacunarity is able to characterise oddities found when vessel organisation has been disturbed significantly, and may be useful to characterise and quantitatively analyze vascular networks in drug treated specimens or pathological vasculature as has been shown in patients'lung tumours where this parameter correlates with stages of aggressiveness Arap3 mutant embryos, we devised a computational analysis method, which evaluated the “branching index”, the number of vessel branch points per unit area. We have since developed this into a standalone application named AngioTool, which allows performing comprehensive quantitative assessments of angiogenesis in a visually user-friendly fashion. AngioTool computes the branching index and several other morphometric parameters such as total explant area, average and total vessel length, number of endpoints, vessel density, and average lacunarity.We previously analysed the function of ARAP3, a PI3K regulated dual GTPase activating protein, in angiogenesis AngioTool's graphical user interface (GUI) contains a top row with buttons and two tabs containing controls to run analysis and to customize the numeric and graphical output . The basWe tested AngioTool using three different sets of images: murine embryonic hindbrains, post-natal retinas and allantois explants. All of these images are available as supplemental files.Endomucin-stained E11.5 embryonic hindbrains  since they are normalised to the area of the analysed network. To our minds, these features make AngioTool particularly useful.To ascertain the reproducibility of the results obtained, we routinely compared analyses performed by different users. Trends and ratios in the results obtained from user independent analysis were consistent throughout, demonstrating the robustness of the software. In http://rsbweb.nih.gov/ij/) and several ImageJ plugins, the Mines Java Toolkit , Apache POI , and JIDE Common Layer  libraries. AngioTool is open source and is distributed as a Windows executable.AngioTool is written in Java and leverages the open source ImageJ .R microscope (Olympus).Postnatal P6 eyes were briefly fixed with 2% PFA in PBS at 4°C, then retinas were dissected and stored in methanol at -20°C. Immediately prior to staining retinas were re-fixed in 4% PFA for 20 minutes. PBS-washed retinas were stained with biotinylated isolectin B4 (Vectorlabs) followed by streptavidin-alexafluor 568 (Molecular Probes) and flat mounted for epifluorescence analysis on a CellR, Olympus). Images were assembled using Photoshop software.Hindbrains were dissected clean from surrounding tissue followed by fixation steps, dehydration and rehydration steps as described 2 (Sanyo). Inhibitors were made up as per manufacturer's instructions and added into the growth medium when setting up the culture. Control explants were vehicle treated. Inhibitors were obtained from Calbiochem and used at the following concentrations: LY294002, 10 µM; Y-27632, 10 µM. Allantois explants were fixed with 4% paraformaldehyde, followed by wholemount staining with rat anti-VE-cadherin antibody (BD Biosciences) together with rat anti-endomucin. Overlapping pictures of the explants were taken with an inverted epifluorescence microscope (Olympus CellR), making sure that no area was over-or underexposed. Photographs were assembled using the panorama function of Photoshop software.Allantoises were dissected from E8.5 embryos and cultured for 18 hours in 8-well µ-slides (Ibidi) coated with bovine fibronectin (Sigma) in DMEM (Gibco) supplemented with 10% fetal calf serum (Invitrogen) in a humidified tissue culture incubator at 37°C and 5% COFigure S1Microscopic hindbrain image used for analysis shown in .(TIF)Click here for additional data file.Figure S2Microscopic hindbrain image used for analysis shown in .(TIF)Click here for additional data file.Figure S3Microscopic hindbrain image used for analysis shown in .(TIF)Click here for additional data file.Figure S4Microscopic retina image used for analyses shown in .(TIF)Click here for additional data file.Figure S5Microscopic retina image used for analyses shown in .(TIF)Click here for additional data file.Figure S6Microscopic retina image used for analyses shown in .(TIF)Click here for additional data file.Figure S7Microscopic retina image used for analyses shown in .(TIF)Click here for additional data file.Figure S8Microscopic image of a DMSO treated control allantois explant used for analysis shown in  and .(TIF)Click here for additional data file.Figure S9Microscopic image of a DMSO treated control allantois explant used for analysis shown in  and .(TIF)Click here for additional data file.Figure S10Microscopic image of a DMSO treated control allantois explant used for analysis shown in  and .(TIF)Click here for additional data file.Figure S11Microscopic image of a DMSO treated control allantois explant used for analysis shown in  and .(TIF)Click here for additional data file.Figure S12Microscopic image of a DMSO treated control allantois explant used for analysis shown in  and .(TIF)Click here for additional data file.Figure S13Microscopic image of a DMSO treated control allantois explant used for analysis shown in  and .(TIF)Click here for additional data file.Figure S14Microscopic image of a DMSO treated control allantois explant used for analysis shown in  and .(TIF)Click here for additional data file.Figure S15Microscopic image of a DMSO treated control allantois explant used for analysis shown in  and .(TIF)Click here for additional data file.Figure S16Microscopic image of a LY294002 treated allantois explant used for analysis shown in  and .(TIF)Click here for additional data file.Figure S17Microscopic image of a LY294002 treated allantois explant used for analysis shown in  and .(TIF)Click here for additional data file.Figure S18Microscopic image of a LY294002 treated allantois explant used for analysis shown in  and .