A number of significant SNP variants were identified for both traits. Many of the SNP variants associated either with susceptibility to – or recoverability from mastitis were located in or very near to genes that have been reported for their role in the immune system. Genes involved in lymphocyte developments  and genes involved in macrophage recruitment and regulation of inflammations (PDGFD and PTX3) were suggested as possible causal genes for susceptibility to – and recoverability from mastitis, respectively. However, this is the first GWAS study for recoverability from mastitis and our results need to be validated. The findings in the current study are, therefore, a starting point for further investigations in identifying causal genetic variants or chromosomal regions for both susceptibility to – and recoverability from mastitis.Because mastitis is very frequent and unavoidable, adding recovery information into the analysis for genetic evaluation of mastitis is of great interest from economical and animal welfare point of view. Here we have performed genome-wide association studies (GWAS) to identify associated single nucleotide polymorphisms (SNPs) and investigate the genetic background not only for susceptibility to – but also for recoverability from mastitis. Somatic cell count records from 993 Danish Holstein cows genotyped for a total of 39378 autosomal SNP markers were used for the association analysis. Single SNP regression analysis was performed using the statistical software package DMU. Substitution effect of each SNP was tested with a  Genome- ≈ 0.03) .Bos taurus autosomes (BTA) 6, 13, 14, and 20 in Nordic Holstein cattle  to determine association with a trait of interest assuming that a marker is in linkage disequilibrium (LD) with, or close to, a causative mutation . In cattn cattle ; BTA 6, n cattle ; 24 chron cattle ; and on n cattle . A revien cattle  indicateThese studies have been useful for identifying genetic variants and genes associated with CM and indicator traits. However, they lack consistency in reporting SNPs or genes implicated in mastitis, and to our knowledge, none of these studies have reported any genetic variant or chromosomal regions associated with recoverability from mastitis. We, therefore, performed GWAS using single SNP regression analysis for two main objectives: (1) to identify new or confirm previously identified regions of the genome for their association with mastitis susceptibility, and (2) identify variants and regions of the genome associated with recoverability from mastitis in Danish Holstein cows.H) and diseased (D). A cow may return to the same state more than once, meaning repeated disease cases are acknowledged by the model  and i had moved from H to D state), and consequently low values of In the above transition probability matrix, the first row consists of the probabilities of being in either of both states at time D state) . Note thThus, for each cow and lactation the sequence of H’s and D’s, indicating whether or not a cow had mastitis on subsequent test weeks, was converted into a new sequence of weekly transitions indicators: 0 if a cow remains in the same state and 1 if the cow changes state. This resulted into two series of transition records: one for healthy to diseased  and the other for diseased to healthy . A more detailed description of the data is given by f(DIM)] was modeled by a combination of Legendre polynomials and a Wilmink term (exp-0.05×DIM) (f(time)] were modeled with Legendre polynomials. The model for the phenotypic analyses included parity (1–3) and herd (1–7) as fixed effects, regression coefficients of a second order polynomial on DIM plus a Wilmink term and regression coefficients of a third order polynomial on time as covariates, and random effects of herd-test-week, cow-parity interaction, and cow effects. The observed transitions were linked to an underlying continues scale called liability  were adjusted for different fixed and random effects before using them in the association analysis. .05×DIM) , to refliability . This rey = 1 if λ > 0 and 0 otherwise. The mean on the liability scale models the average probability for y = 1. Accordingly, the observed transitions and liabilities were modeled with a threshold model as follows:where τ is a fixed threshold and usually set to an arbitrary value (here 0), such that y); b was a vector of all fixed effects (herd and parity) and covariates ; h, p, and c were vectors of random effects of herd-test-week, cow-parity interaction, and cow effects, respectively; X, Z1, Z2, and Z3 were respective incidence matrices, e was a vector of the residual effects. The vectors of random effects  were assumed to be normally distributed, i.e., I were the identity matrices. The residual variance 2, where PSD is the posterior standard deviation of the cow plus cow∗parity effects. Association analyses were carried out on four datasets  to investigate parity specific associations. The number of records, means and SD of the adjusted phenotype used in the association analyses are in Table 1.The phenotypic analyses were performed using the statistical software package MCMCglmm  in R R . Estimatanalysis , residuaFurther bivariate and trivariate analyses were performed to study correlations between traits as well as between parities but within traits. The bivariate model was similar to equation (2) and except the residual, all between traits random effects were assumed correlated. The trivariate model used to calculate correlations between parities was also similar to the model in equation (2) but did not include parity and cow-parity interaction effects. In this trivariate model only the cow effects across parities were correlated. Herd-test-week effects were included and assumed uncorrelated.The raw data contained 1957 cows genotyped with the Illumina BovineSNP50_v2  for a total of 46931 autosomal SNP markers. However, we use phenotypes records from our previous study  and the Multidimensional scaling (MDS) of SNP genotypes was performed for detection of population stratification for the 996 animals that passed genotyping quality control checks. The aim was to assess potential genetic clustering of animals by herd. The cows came from seven herds: one research herd and six commercial herds. In order to visualize possible population stratifications, MDS plots of an identity-by-state (IBS) matrix was generated using PLINK .Figures 1A and 1C, the first five MDS components explained 14.3, 6.2, 5.8, 5.4, and 3.9%, respectively, of the total genomic variance, and therefore, we reasoned that fitting these first five MDS components as clustering factor into our model could improve the GWAS results because it corrects for confounding effect of the population structure . After removing the three genetic outlier animals, the remaining animals formed no distinct cluster .As shown in tructure . The MDSAfter QC and removal of four cows (one for having less than 90% genotype data and three other cows for being genetic outliers), 993 cows and 39378 SNP markers remained and were used for final association analyses.Single SNP association analysis was performed with a linear mixed model in DMU . Each SNy was a vector of posterior mean of summed cow and cow∗parity effects obtained from equation (2); 1 was a vector of ones; μ was the general mean; X was a matrix containing the top five MDS components fitted as covariates, and b was a vector of associated effects; S was vector of SNP genotypes coded as 0, 1, or 2 for genotype copies of one of the alleles at each locus; α was the allele substitution effect; a was a vector of random additive polygenic effects, which was assumed to follow normal distribution, A is the pedigree-based additive genetic relationship matrix; Z was an incidence matrix relating elements of the vector of additive polygenic values a to individual phenotypes; e was a vector of random residuals and the residuals were assumed to follow a normal distribution, W was a diagonal matrix with diagonal element wi is used to account for the different residual variances due to parities. The pedigree for the animals (993) in the genotype data was extended to a maximum depth of 10.9 generations and an average of 4.4 known generations. The extended pedigree contained a total of 12779 animals and were offspring of 2659 sires and 9536 dams.where H0: b = 0 was tested