(TIF)Click here for additional data file.Figure S19Microscopic image of a LY294002 treated allantois explant used for analysis shown in  and .(TIF)Click here for additional data file.Figure S20Microscopic image of a LY294002 treated allantois explant used for analysis shown in  and .(TIF)Click here for additional data file.Figure S21Microscopic image of a LY294002 treated allantois explant used for analysis shown in  and .(TIF)Click here for additional data file.Figure S22Microscopic image of a LY294002 treated allantois explant used for analysis shown in  and .(TIF)Click here for additional data file.Figure S23Microscopic image of a LY294002 treated allantois explant used for analysis shown in  and .(TIF)Click here for additional data file.Figure S24Microscopic image of a water treated control allantois explant used for analysis shown in .(TIF)Click here for additional data file.Figure S25Microscopic image of a water treated control allantois explant used for analysis shown in .(TIF)Click here for additional data file.Figure S26Microscopic image of a water treated control allantois explant used for analysis shown in .(TIF)Click here for additional data file.Figure S27Microscopic image of a water treated control allantois explant used for analysis shown in .(TIF)Click here for additional data file.Figure S28Microscopic image of a water treated control allantois explant used for analysis shown in .(TIF)Click here for additional data file.Figure S29Microscopic image of a water treated control allantois explant used for analysis shown in .(TIF)Click here for additional data file.Figure S30Microscopic image of a water treated control allantois explant used for analysis shown in .(TIF)Click here for additional data file.Figure S31Microscopic image of a Y27632 treated allantois explant used for analysis shown in .(TIF)Click here for additional data file.Figure S32Microscopic image of a Y27632 treated allantois explant used for analysis shown in .(TIF)Click here for additional data file.Figure S33Microscopic image of a Y27632 treated allantois explant used for analysis shown in .(TIF)Click here for additional data file.Figure S34Microscopic image of a Y27632 treated allantois explant used for analysis shown in .(TIF)Click here for additional data file.Figure S35Microscopic image of a Y27632 treated allantois explant used for analysis shown in .(TIF)Click here for additional data file.Figure S36Microscopic image of a Y27632 treated allantois explant used for analysis shown in .(TIF)Click here for additional data file.Figure S37Microscopic image of a Y27632 treated allantois explant used for analysis shown in .(TIF)Click here for additional data file.Figure S38Microscopic image of a Y27632 treated allantois explant used for analysis shown in .(TIF)Click here for additional data file.Figure S39Microscopic image of a Y27632 treated allantois explant used for analysis shown in .(TIF)Click here for additional data file.Figure S40Microscopic image of a Y27632 treated allantois explant used for analysis shown in .(TIF)Click here for additional data file.


The limited ability to vascularize and perfuse thick, cell-laden tissue constructs has hindered efforts to engineer complex tissues and organs, including liver, heart and kidney. The emerging field of modular tissue engineering aims to address this limitation by fabricating constructs from the bottom up, with the objective of recreating native tissue architecture and promoting extensive vascularization. In this paper, we report the elements of a simple yet efficient method for fabricating vascularized tissue constructs by fusing biodegradable microcapsules with tunable interior environments. Parenchymal cells of various types,  were suspended in glycosaminoglycan (GAG) solutions  and encapsulated by forming chitosan-GAG polyelectrolyte complex membranes around droplets of the cell suspension. The interior capsule environment could be further tuned by blending collagen with or suspending microcarriers in the GAG solution These capsule modules were seeded externally with vascular endothelial cells (VEC), and subsequently fused into tissue constructs possessing VEC-lined, inter-capsule channels. The microcapsules supported high density growth achieving clinically significant cell densities. Fusion of the endothelialized, capsules generated three dimensional constructs with an embedded network of interconnected channels that enabled long-term perfusion culture of the construct. A prototype, engineered liver tissue, formed by fusion of hepatocyte-containing capsules exhibited urea synthesis rates and albumin synthesis rates comparable to standard collagen sandwich hepatocyte cultures. The capsule based, modular approach described here has the potential to allow rapid assembly of tissue constructs with clinically significant cell densities, uniform cell distribution, and endothelialized, perfusable channels. Fabrication of 3D constructs that promote cell-cell interaction, extra cellular matrix (ECM) deposition and tissue level organization is a primary goal of tissue engineering In conventional preformed scaffolds, the cell viability depends on diffusion of oxygen, nutrients and growth factors from the surrounding host tissues, and it is limited to 100–200 microns thickness at cell densities comparable to that of normal tissues The bioinspired modular tissue engineering approach has emerged in recent years as a promising fabrication strategy to address the common shortcomings of a preformed scaffold by assembling tissue constructs from the bottom up Here we report the development of a simple yet efficient method for assembling tissue prototypes with embedded, endothelialized channels by fusing microscale capsules. Our method of cell encapsulation was previously developed for perfusion culture of highly metabolic cells All chemical and culture reagents were purchased from Sigma-Aldrich unless mentioned otherwise. The human trophoblast cell line HTR-8/SVneo8 viable cells per liver. Type I collagen was isolated from Sprague dawley rat tail tendons as previous described For co-culture studies, vascular smooth muscle cells (SMCs) were isolated from rat aorta and endothelial cells (AECs) were isolated from sheep aorta using established enzymatic procedures Harvesting of rat hepatocytes and aortic smooth muscle cells for culture was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The cell isolation protocol was approved by the Animal Investigation Committee of Wayne State University (Protocol Number: A-07-16-10). Surgery and liver perfusion were performed under ketamine/xylazine anesthesia, and all efforts were made to minimize suffering.Streptococcus equii, molecular weight 1500–1800 kDa (Sigma); dextran sulfate sodium salt, molecular weight ∼500 kDa (SCBT); heparin sodium salt from porcine intestinal mucosa, molecular weight 17–19 kDa (Celsus); carboxymethylcellulose sodium salt, molecular weight 250 kDa (Sigma); polygalacturonic acid sodium salt (Sigma) and collagen type-I isolated from Sprague Dawley rat tail tendons (Invitrogen).The materials used in preparing our microcapsules and modular scaffolds were: chitosan from crab shells, molecular weight ∼600 kDa (Sigma); chondroitin 4-sulfate sodium salt from bovine trachea, molecular weight ∼50–100 kDa (Sigma); hyaluronic acid sodium salt from Aqueous solutions of the polyanions (chondroitin 4-sulfate (CSA), carboxymethylcellulose (CMC), hyaluronic acid (HA), polygalacturonic acid (PGA)) were prepared in a HEPES-sorbitol buffer containing: 0.4 g/L KC1, 0.5 g/L NaC1, 3.0 g/L HEPES-sodium salt, and 36 g/L sorbitol, pH 7.3. Polyanion solutions were sterilized by autoclaving at 121°C. The two formulations of polyanionic solutions studied for capsule formation were: (a) 4 wt% CSA/1.5 wt% CMC and (b) 1.0 or 1.5 wt% HA. To prepare the polycationic solution, chitosan powder was suspended in water (3 g in 250 ml) and autoclaved at 121°C. Under sterile conditions, 0.6 ml of glacial acetic acid was added to the aqueous suspension and stirred for 4 hours to partially dissolve the chitosan. Likewise, 19 g of sorbitol was autoclaved in 250 ml of water and then mixed with the chitosan solution. Undissolved chitosan was removed by centrifugation at 500 G. PGA (0.1 wt%) in HEPES-sorbitol buffer was used for surface stabilization of capsules. For capsule experiments employing collagen, cold collagen-I solution was diluted to 2 mg/ml in 1 mM HCl, and then neutralized with 10X DMEM (9∶1 ratio). Normal saline (0.9 wt% NaCl) was used for capsule washing immediately after formation.Cells were encapsulated in microcapsules produced by polyelectrolyte complexation between cationic chitosan and polyanions as described in detail previously The interior environment of the capsules could be enhanced with collagen gel or adhesion surface-providing microcarriers when desired. For capsules with an internal collagen matrix, chilled Type I collagen solution (1 mg/ml in 1 mM HCL) was neutralized with 10X DMEM in a 9∶1 ratio, and mixed with an equal volume of double strength polyanionic solution (e.g. 8% CSA/3% CMC). Cells were then suspended in this mixture instead of the regular polyanion solution, and capsules were made as described previously. For microcarrier co-encapsulation, PBS swelled microcarriers were suspended along with cells in normal strength polyanionic solution at a volume ratio of 0.5∶1 (packed cells+microcarriers:polyanion solution). The suspension was then dispensed as droplets to generate capsules as described above. Capsules enhanced with interior collagen or microcarriers were subjected to similar washing and surface stabilization steps as described above prior to culture.6 cells per ml of capsules and incubated at 37°C for 60 minutes with gentle resuspension every 10 min. After incubation, the seeded capsules were transferred to bioreactor chambers or tissue culture dishes for further experiments.Capsules were coated with an adsorbed layer of Type I collagen prior to externally seeding endothelial cells. For coating collagen on the outer surface, non-surface stabilized capsules  were washed in dilute acidic collagen solution (0.2 mg/ml of collagen in 1 mM acetic acid) for 1–2 min and then equilibrated with culture medium for 30 mins. Capsules that had been previously surface stabilized with PGA, were first washed with dilute chitosan solution (0.06% chitosan) prior to the dilute collagen wash. The equilibration culture medium was then removed and an endothelial cell suspension (HUVECs or AECs) in medium was added to settled capsules in a 50 ml centrifuge tube. Cells were seeded at a density of 10The permeability of capsule walls was studied fluorometrically by measuring the rate of diffusion of tetramethylrhodamine-labelled bovine serum albumin (BSA-TMR) from the capsules, as detailed before A precise number of capsules  of similar size from each formulation were counted out and equilibrated in HBSS (pH 7.4) containing 2.5 mg/ml BSA  at 35–50 capsules per cuvette. The HBSS volume was made up to 3 ml and the cuvettes were sealed and mixed horizontally on a linear shaker at 100 rpm. The outward diffusion of BSA was followed by measuring the fluorescence of the external HBSS at exitation/emission wavelengths of 541/572 nm at regular time intervals for 3 h. The BSA concentration was determined using a standard curve covering the range of 0–50 µg/mL total albumin. The overall mass-transfer coefficient for diffusion across the capsule wall (K) was calculated by solving the differential equation obtained through an unsteady state mass balance on the external solution r (V+NVc)); V and Vc are the volume of external solution and volume of capsules, respectively; N