with a t-test. A genome-wide Bonferroni corrected significance threshold can be obtained by dividing the nominal significance threshold (0.05) by the number of independent tests  in the analyses. The P-value after Bonferroni multiple testing corrections was 1.26974E-06, equivalent to -log10  = 5.90. However, in the current study no P-value exceeded the Bonferroni threshold and hence a liberal threshold of P-value < 10-4 was used to declare significant SNP-trait associations.The association analysis was conducted using the statistical software package DMU . The nul2 gene annotation system , which translates to less variation in susceptibility to- than in recoverability from mastitis within year across time. On the other hand, animal related variation was higher for susceptibility to mastitis (HD) than for recoverability from mastitis (DH) (Table 2). The between traits posterior mean of cow effect correction [rc = -0.90 (0.07)] was much higher than the cow-parity interaction correction [rcp = -0.29 (0.11)].Herd-test-week variation was lower for the HD trait than for the DH trait  across parities. As expected, for the HD trait, the smallest between parities cow effect correlation was observed between the non-consecutive parities 1 and 3 [rp13 = 0.08 (0.12)], whereas for the DH trait, the smallest between parities cow effect correlation was observed between the consecutive parities 2 and 3 [rp23 = 0.10 (0.20)] (Table 2).The variance components presented in P-value < 10-4 was considered to determine genome-wide significant SNP-trait association. For the full data , significant SNP-trait associations were detected on BTA3 and 7 for mastitis susceptibility and on BTA7 and 15 for recoverability from mastitis (Table 3). Parity specific significant SNP-trait association were also detected on the same chromosomes but no overlapping association signals were observed. Significant association signals in parity 1 and in the other parities (parity 2 or 3) detected on the same chromosome did not overlap, for either of the traits. For susceptibility to mastitis, the highest number of significant associations were observed on BTA7 and BTA13. For recoverability from mastitis, the highest number of significant associations were observed on BTA12 and BTA13. In total, 29 significant SNPs (14 for susceptibility to mastitis and 15 for recoverability) were detected.The nominal significance level of Figure 2) and Manhattan plot , only 6 SNPs (2 for susceptibility and 4 for recoverability) reached the defined genome-wide significance threshold when all parities were analyzed together. The QQ plots and Manhattan plots for the parity specific associations are provided in the Supplementary Material  further demonstrated that the GWAS results were not substantially influenced by the observed population stratification. Influence of population stratification was adequately controlled by including the MDS components in the model and this was quantified by a calculated genomic inflation factor ≤ 1.05 .As shown in the quantile–quantile (QQ) plot  indicates that highly susceptible cows are in general less likely to recover fast. Relatively, the trait correlation between cow-parity interaction effects was much smaller than the cow effect correlation, indicating even highly susceptible cows in parity one could recover relatively faster in the other next parity.The lower posterior mean of herd-test-week variance for HD than for DH could indicate that management to prevent mastitis (and other factors influencing susceptibility) is fairly stable across time, whereas management (and other factors) related to recoverability is more variable over time. This could suggest that there are possibilities to enhance recoverability by improvements in herd management . The posTable 2. The cow-parity interaction variance is larger than the cow variance, i.e., the larger part of cow variance is parity specific. Results from the trivariate analysis where parities are considered as different traits, also point to relatively low agreement between parities (Table 2). So, the data does not support the general expectation that parities may be similar, and this can explain the absence of common association signals across parities in the association results. A possible confounding effects of multiple mastitis pathogens may have contributed to the lack of consistent association signals across parities and traits. The cow effect correlations across parities but within traits were positive and ranged from low to moderate. For the HD trait, the weakest posterior mean of cow effect correlation across parities was observed between parities 1 and 3, indicating that susceptibility in parity 1 is distinct from susceptibility in parity 3. For the DH trait, the weakest posterior mean of correlation across parities was observed between parities 2 and 3, suggesting recoverability in parities 2 and 3 should not be considered as the same trait. In general, we would expect parities to be similar, but this data shows larger variation in cow-parity interaction than the cow effect variation as we presented it in TNF-α was associated with a lower number of mastitis cases in lower parities and a higher number of mastitis cases in higher parities. Similarly, the causal variants or genes could be different for susceptibility to – and recoverability from mastitis as association signals present on the same chromosome do not overlap. Our results support the findings that QTLs even for highly correlated traits  present on the same chromosome do not overlap  detected on the same chromosome do not overlap, suggesting that genetic regulation of mastitis could also be parity dependent. Effect of genes has been shown to vary depending on parity or age of cows . Wojdak- overlap . Most ofMARCH3 (E3 ubiquitin-protein ligase MARCH3). The gene MARCH3 encodes an E3 ubiquitin-protein ligase enzyme that may be involved in regulation of the endosomal transport pathway and it has been implicated in diverse biological functions, such as immune regulation, protein quality control, and membrane trafficking (STAB2 (stabilin-2 precursor). Studies in pigs . SLX4-deficient mice have been shown to develop epithelial cancers and to have a contracted hematopoietic stem cell pool  for their association with mastitis traits defined from SCCs. The SNP rs43732911 on BTA7, an intron variant and in significant association with susceptibility to mastitis in the full dataset, is located within a potential candidate protein coding gene fficking . The SNP in pigs  and in m in pigs  showed tell pool .ATG16L2 (autophagy related 16 like 2). The gene ATG16L2 has been reported to play a role in autophagy (PDGFD (platelet derived growth factor D). The protein encoded by the gene PDGFD plays an important role in wound healing and macrophage recruitment (PTX3 (pentraxin 3). The gene PTX3 may involve in the innate immune response against intra-amniotic infection and inflammation  and IFI30 . Both genes have been reported for their role in antigen processing. In a gene expression assays . The enzyme also facilitates MHC class I-restricted recognition of exogenous antigens containing disulfide bonds by CD8+ T-cells or cross-presentation (GeneCards).We have also identified some potential candidate genes from the nearby gene cluster, in addition to the genes identified by within gene SNP variants. The SNP rs41255569 on BTA7, an upstream gene variant and in significant association with susceptibility to mastitis in parity 3, is mapped very near to a functionally known candidate genes n assays , the geninvasion . The enzFigure 4 we present a 1Mb gene cluster surrounding the significantly associated variant in the region. This illustrates that complex traits could be regulated by several clusters of genes surrounding association signals and confirms the fact that mastitis is a complex trait and is determined by many genes with small effect limiting the ability of GWAS to identify causal genes . The gene EPS15L1 has been reported as an essential gene for T lymphocyte development in Zebrafish , MAST3 (highly expressed in antigen-presenting cells and in lymphocytes) and STAB2  as candidate for the causal genes for susceptibility to mastitis. For recoverability from mastitis we suggest EPS15L1 , PDGFD , and PTX3 (involved in regulating inflammation) as candidates for the causal genes. Regardless of the