is the number of capsules; A is the total surface area (A  =  N*surface area of single capsule); C is the concentration of solute in external solution; Co is the initial extracapsular concentration; Cc is the concentration of solute in the capsules; C∞ is the initial intracapsule concentration; Cr is the final concentration after end of 3 hours; and t is time. The solution to the Where: K is the overall mass transfer coefficient for membrane diffusion; M is the total mass of solute present in the cuvette  vs. time and determining the slope of the linear portion of the curve by linear regression. The intrinsic permeability, P, of each capsule wall was determined from the relation:Where δ is the thickness of the capsule wall.Individual capsules were fused into 3D constructs in one of two ways. In the first method, freshly formed capsules were fused by allowing them to sit in contact with each other after the second saline wash, but before the surface stabilizing PGA wash. Freshly formed capsules were washed once with normal saline and then transferred in saline to a cylindrical mold with a 250 micron mesh at the base. Capsules were allowed to settle within the mold and held stationary for 2–3 minutes to allow inter-capsule adhesion. The excess saline was then drained and the capsule surfaces in the fused construct were then stabilized by briefly rinsing with saline, followed by a diluted polyanion solution (i.e. 0.1% PGA or 0.4% CSA/0.15% CMC), followed by a final PBS rinse.In the second fusion method, previously stabilized and cultured capsules were first reloaded with a polyanion by incubation in a diluted polyanion solution (0.1% heparin or 0.4% CSA/0.15% CMC). The capsules were then transferred to a cylindrical mold with the mesh base. After draining excess polyanion solution, the mold with reloaded capsules was perfused with 0.06 wt% chitosan solution to ionically fuse the capsules. Excess chitosan solution was drained, the capsules were rinsed with normal saline and surface stabilized by a brief perfusion with a dilute polyanion solution. The fused modular construct was then removed from the mold for further culture or analysis.Cell proliferation inside capsules was characterized using either a Hoechst DNA quantification assay 0 and A are initial and final absorbance or fluorescence readings respectively, t0 and t are initial and final time points, and μ is the specific growth rate in time−1.Where ACell viability was assessed using Calcein-AM and ethidium homodimer . The cell laden capsules were washed with PBS and incubated in serum free DMEM containing 4 µM Calcein-AM and 4 µM ethidium homodimer for 20 min at 37°C. For long-term tracking of HUVECs on capsules and fused capsule constructs, CellTracker™ Green CMFDA (Invitrogen) was used. Briefly, adherent cells were rinsed with PBS and incubated in a serum free culture medium containing 5 µM CellTracker Green probe for 45–60 min. After the incubation the medium was replaced with pre-warmed normal medium and incubated for another 30 min for the dye to undergo modification due to intracellular esterases. The cells were then trypsinized and seeded onto capsule outer surface. Cell fluorescence was then observed using wide-field fluorescence microscopy and laser scanning confocal microscopy (Zeiss LSM-410).The distribution and organization of cells and matrix inside the encapsulated cultures were investigated by histology. Cell laden individual capsules and fused capsule constructs were washed in PBS, fixed in 10% buffered formalin, dehydrated in an ethanol series, paraffin embedded, sectioned (4–6 µm) and stained using Hematoxylin and Eosin (H&E) or Masson's trichrome stains (Sigma-Aldrich). The stained sections were observed using bright field microscopy.6 cells/mL of CSA/CMC) were maintained in perfusion cultures under both packed bed and fluidized bed conditions as previously described 2) and recirculated using a peristaltic pump. The flow rates were adjusted to maintain physiological pressure differences (<100 mmHg) across the chamber (4–5 mL per minute). The perfusion system was maintained at 37°C for 1–2 weeks and medium was changed every 2–3 days. Medium samples were collected daily for evaluation of urea and albumin synthesis by the hepatocytes.Encapsulated primary rat hepatocytes  and a peroxidase conjugated anti-rat albumin antibody (Bethyl). Urea production was quantified using the diacetylmonoxime method as previously described Measurements were performed in triplicate (n = 3). Data are plotted as means with error bars representing standard deviation. Statistical comparisons were done using Student's t-test with a 95% confidence limit. Differences with p<0.05 were considered statistically significant.We investigated the effects of different polyanions and polyanion blends on the capsule characteristics and encapsulated cell growth patterns. The 4%CSA/1.5%CMC capsules  and the 7 cells/cm3, assessed via image analysis) at day-30 was high enough to replicate the cell density in many tissues. By the end of week-3, HTBs had invaded the capsule wall as seen in Encapsulated HTBs grew rapidly, eventually filling the capsules, and most cells appeared viable with a distinct nucleus up to at least day 30 . This inThe growth of sheep aortic endothelial cells (AEC) and HUVECs was investigated by seeding these cells onto the outside surfaces of CSA/CMC capsules. Endothelial cells attached poorly to surface stabilized capsule surfaces. However, their attachment and growth greatly improved when type-I collagen was coated onto the outer surface of the CSA/CMC capsules. HUVECs seeded on the collagen coated CSA/CMC capsules attached well and formed a viable monolayer within 24 hours of seeding . SEM imaSheep aortic smooth muscle cells were encapsulated for purposes of evaluating the performance of a normal parenchymal cell type. Use of these cells also allowed indirect evaluation of their interaction with endothelial cells in a subsequent study. SMC specific growth rate data showed that the cells proliferated significantly better in HA than in CSA/CMC capsules (p<0.05) during the first 36 hours of culture . HoweverResults of the diffusion studies on encapsulated BSA are shown in The hollow nature of the GAG-based microcapsules allowed us to tune the inner microenvironment by co-encapsulating additional materials. We investigated the effect of incorporating a collagen gel matrix and the inclusion of microcarriers by co-encapsulating them in microcapsules along with cells. When a collagen matrix was included along with the HTBs, the aggregates formed were smaller, more numerous, loosely organized and distributed inside the CSA/CMC capsules . The colTo further modulate the intracapsule environment, we investigated the effect of gelatin coated dextran microcarriers  encapsulated in HA and CSA/CMC capsules, on aortic smooth muscle cell proliferation and viability. The inclusion of microcarriers in the capsules reduced the formation of large aggregates and dispersed the cells more evenly across the capsule. Interestingly, the inclusion of the microcarriers in the polyanionic solution resulted in greatly reduced swelling and smaller sized capsules  comparedSome cell types may not grow well in a GAG-only ECM environment and hence attaining high cell densities could be a challenge. To address this, we developed a capsule formulation that promoted the cell-mediated contraction of capsules and the rapid elimination of excess capsule volume. We investigated the ability of collagen to promote contraction of the capsules by encapsulating smooth muscle cells with varying volume ratios of collagen gelling solution to GAG solution. Initially when collagen was encapsulated along with HTBs in CSA/CMC capsules at a final concentration of 1.5 mg/ml, the growing cells formed aggregates which were loosely packed as discussed earlier . When coMany studies have demonstrated functional relationships between endothelium and adjacent cell types Assembly of capsules into three-dimensional modular constructs is fabrication critical step in our modular tissue engineering approach to generating vascularized tissue. We investigated various methods for assembling larger 3D constructs from pre-cultured individual capsules. The most successful method involved reloading cultured capsules with a polyanion, followed by perfusion with a diluted polycation solution. Outward diffusion of reloaded GAG during the polycation perfusion step deposited a polyelectrolyte complex that effectively fused capsules together around points of contact. This method yielded self-supporting structures with interconnecting, perfusable spaces as shown in Capsules that were surface-seeded with HUVECs and subsequently fused showed well endothelialized, interconnected channels as seen in phase contrast  and conf6 cells/ml of the HA/collagen solution, followed by heparin reloading, and centrifugation for 10 seconds at 50G to expel excess intracapsular liquid. H&E stained sections of the resulting construct showed a dense cell mass  with reduced, but still significant intercapsule spaces  with more and larger intercapsule spaces  produced a lower osmotic pressure and resulted in contraction of the capsule membrane around the cell+microcarrier mass. In general, the inclusion of microparticles provides a wide range of options for tuning the cellular organization and overall mechanical properties of modular constructs.Fused capsule modules yielded 3D constructs with inter-capsular spaces that are perfusable in vitro and potentially vascularizable in vivo. The urea and albumin synthesis rates of the perfused cultures indicate that mass transfer rates were sufficient to maintain the encapsulated hepatocytes in our modular constructs. In addition, the interconnected endothelilized channels may provide a foundation for a vascular network and thereby accelerate the process of neovascularization by anastomosing with the host vasculature post-implantation. At the very least, intercapsular endothelial cells are likely to participate in vessel formation between fused capsules. However, the kinetics of this process, and the relative degrees of transplanted vs. host cell organization in the final structure remain to be characterized through animal studies.Our results also suggest that the capsule membrane can facilitate paracrine signaling as seen by the increase in SMC proliferation during coculture with endothelial cells. This suggests that various other interacting cell types can be cultured in this modular system with a degree of material-based control over cell organization while still allowing substantial paracrine signaling. Several coculture systems have previously been shown to improve morphology and function of engineered tissues including liver Unlike traditional scaffolds, porosity can be either maintained evenly throughout the modular capsule scaffolds or different layers with different capsule sizes and hence different porosity can be easily implemented. GAG-chitosan surfaces can support cell adhesion and proliferation, partly due to GAG-mediated binding of matrix proteins and growth factors In conclusion, we have demonstrated the formation and use of GAG-based microcapsules to generate a variety of tunable, intracapsular microenvironments. These capsules have been shown suitable for fabrication of porous, 3D constructs that have the potential to mimic native tissue architecture with high cell densities, vascular and parenchymal cell types, and perfusable, endothelium-lined channels. This capsule-based modular tissue assembly approach is a promising strategy that provides a wide range of options for the efficient assembly of three-dimensional, engineered tissues.