gene annotations related to immune responses, we were not fully convinced to suggest these genes as strong candidates as we were not able to confirm previously identified SNP variants or regions of the genome. However, this is the first GWAS study for recoverability from mastitis and our results need to be validated. Moreover, most of the functional annotations were derived from gene expression studies in mice and humans and gene functions may be different in cattle. There have also been discrepancies between gene-expression platforms and analyses methods reported to be obstacles in mapping loci that might underlie functional trait variation  determined at a significance level of BW performed the genome-wide association analysis and wrote the draft manuscript. PL and LJ contributed to the research hypothesis and discussion of the results. D-JK contributed to the research hypothesis, analysis, discussion of the results, and writing the manuscript. WF contributed to the research hypothesis, analysis of phenotype data, and discussion of the results. All authors approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Biotinidase deficiency is an autosomal recessively inherited disorder characterized by neurological and cutaneous abnormalities. Untreated individuals with biotinidase deficiency cannot recycle biotin from biocytin (N-biotinyl-ϵ-lysine), the proteolytic digestion product of protein-bound biotin. Biotin therapy can markedly resolve symptoms, or can prevent the development of symptoms if initiated early. To understand better the pathogenesis of the neurological problems in the disorder in humans, we have compared gene transcription changes during the first week post-birth in the brains of biotinidase-deficient, transgenic, knock-out mice at days 1 and 8 and compared to changes in wildtype mice at the same times. The knockout pups that were not supplemented with unconjugated biotin became symptomatic by day 8 and exhibiting failure to thrive. Wildtype pups remained asymptomatic under the same experimental conditions. We compared all four possible combinations and noted the most significant up- and down-regulated genes in the knockout animals at Day 8 compared to those at Day 1, reflecting the changes in gene expression over the first week of development. These alterations involved neurological development and immunological function pathways and provide some clues to avenues for further research. At this time, these preliminary analyses provide us with limited, but new information. However, with the development of new algorithms and programs examining various mechanisms and pathways in the central nervous system, these analyses may help us to understand better the role of biotinidase and the pathogenesis of biotinidase deficiency. After determining that we had satisfactory quality control of the RNA samples, we analyzed the differential gene expression data.2.2WT and KO dams were maintained on a normal, biotin-containing diet. KO pups born to KO dams were nurtured by their mother and did not receive any additional unconjugated biotin. Similarly, WT pups born to WT dams were nurtured by their mother and did not receive any additional unconjugated biotin. The KO pups failed to thrive, became symptomatic, and died in 8 to 12 days under these conditions. On the other hand, WT pups fed by their mother thrived and remained asymptomatic over this same time period.The pups were sacrificed and their brains were harvested and stored in RNAlater  at − 80 °C for subsequent RNA isolation, as described previously in the manual provided with the kit. RNA was isolated using the miRNeasy mini kit (Qiagen) from each of the 12 stored brains, and samples were quantitated by Nanodrop . Quality was assessed by running samples on Agilent TapeStation and using RNA Integrity Number (RIN-Agilent 2200 TapeStation). 500 ng of RNA from the three pups in each of the four groups  were pooled and subjected to Gene Expression Analysis on Agilent platform.2.3Differential gene expression of RNA samples was compared in each of the four arrays using Agilent Mouse Gene Expression V.2 4X44K array . Each chip is capable of detecting for 39,430 Entrez Gene mRNAs. The quality of the results were assessed using positive control probes and spike-in control probes 31 × 10 E1A  at the Functional Genomics and Bioinformatics Core Facility at Wayne State University, Detroit, MI. These probes establish baseline criteria against which experimental signals were compared. Agilent's microarray design is based on two-color microarray-based gene expression analysis (quick amp labeling) with Tecan HS Pro Hybridization. To obtain fluorescent intensities for each spot on the array, the Agilent dual laser scanner with SureScan High Resolution Technology was used.2.4BTD, were deleted. The promoter of BTD was intact, and transcription of the gene thus resulted in a truncated, but detectable transcript. However, the transcript resulted in no biotinidase protein on western blot analysis against anti-biotinidase antibody and, therefore, no biotinidase activity in the preparation In preparing a transgenic, knock-out mouse with biotinidase deficiency, part of intron 1, all of exon 2, intron 2, exon3 and part of intron 3 of the biotinidase gene, BTD probe was used that hybridizes to within the 3′-region of the transcript for the remainder of intron 3 and exon 4. Based on the results found in KO Day 1 and WT Day 1 mice, it appears that there is about 50% of the transcript in KO mice compared to that of WT mice. This may indicate that the transcript in the KO mice is less stable.In the expression studies, a 2.5p-value was then calculated for these fold changes, reflecting the probability of obtaining a given fold change for a probe by chance, and incorporated the control measurements from the spike-in controls and dye-swap variation. In p-values were normalized by a (− log10) conversion. mRNA signals required a mean fold change of >|2 |, and a p-value of < 0.05 in order to be considered experimentally significant.Dye-swapped duplicate data were mean averaged to control for color biases, and this value was used for subsequent analyses . The absolute fold change was converted by a (log2) transformation. 2.6https://david.ncifcrf.gov/ and using Ingenuity Pathway Analysis (http://www.ingenuity.com/products/ipa) to identify functional annotations, pathways, and themes.Processed data sets were analyzed using DAVID (31.Comparisons of expression of KO pups at day 1 with WT pups at day 1 (KO/WT Day 1) examine the baseline differences in gene transcription between KO and WT animals.2.Comparisons in expression of WT pups at day 8 with WT pups at day 1 (WT Day 8/Day1) examine the differences in gene transcription of WT animals during the early developmental phase post-birth. KO animals develop symptoms during this time period, whereas WT animals remain asymptomatic. This scenario compares developmental gene changes from Day 1 to Day 8, but not changes due to the decreased intake of biotin, because these animals remain asymptomatic and have biotinidase capable of recycling endogenous biotin.3.Comparisons of expression of KO pups at day 8 with WT pups at day 8 (KO/WT Day 8) examine differences in gene transcription due to biotinidase deficiency. In this period, KO animals become symptomatic, whereas WT animals remain asymptomatic.4.Comparisons in expression of KO pups at day 8 with KO pups at day 1 (KO/WT Day 8) examine the differences in gene transcription of KO animals when they become symptomatic versus asymptomatic KO animals at baseline representing the developmental phase affected by biotinidase deficiency. Not only does this scenario compare developmental gene changes from Day 1 to Day 8, but also genes that are up- and down-regulated in response to the stresses of biotinidase deficiency and consequential biotin deficiency.Our study design allowed us to evaluate and compare four different differential expression scenarios. The following are the comparisons and their respective rationales:p-value)) (y-axis) for mRNA transcript signals. Note that the differences in magnitude of both the x- and y-axes are to accommodate major out-lying values. Significant probe signals shown within the non-shaded regions were defined as having a mean fold change of >|2 |, and a p-value of < 0.05 (where − log10(P) = 1.301). Shaded regions indicate mRNA