A large number of highly pathogenic avian influenza (HPAI) H5N1 outbreaks in poultry and wild birds have been reported in Europe since 2005. Distinct spatial patterns in poultry and wild birds suggest that different environmental drivers and potentially different spread mechanisms are operating. However, previous studies found no difference between these two outbreak types when only the effect of physical environmental factors was analysed. The influence of physical and anthropogenic environmental variables and interactions between the two has only been investigated for wild bird outbreaks. We therefore tested the effect of these environmental factors on HPAI H5N1 outbreaks in poultry, and the potential spread mechanism, and discussed how these differ from those observed in wild birds. Logistic regression analyses were used to quantify the relationship between HPAI H5N1 outbreaks in poultry and environmental factors. Poultry outbreaks increased with an increasing human population density combined with close proximity to lakes or wetlands, increased temperatures and reduced precipitation during the cold season. A risk map was generated based on the identified key factors. In wild birds, outbreaks were strongly associated with an increased Normalized Difference Vegetation Index (NDVI) and lower elevation, though they were similarly affected by climatic conditions as poultry outbreaks. This is the first study that analyses the differences in environmental drivers and spread mechanisms between poultry and wild bird outbreaks. Outbreaks in poultry mostly occurred in areas where the location of farms or trade areas overlapped with habitats for wild birds, whereas outbreaks in wild birds were mainly found in areas where food and shelters are available. The different environmental drivers suggest that different spread mechanisms might be involved: HPAI H5N1 spread to poultry via both poultry and wild birds, whereas contact with wild birds alone seems to drive the outbreaks in wild birds. Highly pathogenic avian influenza (HPAI) H5N1 can spread rapidly over a large geographic area among poultry and wild birds and has also been transmitted from birds to mammals including humans, with high mortality rates Environmental determinants can provide clues to the spread mechanisms underlying the outbreak patterns. Three spread mechanisms are currently under investigation: poultry transport , wild bird movement (mainly waterfowl), and poultry-wildfowl interactions. Transport of poultry could be the main cause if the disease pattern is closely related to anthropogenic environmental factors A large number of HPAI H5N1 outbreaks in both poultry and wild birds have been reported in Europe since 2005. Outbreaks in wild birds are concentrated in the central part of Europe, while outbreaks in poultry are mainly found in the eastern Europe such as the Black Sea region, with sporadic infections mostly around wild bird infections . Kernel Williams et al. Si et al. http://empres-i.fao.org/eipws3g/) provided by FAO. Three groups of environmental variables were complied, corresponding with different spread mechanisms: anthropogenic environmental (poultry-dominant), physical environmental (wildfowl-dominant), and (poultry-wildfowl) interaction variables.The time and locations of 170 confirmed events of HPAI H5N1 outbreaks in poultry in Europe, reported from 2005 to 2008, were acquired from the Global Animal Disease Information System EMPRES-I  and derived slope and aspect, mean annual potential evapotranspiration, mean annual aridity index, mean monthly precipitation, mean monthly minimum and maximum temperature, and monthly Normalized Difference Vegetation Index (NDVI). Monthly data were included to consider potential seasonal effects. Four 12-month NDVI data sets from 2005 to 2008 were smoothed by employing an adaptive Savitzky-Golay smoothing filter, using the TIMESAT package Localities where HPAI H5N1 outbreaks had been reported before tend to be more intensively surveyed/sampled than localities where no outbreaks were reported. Duplicated outbreaks of HPAI H5N1 from the same locality were discarded to reduce this sampling bias, resulting in 133 unique geographic coordinates. A disease presence area was constructed by generating 10 km radius buffers around each presence location, as Europe adopted a 10 km surveillance zone policy Logistic regression analyses were utilized to examine the relationship between explanatory variables and the HPAI H5N1 outbreaks in poultry. A bootstrapping procedure was adopted in which, together with the 133 presence locations, 133 absence locations were randomly selected with replacement from the 5000 absence locations. This process was repeated 1000 times, creating 1000 subsets for model training , and the area under the Receiver Operating Characteristic curve (AUC) calculated from the 1000 training subsets. Variables with P-values ≥0.1 were excluded from further analyses. Variables with relatively high collinearity or high spatial autocorrelation were dropped by examining the variance inflation factors (VIF) and Moran’s I, respectively. Variables were removed by sequentially dropping the variable with the lowest impact, recalculating the VIFs and repeating this process until all VIFs were <10 Univariate logistic regression analyses were firstly applied to investigate both linear and quadratic effects of each variable on the outbreak of HPAI H5N1 in poultry. The relationship was evaluated by the mean odds ratios (OR), 95% confidence intervals (CIs) of OR, the P-value was calculated for each selected variable, and variables yielding non-significant effects  were discarded.Multiple backward stepwise logistic regression was carried out to select the significant independent variables. This stepwise process was repeated 1000 times using the different training subsets. The frequency of each variable being selected was calculated on the basis of applying 1000 best stepwise logistic regression models, ranked by AIC. A mean P-value, AIC, and AUC were used as indicators of model performance. Only significant variables  were retained in the final model.Multiple logistic regression was then carried out using the remaining variables. This process was repeated 1000 times using the different training subsets. The mean values of coefficients, OR, 95% CIs of OR, www.r-project.org). R codes are available in All variables were standardized (z-score) prior to the analyses. All statistical analyses were conducted using R statistical software , and several interaction variables . Negative linear associations were recorded between poultry outbreaks and DEM, slope, potential evapo-transpiration, precipitation and NDVI (Apr to Sep). Precipitation and maximum temperature (Sep-Nov) had significant positive quadratic terms (upward parabola) while maximum temperature (Mar) and NDVI (Jan) showed significant negative quadratic terms (downward parabola).P-value ≤0.05) (Seven variables were kept after the stepwise selection (mean e ≤0.05) . The minIn the final analysis, three variables were identified as key risk factors strongly influencing HPAI H5N1 occurrence in poultry in Europe. Reduced precipitation (Nov), increased maximum temperatures (Nov) and an increased human population density combined with close proximity to lakes or wetlands all increased the probability of HPAI H5N1 outbreaks in poultry. A predictive risk map of HPAI H5N1 outbreaks in poultry was generated based on the final multiple logistic regression analysis . High-riDifferent environmental drivers operate on HPAI H5N1 outbreaks in poultry and wild birds in Europe. The probability of HPAI H5N1 outbreaks in poultry increases in areas with a higher human population density and a shorter distance to lakes or wetlands. This reflects areas where the location of farms or trade areas and habitats for wild birds overlap. In wild birds, HPAI H5N1 outbreaks mostly occurred in areas with increased NDVI and lower elevations which are typically areas where food and shelter for wild birds are available These findings are in contrast with a previous study Human population density combined with the proximity to lakes or wetlands consistently showed a positive relationship with HPAI H5N1 outbreaks in poultry. Both human population density and the presence of water were identified as one of the most common environmental factors influencing HPAI H5N1 outbreaks across different regions and spatial scales This study emphasizes the substantial contribution of climatic factors, in particular, temperature and precipitation, on HPAI H5N1 outbreaks in wild birds and poultry. The association between poultry infections and climatic factors could be explained by the direct or indirect contact (e.g. through contaminated environment) with wild birds. The link between disease outbreaks in wild birds and climatic factors has been reported by previous studies. For instance, Ottaviani et al. Some physical environmental factors, such as elevation and NDVI, consistently correlated with the outbreak patterns of wild birds, but were dropped from the final risk model of poultry infections . These tThe level of bio-security, preventive measures designed to reduce the risk of disease transmission, may negatively influence the sensitivity of the model to environmental factors. Poultry infections would be decreased if poultry are quarantined from contact with other birds. In this case, areas with a higher poultry density might not necessary have a higher risk. Hence, in line with previous findings in China We suggest that areas with a relatively high human population density and also close to wetlands or lakes, facilitated by increased temperatures and reduced precipitation during the cold seasons  should bTable S1Summary of the links to the data sets used in this study.(DOCX)Click here for additional data file.Appendix S1R scripts used to perform the analysis.(R)Click here for additional data file.