signals that do not meet significance and magnitude criteria.First, we performed an overview of the entire complement of transcriptomes for each of the above comparisons . These vIn general, most of the genes expressed in KO/WT Day 1 showed few differences in up-regulations or down-regulations which represents baselines for both the KO and WT animals, whereas there are more differences noted in WT Day 8/Day 1 which may be indicating changes in development or other processes. Because the WT animals are able to recycle biotin, it unlikely that the differences represent differences in expression due to biotin restriction or related metabolic pathways. Interestingly, there are fewer changes noted in KO/WT Day8. This is possibly because many of the gene differences are likely common to developmental processes. However, there are many more differences in gene expression noted in the KO Day8/Day1 that are comparing the differences when the KO animals are symptomatic. The changes may be indicative of major, mostly up-regulatory transcription changes in response to the metabolic stresses caused by their secondary, biotin-deficient status, and that deficiency's effects on other metabolic pathways.BTD, a constitutive enzyme, did not up- or down-regulate significantly in any of the four scenarios. The various enzymes did not significantly vary more than a fold up- or down-regulation of expression in any of the four comparisons, except in the Day8/Day1 comparison for up-regulation of between 2- and 3-fold of pyruvate dehydrogenase complex (PDA2 and PDHX) and the in the enzyme responsible for the initial enhancer step in the urea cycle, N-acetylglutamate synthetase.However, these plots only provide a comparative overview of metabolic activity differences under the various conditions and do not address specific gene changes. To evaluate specific genes, we have specifically chosen to examine the up-regulations and down-regulations of genes of the biotin-dependent carboxylases and in pathways related to their specific metabolites and those involved with the urea cycle, because individuals with untreated biotinidase deficiency often exhibit hyperammonemia . As expeIn 4Symptomatic mice with biotinidase deficiency developed neurocutaneous abnormalities similar to the clinical features seen in children with untreated profound biotinidase deficiency BTD. This is expected, because biotinidase is a constitutively expressed enzyme N-acetylglutamate synthetase  that these changes in transcription have on a pathway will require a comprehensive understanding of biochemistry and physiology of the involved pathways. Although we are currently in a position to examine the degree of up- or down-regulation of specific genes or common pathways, interpretation of their effects will require more powerful integrative computer programs to analyze these complex metabolic pathways and their interactions.Biotinidase deficiency is a monogenic deficiency that in the untreated individual can produce clinical features involving primarily the neurological and cutaneous systems. We have only examined the neurological system in this study, but clearly, we see that multiple genes and pathways are affected. We may have been naïve in believing that we would uncover or discover a specific gene or group of genes that would help answer some important questions, such as why there is such variability in expression of the disorder in the same family or why some untreated individuals develop symptoms during adolescence, adulthood or even remain asymptomatic.We hoped that these preliminary studies of gene expression in the various scenarios would provide us with clues or directions for areas of further study. However, the changes in expression of various genes or gene pathways have not necessarily provided us with specific answers. Moreover, the gene expression alterations may not indicate direct effects, but possibly more down-stream effects, thereby making interpretation even more difficult.These studies together with appropriate computerized programs may also provide us with a method for understanding the biochemistry and pathophysiology in various organ systems of other monogenetic inherited metabolic disorders Our intent in performing these studies was to determine if we could find any genes or pathways that could provide us with further insights into changes that are occurring when KO animals become symptomatic. As we have stated, this is a preliminary study. However, we believe that this study is important in informing other investigators of inherited metabolic diseases about some of the strengths and weaknesses of this approach for their research.This study represents an attempt to narrow the search for mechanistic explanations of biotinidase deficiency. We are certainly not the first to use such an approach with the understanding that the results may not provide specific answers without further advancements in bioinformatics. These preliminary analyses have provided us with some new information that confirms existing knowledge, and it may serve as a guide for future studies targeted at pathways implicated here. The development of new algorithms and programs examining these mechanisms and pathways of the central nervous system and immune system may better explain the role of biotinidase and the pathogenesis of biotinidase deficiency.


Km values increased slightly, but the change was not statistically significant. The effect of BSA on substrate uptake was dependent on the vesicle amount, while increasing BSA concentration did not significantly improve substrate uptake. These results indicate a minor effect of albumin on MRP2 and BCRP, but it should be considered if albumin is added to transporter assays for example as a solubilizer, since the effect may be substrate or transporter specific.The ABC transporters multidrug resistance associated protein 2 (MRP2) and breast cancer resistance protein (BCRP) are of interest in drug development, since they affect the pharmacokinetics of several drugs. Membrane vesicle transport assays are widely used to study interactions with these proteins. Since albumin has been found to affect the kinetics of metabolic enzymes in similar membrane preparations, we investigated whether albumin affects the kinetic parameters of efflux transport. We found that albumin increased the V In the case of E217βG, which follows sigmoidal kinetics with MRP2  is the concentration of inhibitor (μM), IC50 is the inhibitor concentration required for a response half-way between minimal and maximal response (μM) and h is the Hill coefficient. The Responsemin term was constrained to be larger than zero, where zero indicates full abolishment of uptake.The oleic acid concentration required for 50% inhibition . The presence of 0.1% BSA produced a small, but statistically significant increase of 28% in maximal LY uptake by BCRP , Table 3ransport , we usedOur in-house vesicle transport assays have been optimized to use lower protein amounts in the MRP2 assay than in the BCRP assay. In order to examine the sensitivity of the BSA effect to protein concentration, we tested the MRP2 and BCRP vesicles using three different protein amounts. For both transporters, the magnitude of BSA effect decreased as the protein amount increased .To determine whether the albumin effect is dependent on the BSA concentration, we compared CDCF and LY uptake in presence of 0.1 and 1.0% BSA. The higher BSA concentration resulted in a larger uptake only at the highest tested CDCF concentration  Km with albumin has earlier been noted for UGT1A1 3-glucuronidation of estradiol in human liver microsomes [Our results show that the BSA effect for MRP2 and BCRP is displayed mainly through changes in V albumin , 45, 46.crosomes  and UGT1crosomes , but them of 6-hydroxyindole metabolism by UGT2A1 [max of entacapone metabolism by UGT1A7. The effect of albumin that is observed on the efflux transporter activity is more comparable to the results obtained with CYPs, where albumin decreased the Km of phenytoin and paclitaxel hydroxylation in human liver microsomes by 4- and 3-fold respectively [The effect of BSA in this study was lower for MRP2 and BCRP (maximum 3-fold) than what has previously been reported for metabolic enzymes. Albumin caused a > 20-fold decrease in the Ky UGT2A1  as well y UGT2A1 , 45. Many UGT2A1  also repectively , 41. Theectively , 46, 48.BSA has a slightly larger effect with the studied MRP2 assay than the BCRP assay, but that might be due to the different protein amounts used in the assays. In our assays, the total amount of protein in the MRP2 and BCRP vesicles were 7.5 μg and 50 μg, respectively. However, the direct comparison of vesicle amounts between MRP2 and BCRP would not be feasible since the vesicle preparations are not the same. Moreover, we investigated if the cholesterol treatment could cause the observed differences. Apart from changing the properties of the membrane, cholesterol addition may remove some of the free fatty acids or other lipophilic compounds in the vesicle preparation through binding to the cyclodextrin complex used to solubilize cholesterol during cholesterol loading. However, as the cholesterol loading does not appear to influence the observed BSA effect in BCRP vesicles, it is unlikely that it would cause the difference between BCRP and MRP2 either. It is possible that the difference in the BSA effect on the MRP2 and BCRP assays are due to substrate- or transporter-dependency, as has been observed for UGTs .50 value for BCRP, 28.2 μM, should be considered as an apparent value, because the concentration of oleic acid inherently present in the vesicle preparation is unknown and high concentrations of oleic acid interferes with the membrane structure of the cholesterol-loaded BCRP vesicles. High concentration (> 1 mM) of oleic acid was previously reported to decrease BCRP-mediated transport of mitoxantrone in Caco-2 cells [Albumin is proposed to influence the activity of metabolic enzymes through binding of inhibitory free fatty acids that originate from the vesicles. Oleic acid, the most abundant fatty acid in Sf9 cells, was able to inhibit BCRP transport activity, but our assay was unsuitable to investigate the effect of oleic acid on MRP2, since oleic acid interfered with the membrane integrity of MRP2 vesicles. The calculated IC-2 cells  and rosumax and Km values of the substrates increased in our study, the calculated transporter mediated clearance would be only slightly altered (30% higher) compared to conditions without BSA, considering linear kinetics where clearance is the ratio of Vmax to Km. Such small alterations in clearance are unlikely to have a major role in in vitro—in vivo predictions of the MRP2- and BCRP -mediated transport, as they are easily lost in other variability such as inter-laboratory variation. However, as the albumin effect has been observed to be both transporter- and substrate-dependent for the metabolic enzymes, it may have a larger impact with other substrates and/or transporters and should therefore be kept in mind when BSA is included in the vesicular assay for example as a solubilizer.In summary, we have shown that the addition of BSA to the vesicular transport assay has an effect on the maximal uptake rate of MRP2 and BCRP substrates. Oleic acid inhibited BCRP, which is in line with the proposed mechanism behind the albumin effect on metabolic enzymes. The inhibitory effect of oleic acid on MRP2 could not be investigated, as oleic acid damaged the MRP2 vesicles. The effect of BSA on substrate uptake was dependent on the vesicle amount, determined by protein concentration, while increasing BSA concentration did not significantly improve substrate uptake. Even though albumin clearly has an effect in the vesicle transport assay, it may not have a very large effect on the estimated clearance of transporter substrates. Since both V


The causal effect is confirmed in sensitivity analyses. In identifying this positive causal effect of age at menarche on time spent in education, we offer further insight into the social effects of puberty in girls.Menarche signifies the primary event in female puberty and is associated with changes in self-identity. It is not clear whether earlier puberty causes girls to spend less time in education. Observational studies on this topic are likely to be affected by confounding environmental factors. The Mendelian randomization (MR) approach addresses these issues by using genetic variants  as proxies for the risk factor of interest. We use this technique to explore whether there is a causal effect of age at menarche on time spent in education. Instruments and SNP-age at menarche estimates are identified from a Genome Wide Association Study (GWAS) meta-analysis of 182,416 women of European descent. The effects of instruments on time spent in education are estimated using a GWAS meta-analysis of 118,443 women performed by the Social Science Genetic Association Consortium (SSGAC). In our main analysis, we demonstrate a small but statistically significant causal effect of age at menarche on time spent in education: a 1 year increase in age at menarche is associated with 0.14 years (53 days) increase in time spent in education  contains supplementary material, which is available to authorized users. Puberty is a time of physiological change in the human body, and its effects extend into the social domains of life  technique can often be used to overcome these limitations by using single nucleotide genetic polymorphisms (SNPs) as instrumental variables (IVs) to explore the direction and magnitude of any causal effect of age at menarche on time spent in education . We measure the strength of the instruments using the F statistic, which is a function of the magnitude and precision of their genetic effects , the summary estimates for which can be downloaded from Individual MR estimates for each of the 122 SNPs were derived using the Wald estimator, which is the ratio of the estimates of the two genetic associations (i.e. SNP-time spent in education estimate over SNP-age at menarche estimate)  describes the percentage of total variation in MR estimates across instruments that arises because of heterogeneity rather than chance, and can be used as a proxy for pleiotropy , is used to quantify any potential bias arising in the MR-Egger analysis due to measurement error. An I2GX estimate close to 100% would suggest that such a phenomenon is not creating bias, as greater heterogeneity reduces regression dilution with MR-Egger  Age at menarche has been previously demonstrated to be associated with obesity   and R version 3.3.2 (R Core Team).Supplementary Tables 2 and 3 report individual SNP estimates of the per-allele effects on age at menarche (years) and time spent in education for women , respectively, while Supplementary Table 4 reports individual SNP MR estimates for the causal effect of age at menarche on time spent in education . The considered SNPs are all strong instruments for age at menarche, with F statistics ranging from 25 to 576 (Supplementary Table 2), which are all greater than the recommended threshold of 10 . With the standard deviation of time in education reported as 3.6 years . Further sensitivity analyses were performed, and MR-Egger regression analysis estimated that a 1 year increase in age at menarche is associated with 0.10 standard deviation increase in time spent in education  . The I2GX statistic is 85%, suggesting that there is no major evidence of measurement error biasing MR-Egger analysis , with a p value of 3.5 × 10−5) . Fixed-effect IVW meta-analysis after excluding the 12 SNPs associated with BMI (Supplementary Table 1) also shows a statistically significant causal effect of age at menarche on time spent in education: a 1 year increase in age at menarche is associated with 0.05 standard deviation increase in time spent in education , although evidence of pleiotropy persists . Thus, removing these 12 SNPs that are associated with BMI did not have any major effect on the results obtained, and for this reason, the results of the original IVW approach are reported as the main analysis.The weighted median approach estimates that a 1 year increase in age at menarche is associated with 0.05 standard deviation increase in time spent in education . This is reassuring that the causal effect of age at menarche on time spent in education shown by our main analysis is not attributable to bias due to use of SNP-age at menarche estimates from discovery stage results. The unweighted allele score is only used here to test for a causal effect of age at menarche on time spent in education and not to estimate the magnitude of this effect.Use of an unweighted allele score for the 