Here we observed that estrogen treatment resulted in a significant increase of trabecular and cortical bone mass in both, wildtype and −/−Fshb mice. Unexpectedly, this FSH-independent pharmacological effect of estrogen was not caused by influencing bone resorption, but primarily by increasing bone formation. To understand the cellular and molecular nature of this osteo-anabolic effect we next administered estrogen to mouse models carrying cell specific mutant alleles of the estrogen receptor alpha (ERα). Here we found that the response to pharmacological estrogen administration was not affected by ERα inactivation in osteoclasts, while it was blunted in mice lacking the ERα in osteoblasts or in mice carrying a mutant ERα incapable of DNA binding. Taken together, our findings reveal a previously unknown osteo-anabolic effect of pharmacological estrogen administration, which is independent of FSH and requires DNA-binding of ERα in osteoblasts.Postmenopausal osteoporosis is characterized by declining estrogen levels, and estrogen replacement therapy has been proven beneficial for preventing bone loss in affected women. While the physiological functions of estrogen in bone, primarily the inhibition of bone resorption, have been studied extensively, the effects of pharmacological estrogen administration are still poorly characterized. Since elevated levels of follicle-stimulating hormone (FSH) have been suggested to be involved in postmenopausal bone loss, we investigated whether the skeletal response to pharmacological estrogen administration is mediated in a FSH-dependent manner. Therefore, we treated wildtype and FSHβ-deficicent ( First, although estrogen supplementation at pharmacologic doses has been shown to positively affect bone mineral density and reduce fracture risk in postmenopausal women in vivo.In the current study, we have investigated the cellular and molecular mechanisms of the osteo-anabolic effect of pharmacological estrogen administration. For that, we implanted mice with subcutaneous estrogen pellets and analyzed their bone phenoype after 4 weeks of treatment. Our data show that estrogen treatment results in a FSH-independent increase in bone mass, which is mediated by the ERα in osteoblasts and primarily affects bone formation, not bone resorption. Furthermore, applying a mouse model carrying a mutation specifically disrupting DNA-binding of ERα, we demonstrate that the bone-anabolic effects of estrogen administration rely on this function of the ERα −/−Fshb mice with estrogen for 4 weeks. As expected, −/−Fshb mice displayed a marked atrophy of the uterus and ovaries, which was fully normalized by estrogen treatment  LysM-CreRunx2-CreCre negative ERαfl/fl controls, ERαLysMCre showed no changes in the trabecular bone volume, whereas ERαRunx2Cre displayed significantly decreased bone mass in spine and tibia sections relative to controls  in the promoters of target genes, but the ERα protein is still recruited to AP-1 binding sites EAAE mice displayed a reduced trabecular bone volume in spine and tibia sections  the different doses and timing of estrogen treatment in these cultures and iii) the lack of bi- or multidirectional communication between osteoblasts and osteoclasts and other cell types in most culture systems, potentially omitting cellular mediators important for estrogen action in vivoFew studies so far have investigated the influence of estrogen treatment on osteoblast function in vivo, we thus chose to employ genetically modified mouse models to elucidate the underlying cellular mechanism. Applying transgenic mice expressing Cre recombinase under the control of the LysM or Runx2 promotor, respectively, two previously well characterized and specific deleter strains Runx2Cre mice displayed a significantly decreased trabecular bone mass in the spine and tibiae. Most importantly however, the increase in bone mass following estrogen administration, which we observed in the corresponding control groups, was completely abolished in ERαRunx2Cre mice. These data clearly demonstrate that the ERα in osteoblasts is the responsible mediator of the osteo-anabolic effect caused by pharmacological estrogen administration.Given the striking osteo-anabolic effect of pharmacological estrogen administration observed EAAE mouse model in order to evaluate, whether estrogen-induced bone formation would also be independent of DNA-binding. Here we found that ERαEAAE mice did not respond to the osteo-anabolic effect of estrogen administration. Therefore, although monomeric ERα signaling may be of relevance in some tissues We further addressed the question, whether intact DNA-binding of ERα is required for the osteo-anabolic effect of pharmacological estrogen administration. Although ERα is a nuclear receptor mediating most of its pleiotrophic effects as a DNA-binding transcription factor, several reports have suggested that estrogen receptors are also capable of associating with the cell membrane to conduct rapid responses independent of gene expression Of interest, the applied treatment regimen in this study positively affected bone volume in both spine and long bones. This is clinically relevant, since fractures of the femur are associated with higher mortality than vertebral fractures +/−Fshb mice were obtained from the Jackson Laboratory (# 003283) and genotyping was performed as described previously −/−Fshb females, we only used wildtype and −/−Fshb littermates derived from +/−Fshb matings for our analysis. The generation and genotyping of ERαfl/fl and ERaEAAE mice has been described previously −/−Fshb mice, synchronization of the reproductive status was not performed prior to surgery. To assess dynamic histomorphometric indices estrogen-treated and sham-operated mice were given two injections of calcein 9 days and 2 days before sacrifice. Radiographic and histologic analyses were performed after a treatment period of 4 weeks. To evaluate the impact of estrogen treatment on the gonads, the size of the ovaries was monitored immediately after sacrifice.For estrogen treatment 12 week old female mice of each genotype were implanted with an estrogen pellet  in the scapular region behind the neck. Due to the preantral stage block in folliculogenesis in female After removal of internal organs, the whole skeletons were fixed in formalin as described previously After an incubation in 70% ethanol for 24 hours, the lumbar vertebral bodies L2 to L5 and one tibia of each mouse were dehydrated in ascending alcohol concentrations and embedded in methylmethacrylate as described previously Histomorphometric quantification was performed on toluidine blue–stained sections. Analysis of the trabecular bone volume (BV/TV), trabecular thickness (TbTh), trabecular number (TbN), trabecular spacing (TbSp), and the determination of osteoblast and osteoclast numbers  were carried out using the Osteo-Measure histomorphometry system  according to the guidelines of the American Society for Bone and Mineral Research Estrogen levels were determined by the Exciting Systems  at the Institute of Clinical Chemistry, University Medical Center Hamburg-Eppendorf, Hamburg, Germany. To evaluate the rate of bone resorption we quantified the serum concentrations of collagen degradation products (Crosslaps) by ELISA .+SD. Statistical analyses of the results were performed using two-way-ANOVA (IBM SPSS Statistics 20). Statistical differences were considered significant when p<0.05.All data are presented as mean