122 instruments in women shows a statistically significant positive association with time spent in education , thus strengthening our belief that the observed association in women is driven by mediation through age at menarche.In summary, all sensitivity analyses support our findings of a statistically significant causal effect of age at menarche on time spent in education.We have used MR to investigate the causal effect of age at menarche on time spent in education. Under the required assumptions, this technique circumvents the classical confounding seen in observational studies, and our main  analysis suggests that for every year increase in age at menarche, women spend an extra 53 days in education on average (95% CI 37–78 days). The main limitation of the MR approach is possible bias due to pleiotropic instruments, and we have addressed this using several sensitivity analyses.The physical, behavioural and cognitive aspects of development that are associated with puberty vary in their timing. A lower age at menarche has been hypothesized to result in earlier physical and physiological development, but without matching levels of cognitive and behavioural development. This delay can lead to inadequate coping strategies, greater risk-taking behaviour, lower social competence, and higher rates of internalizing and affective disorders  and SNP-age at menarche  estimates for all 122 SNPs. The red line depicts the IVW meta-analysis estimate, the dashed blue line the MR-Egger estimate and the dashed black line the weighted median estimator. (BMP 1122 KB)Supplementary Figure 2. Funnel plot of 1/standard error of MR estimate (y-axis) by the MR estimate (x-axis), to highlight any evidence of directional pleiotropy . There is no major asymmetry around the fixed-effect IVW meta-analysis causal estimate (dashed red line) to suggest directional pleiotropy. The blue line depicts the MR-Egger causal estimate. (BMP 1181 KB)Supplementary material 3 (DOCX 648 KB)Supplementary material 4 (XLSX 20 KB)Supplementary material 5 (XLSX 13 KB)Supplementary material 6 (XLSX 14 KB)Supplementary material 7 (XLSX 15 KB)


The advantage of using highly soluble esters rather than carboxylic acids as starting compounds became evident when the reactions were performed under flow conditions. On the other hand, when 2-vinylbenzoic acid derivatives were employed as reagents, isobenzofuranones were obtained together with unprecedented benzo[ Unfortunately, reactions performed in inert solvents with isocyanides, carboxylic acids, and mixtures of both, alcohols or amines never succeeded in affording the desired multicomponent adducts. It was rather curious instead to find out that many solvents or additives would attack the radical/cationic intermediate while reactants rationally and appositely selected would not. Some products unexpectedly obtained during various attempts are shown in Based on our experience on multicomponent reactions, we postulated that intermediate A could be more favourably attacked in an intramolecular fashion, with the nucleophilic group appropriately displayed by one of the reagents. By inspection of the literature data, we were surprised to find only one example where benzothiophenes were obtained by reacting o-methylthioarenediazonium salts with alkynes under green light irradiation [At this stage we postulated that adiation .We therefore designed and synthesized reagents bearing ester or carboxylic functions in a convenient position, for a possible intramolecular attack, to investigate their reactivity. While working on this project, König and Fagnoni reported the photoredox-catalyzed reaction of 2-carboxybenzenediazonium salts with various alkenes , which p2a with methyl methacrylate (3a) in acetonitrile in the presence of a catalytic amount of Ru(bpy)3Cl2 under blue laser irradiation, we could isolate isochromanone 4a in 72% yield  without any other detectable product. However, the TLC analysis of the crude material, after staining with cerium molybdate, displayed a plethora of red-colored spots typical for 4-methoxybenzyloxycarbonyl (MeOZ) cleavage with trifluoroacetic acid, thus suggesting the formation of a 4-methoxybenzyl carbocationic species.As no other detectable products were isolated from the reaction mixtures, we were intrigued by the fate of the alkyl group of the ester functionality that was not incorporated in the final products. Diazonium salt tamide 5 . This ou2i was reacted with styrene (3b), the formation of dihydroisoquinolinone 10 was not observed, while relatively unstable imidate 9 was detected. This observation supports our hypothesis that the nucleophilic attack to the intermediate A is taken by the carbonyl oxygen. Compound 9 immediately afforded isochromanone 4d (60%) upon exposure to water .In addition, when diazonium salt to water . Carefulto water  allowed The interest of both academia and industry in flow chemistry has recently increased. In particular, photochemical reactions performed under continuous flow have been proven particularly effective , as the Having solved the issue of partial insolubility of diazonium salts by replacing anthranilic acid derivatives with the corresponding esters and having demonstrated that an inert atmosphere was not essential, we therefore decided to test our synthetic methodology with an in house-made flow apparatus. The reaction coil was formed with 1 m of FEP tubing  wrapped around a glass cylinder. Irradiation was obtained with strips of blue LEDs attached inside a hollow cylinder. The reagents were supplied by means of a single solution via a syringe pump .4c . All reagents were supplied by the same syringe, which was kept shielded from the light source. In fact, we verified that no reaction was taking place in the dark, even prolonging the experiment for several hours.The best results were obtained with a 130 mM solution of the diazonium salt in acetonitrile, 2 equiv of alkene and 0.5% of catalyst, and a residence time in the reaction coil (1 mL) of 10 h. Although nitrogen evolution started within the first few minutes of irradiation, shorter residence times resulted in lower conversions, as shown in Although in the past we observed improvements in yields and purities switching from batch to flow conditions , in this13 by introducing an ester functional group in the alkene building block, according to the pathway depicted in Having set up the conditions for the synthesis of isochromanones, we moved to explore the possibility to obtain isobenzofuranones 11a) and 2-ethoxycarbonylstyrene (12a) under the conditions previously developed for isochromanones. The reaction, together with isobenzofuranone 13a (31% yield), unexpectedly afforded also compound 14a in 17% yield . Nitrilium ion B is then intramolecularly intercepted by the ester moiety and a seven-membered ring is formed, with loss of an ethyl group.The reaction was initially tested with 4-methoxybenzenediazonium tetrafluoroborate (7% yield . Benzoxa13a was selectively obtained in 40% yield (12b) to our reaction conditions, achieving an overall 41% yield and no selectivity among the two products  but with an opposite selectivity , affording the major product 14b in 41% yield together with traces of isobenzofuranone 13b , furnished only traces of the products, showing a dramatic dependence of the reaction course from the substrates employed.Apparently, the electronic effects of the substituents of the diazonium salt play a major role, especially when considering the reactivity of benzenediazonium tetrafluoroborate 11a and 12a was also performed under flow conditions, with no apparent improvement (The reaction of rovement , entry 92c with 12a. In this case both substrates bear an ester functionality and in principle both six- and five-membered rings could be obtained. Unexpectedly, upon exposure to standard reaction conditions, isobenzofuranone 13c was isolated in 58% yield, while only traces of isochromanone 4l were detected in the crude material (We were expecting to find further demonstration that cyclization to isochromanone would be favored with respect to benzofuranone from the reaction of compound material . This fiBenzopyranones are widespread in nature, being found in a wide variety of organisms, including fungi, and are endowed with a broad-spectrum of biological activities . Also beFile 1Experimental details and detailed spectroscopic data.


Given the astounding rates of diabetes and related complications, and the barriers to providing care present in Indigenous communities in Canada, intervention strategies that take into account contextual factors such as readiness to mobilize are needed to maximize improvements and increase the likelihood of success and sustainment. As part of the national FORGE AHEAD Program, we sought to develop, test and validate a clinical readiness consultation tool aimed at assessing the readiness of clinical teams working on-reserve in First Nations communities to participate in quality improvement (QI) to enhance diabetes care in Canada.A literature review was conducted to identify existing readiness tools. The ABCD – SAT was adapted using a consensus approach that emphasized a community-based participatory approach and prioritized the knowledge and wisdom held by community members. The tool was piloted with a group of 16 people from 7 provinces and 11 partnering communities to assess language use, clarity, relevance, format, and ease of completion using examples. Internal reliability analysis and convergence validity were conducted with data from 53 clinical team members from 11 First Nations communities (3–5 per community) who have participated in the FORGE AHEAD program.r = 0.45, p < 0.05.The 27-page Clinical Readiness Consultation Tool (CRCT) consists of five main components, 21 sub-components, and 74 items that are aligned with the Expanded Chronic Care Model. Five-point Likert scale feedback from the pilot ranged from 3.25 to 4.5. Length of the tool was reported as a drawback but respondents noted that all the items were needed to provide a comprehensive picture of the healthcare system. Results for internal consistency showed that all sub-components except for two were within acceptable ranges (0.77–0.93). The Team Structure and Function sub-component scale had a moderately significant positive correlation with the validated Team Climate Inventory, The testing and validation of the FORGE AHEAD CRCT demonstrated that the tool is acceptable, valid and reliable. The CRCT has been successfully used to support the implementation of the FORGE AHEAD Program and the health services changes that partnering First Nations communities have designed and undertaken to improve diabetes care.NCT02234973. Date of Registration: July 30, 2014Current ClinicalTrial.gov protocol ID The online version of this article (doi:10.1186/s12913-017-2175-6) contains supplementary material, which is available to authorized users. The burden of diabetes on both families and the healthcare system in Canada is growing at an astonishing pace with prevalence rates that have more than doubled in the last decade . In 2010It has been suggested that barriers to optimal diabetes care are different and sometimes more pronounced in Indigenous communities in Canada than those experienced in non-Indigenous communities due to geographic isolation, cultural differences and the disjointed healthcare provided by a combination of federal government, provinces and territories , 11. GivWhen evaluating readiness relative to the healthcare setting in Indigenous communities in Canada, a valuable assessment is possible when all individuals involved in the prevention and treatment process participate . The results of these evaluations can be used to guide care and maximize health improvements . Holt etDetermination of health teams’ degree of readiness is advantageous with regards to maximizing the success of interventions through the identification of available resources and the present knowledge of a health issue . A low rTransFORmation of IndiGEnous PrimAry HEAlthcare Delivery (FORGE AHEAD): Community-driven Innovations and Strategic Scale-up Toolkits is a 5-year national research program that partners with Indigenous communities in Canada to improve chronic disease care and access to available resources by developing and evaluating community-driven, culturally-relevant primary healthcare models through a quality improvement (QI) process [The  process . The pro process , 22; andwith communities rather than on communities is consistently recognized as a critical factor in research with Indigenous partners [The objective was to develop a validated Clinical Readiness Consultation Tool (CRCT) to assess the readiness of clinical teams working on-reserve in Indigenous communities before they engage in QI efforts to enhance diabetes care in Canada. A consensus approach  within tpartners , 26, 27.partners .FORGE AHEAD partners with 11 First Nations communities across 6 provinces  and three isolation levels .A literature review was conducted in March 2014 using PubMed to identify existing clinical readiness tools published prior to January 2014. Key search terms included “Indigenous” or “First Nations” or “Aboriginal AND “healthcare” or “primary healthcare” or “chronic disease” AND “clinical readiness”, or “clinical change readiness” or “organizational readiness” or “organizational change” or “change management”. With no tools found in the Canadian Indigenous domain, the search was expanded internationally, included all populations, and examined grey literature for key content. A total of 13 PubMed articles and four grey literature articles/sources were found. Inclusion criteria included use in Indigenous populations, a health domain tool, and restricted to clinicians (versus patients).Members of the Working Group which consisted of experts in the fields of readiness, Indigenous research, survey development and chronic disease care/epidemiology assessed the literature to identify the most appropriate tools for use within FORGE AHEAD. From the review of literature, the Chronic Care Model , ExpandeThe aforementioned existing tools  were considered for the FORGE AHEAD program; however the following drawbacks were identified: i) reliance on costly resources external to the community for tool implementation and/or interpretation, ii) not tailored to the unique Canadian setting and First Nations primary healthcare setting, iii) not focused on type 2 diabetes, iv) lack of a qualitative component where Indigenous team members’ can add information regarding their insights and perceptions regarding diabetes care, and, v) no built-in mechanism to integrate individual perceptions to be used to support clinical readiness at a small group level. Therefore, the FORGE AHEAD Program aimed to develop and test a CRCT relevant to diabetes care in the Canadian Indigenous context that could be applied by primarily using existing internal community resources without the need for external interviewers and transcription. The ABCD-SAT (Version 2) was selected by the Working Group as the basis for conceptualizing and developing the CRCT. The overall goal of the Working Group was to develop a comprehensive, user-friendly, understandable, and stand-alone questionnaire that could be administered and used by community-based facilitators  in First Nations communities within the FORGE AHEAD Program to assess readiness to adopt chronic disease interventions.Adapting the ABCD-SAT into the CRCT involved a thorough review by Working Group members over a series of six rounds from January to July 2014. Each of the components and sub-components as well as the individual items that encompass the sub-components of the ABCD-SAT were reviewed for relevance to the context of Canadian First Nations primary healthcare delivery systems, redundancies, cultural relevance, and language. One item was added on cultural competence and two questions about registries were removed. Type 2 diabetes specific examples were inserted to enhance clarity in most items. All items were revised to reflect the Canadian First Nations context and the language was adjusted for improved clarity and ease of understanding.In June 2014, 15 people from 7 provinces comprised of 3 co-investigators and 12 Indigenous and non-Indigenous community members from 11 partnering communities received the pilot CRCT. This group was selected given their prior involvement in the planning and development of the FORGE AHEAD Research Program proposal. Community members included family physicians, nurses, health managers and administrators, health promoters, and adult educators. The draft CRCT was distributed electronically along with a feedback form  completed the CRCT at three time points: pre, during, and post program implementation. For this study, only pre-program implementation data was used to avoid a learning effect bias.α) which measures whether a group of items is related to a single construct or sub-component [α but an α greater than 0.9 may also be indicative of a redundant item. An α greater than or equal to 0.7 is considered satisfactory (34). Sub-components with a single item were excluded from the internal consistency analysis. Overall α for the total scale (CRCT) was not calculated because each sub-component is intended to measure a distinct factor. Missing data was handled through pairwise deletion  of cases. Descriptive statistics were generated to evaluate the score distribution and proportion of missing data per item. Items with high levels of missing data (>10%) were identified and considered for further analysis and interpretation.The internal reliability assessment was based on the correlations between the individual items that make up the sub-components, relative to the variances . The intomponent . The higAs part of FORGE AHEAD, participants also completed the Team Climate Inventory (TCI) to examine team functioning of clinical teams working on-reserve. The TCI  has been validated  and usedThe 27-page CRCT  completed the feedback form for an overall review and response rate of 53.3%. The CRCT took an average of 118 min to complete and ranged from 53 to 240 min. Based on the five-point Likert scale feedback form, average respondent scores were: appropriate language use for First Nations health clinics in Canada (3.75); questions clearly written (3.25); questions are relevant to health clinics in First Nations communities (4); format of the tool is appropriate (4.5); and, helpfulness of examples to complete the clinical readiness assessment (4). The 12-point Likert scale of the CRCT was maintained based on the qualitative feedback. Analysis of the written answers to open-ended questions revealed that although the questionnaire was long, respondents found that all the questions were important and necessary to obtain a comprehensive understanding of the current services available in communities. Some respondents noted that the language was geared for higher education/reading and comprehension skills. Revisions to the tool were made following the pilot to improve clarity to address this concern.Of the 53 people who completed the CRCT, 87% were female and 62% were Indigenous   to calculate sub-component 3) Table . Two subα < 0.7, a low level of correlation (<0.4), and where removal of the item did not affect the total α  were assessed by the Working Group members based on the internal consistency results and the theoretical concepts to ensure acceptability of the level of consistency and validity. Of the 68 items, only one was found to increase the reliability of the tool if removed. The Appointment and Scheduling sub-component had three items and removing the third item (Is it routine practice for the diabetes community based activities and programs to be planned or scheduled ahead of time?) improved the internal consistency of the sub-component (from 0.66 to 0.69). The two-item Patient Access sub-component was retained given that information related to patients’ barriers to access are an important aspect of chronic disease care. Two items in the Communication and Cooperation subcomponent were removed due to redundancy (α = 0.91),  and .The CRCT items with r = 0.45, p < 0.05 , which demonstrated adequate internal consistency for standard scale development criteria. Based on a rigorous review guided by theoretical and statistical principles, only three out of 74 items were removed.Examination of the CRCT response data also demonstrated good acceptability of the tool with a high completion rate for all items except 8 that were left unanswered by more than 10% of the participants. Given the relatively high number of items in the CRCT, the proportion of unanswered items represents a small number. In addition, all items with higher proportions of missing data were those asking about the regional health planning and development, organizational commitment, or existing QI strategies at the health center. Given the fact that participants had a wide range of professional roles, this could be due to difficulty in understanding those questions and/or less knowledge about the issues that may have been more external to their role. In fact, of the four Health Directors/Program Managers who completed the CRCT, all four answered all the questions about regional health planning and development, organizational commitment and existing QI strategies. Reliability analysis demonstrated an acceptable As expected, the TSF sub-component of the FORGE AHEAD CRCT showed evidence of convergent validity when compared to the validated TCI tool. This suggests that the sub-component of the CRCT intended to examine team functioning measures the same construct as the TCI. The moderate level of correlation is acceptable and can partly be explained by the fact that the TSF and TCI are structured differently with varying scales and types of questions. While the CRCT instrument has demonstrated validity and reliability, there are limitations. The small sample size did not allow for a confirmatory factory analysis to test whether the data collected from First Nations communities in Canada fits with the hypothesized measurement of readiness using the CRCT. Future confirmatory factor analysis studies with larger samples are suggested to better establish the validity of the FORGE AHEAD CRCT. Also, the psychometric properties of convergent validity for the entire CRCT could not be evaluated due to the non-existence of validated tools that measure a similar concept. As such, only the TSF sub-component was tested for convergent validity against the validated TCI.The FORGE AHEAD Program is still in its implementation phase and as such, complete data are not yet available on the recruitment and retention of partnering communities, community and clinical team members QI initiatives, clinical outcomes, and cost analyses. Data on the use of the CRCT to facilitate QI initiatives and the impact of the CRCT on health-related outcomes will be available upon completion of the program. Detailed process evaluations including stakeholder interviews will inform further revisions to the CRCT as well as best practices for the successful implementation and scale-up of the tool. Process evaluations will also highlight how the CRCT enabled understanding and relationship building among healthcare team members working in Indigenous communities and between researchers and community stakeholders.In conclusion, the validated CRCT has been successfully used to support the implementation of the FORGE AHEAD Program and the health services changes that partnering First Nations communities have designed and undertaken to improve diabetes care.Additional file 1:FORGE AHEAD: Clinical Readiness Consultation Tool – Community Feedback Form. The Community Feedback Form was used during the pilot of the CRCT and was includes a qualitative (open-ended) section for suggestions/comments and a five-point Likert scale to gather responses on the following: (1) appropriate language use for First Nations health clinics in Canada, (2) clarity of questions, (3) relevance of questions to health clinics in First Nations communities, (4) appropriate format for the tool, and, (5) helpfulness of examples provided for each question to complete the clinical assessment tool. (DOCX 43 kb)Additional file 2:FORGE AHEAD: Clinical Readiness Consultation Tool. The 27-page tool has 4 main sections: 1) 1-page introduction describing the background, confidentiality, benefits, risks, reimbursement, consent, and contact information; 2) 1-page brief instruction ; 3) general information (brief 8-item demographic profile); and 4) 5 main components and sub-components of healthcare systems important in chronic disease care. (DOCX 92 kb)