Our work demonstrated that the specific microbial strains used to construct a microbiome could impact the species composition, perturbation responses, and functional outputs of that system. These findings suggest that 16S rRNA gene taxonomic profiles alone may have limited potential to predict the dynamics of microbial communities because they usually do not capture strain-level diversity. Observations from our synthetic communities also suggest that strain-level diversity has the potential to drive variability in the aesthetics and quality of surface-ripened cheeses. Staphylococcus equorum, Brevibacterium auranticum, and Brachybacterium alimentarium) from nine cheeses produced in different regions of the United States and Europe to construct nine synthetic microbial communities consisting of distinct strains of the same three bacterial species. Comparative genomics identified distinct phylogenetic clusters and significant variation in genome content across the nine synthetic communities. When we assembled each synthetic community with initially identical compositions, community structure diverged over time, resulting in communities with different dominant taxa. The taxonomically identical communities showed differing responses to abiotic  and biotic (the fungus Penicillium) perturbations, with some communities showing no response and others substantially shifting in composition. Functional differences were also observed across the nine communities, with significant variation in pigment production (light yellow to orange) and in composition of volatile organic compound profiles emitted from the rinds (nutty to sulfury).Diversification can generate genomic and phenotypic strain-level diversity within microbial species. This microdiversity is widely recognized in populations, but the community-level consequences of microbial strain-level diversity are poorly characterized. Using the cheese rind model system, we tested whether strain diversity across microbiomes from distinct geographic regions impacts assembly dynamics and functional outputs. We first isolated the same three bacterial species (IMPORTANCE Our work demonstrated that the specific microbial strains used to construct a microbiome could impact the species composition, perturbation responses, and functional outputs of that system. These findings suggest that 16S rRNA gene taxonomic profiles alone may have limited potential to predict the dynamics of microbial communities because they usually do not capture strain-level diversity. Observations from our synthetic communities also suggest that strain-level diversity has the potential to drive variability in the aesthetics and quality of surface-ripened cheeses. Through variations in abiotic  and biotic  selection pressures applied during cheese production and aging , Brevibacterium auranticum (hereafter Brevibacterium), and Brachybacterium alimentarium (hereafter Brachybacterium)—from nine different cheeses made across the United States and Europe  and Brevibacterium (ranging from 72 to 213 unique gene clusters), suggesting that these taxa have the most dynamic accessory gene content.The 16S rRNA gene region had 100% pairwise identity within all ains see . C1 was ommunity . The othommunity . The totommunity . Variabi10.1128/mSystems.00149-20.1FIG S1Staphylococcus equorum, (B) Brevibacterium auranticum, and (C) Brachybacterium alimentarium. 16S rRNA gene sequences were extracted from each of the genomes and were aligned with 16S rRNA gene sequences of closely related type specimens using MUSCLE. RAxML 8.2.11 (with a GTR GAMMA nucleotide model and 100 bootstrap replicates) was used to create a 16S rRNA gene maximum likelihood phylogeny. Download FIG S1, TIF file, 2.1 MB.16S rRNA gene phylogenies for (A) Copyright © 2020 Niccum et al.2020Niccum et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.00149-20.2FIG S2Staphylococcus equorum, (B) Brevibacterium auranticum, and (C) Brachybacterium alimentarium. Download FIG S2, TIF file, 1.6 MB.Maximum likelihood SNP consensus trees. Trees were constructed from SNPs identified across the nine genomes of (A) Copyright © 2020 Niccum et al.2020Niccum et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.00149-20.5TABLE S2Table S2, XLSX file, 2.0 MB.Distribution of gene clusters in the three taxa from each of the nine cheese rind microbiomes. Filled cells indicate that a predicted gene belongs to a gene cluster (row). In some communities, multiple genes belong to a single gene cluster. The identifiers in the cells are the gene identifiers (IDs) of each of the genomes based on the RAST annotation of that genome. Rows that are highlighted with a color are referred to in the manuscript text. See column R for details. Download Copyright © 2020 Niccum et al.2020Niccum et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Brevibacterium of C3 has a unique potassium transport system encoded by genes with high similarity to the kdfABCF operon  of the cheese curd, (ii) growth of Brachybacterium in the middle step of succession, and (iii) dominance by Brevibacterium at the end of succession  early colonization of ccession , 43. We in vitro community assembly assays to measure total CFU and community composition (relative abundance of each species)  . Communidynamics , 43. We F8,81 = 2.07, P = 0.05; day 10 ANOVA F8,79 = 0.46, P = 0.88). However, there were substantial differences in community composition across the nine cheese rind communities . Many communities  were dominated by Brevibacterium at the end of succession  grew poorly alone on cheese curd agar and were strongly stimulated by growth in the community. One Brevibacterium strain (C4) was inhibited by growth in the community. All Brachybacterium strains grew well on cheese curd by themselves and were generally inhibited when grown in the community.A simple explanation for differences in community composition across the nine cheese rind communities is that individual bacterial strains have different growth abilities alone and in the community. Those taxa and strains that grow best alone and with the community present should be the most abundant members of the community. To test this, we determined total growth of each of the 27 strains on cheese curd agar and compared growth alone after 10 days to growth in the community. All ommunity . Two strStaphylococcus r2 = 0.166, P = 0.276; Brevibacterium r2 = 0.001, P = 0.923; Brachybacterium r2 = 0.020, P = 0.716). A somewhat better predictor of mean relative abundance was how growth of each strain was impacted by the community . This suggests that interactions between each of the strains and their communities may contribute to differences in community composition across the nine synthetic communities. For example, the inhibition of Brevibacterium and lack of inhibition of Brachybacterium in C4 may partly explain why this community was the only one to be dominated by Brachybacterium.For all three taxa, mean growth alone was a very poor predictor of mean relative abundance in the community  to molds   , 25, 46.bacteria , 43, 47,Penicillium (+Penicillium). We used a strain of Penicillium that was isolated from a natural rind cheese and was previously demonstrated to inhibit cheese rind bacterial growth , the presence of Penicillium caused an increase in the relative abundance of Staphylococcus. The 6% salt treatment caused fewer shifts in community composition, with only two communities (C5 and C6) responding to the higher-salt environment. In both cases, Brevibacterium increased in relative abundance.To determine how the nine synthetic cheese rind communities would respond to salt and fungal perturbations, we used the community assembly assay described above with the addition of two treatments: a 6% NaCl treatment and a treatment with added l growth . Across hibition . Rind miposition  and C. IOur experiments described above demonstrated that strain-level diversity of cheese rind taxa drove divergence in community composition across the nine microbiomes. Does this divergence lead to the production of cheeses with different properties that could be perceived by consumers? Differences in community composition may not necessarily translate into differences in functional outputs. Many studies of the microbiome have suggested that communities with different compositions may have similar functions due to functional redundancy across community members –50. Whil–F9,39 = 524.9, P < 0.0001), with C3, C4, C6, C7, and C9 having significantly greater a* values (where a* represents one of two chromatic coordinates) than the control, indicating more red pigmentation , with C3 and C4 having the highest values and appearing the most orange . The compositions of VOCs across the nine cheese communities were significantly different   . As dete< 0.001) –65. C5 hBrevibacterium would potentially have higher red and yellow pigmentation due to orange carotenoid production by Brevibacterium. We do not believe that this explains pigmentation differences across the communities because the two communities with the highest Brevibacterium abundances  in the dark (representing the conditions under which the cheese communities were incubated), the results showed considerable variation in pigment production in Brevibacterium strains, some pigment variation in Staphylococcus strains, and no observable pigment variation in Brachybacterium strains (Brevibacterium strains that showed high levels of orange pigmentation (Brevibacterium strains harbored predicted genes corresponding to the carotenoid biosynthesis operon previously characterized in Brevibacterium species (Staphylococcus strain (908_10 from C7) was noticeably yellow in color on CCA  in just one species in the communities, irrespective of the final abundance of that species in the community. For example, when  strains . Interesentation . Genomes species , 66, 67,r on CCA  and corrs genome . Further reasons  and C. T strains  and D, w strains . These p10.1128/mSystems.00149-20.3FIG S3Brachybacterium alimentarium, Brevibacterium auranticum, and Staphylococcus equorum. (B) Lawns of strains of Brachybacterium alimentarium, Brevibacterium auranticum, and Staphylococcus equorum from the nine cheese communities demonstrating strain differences in pigmentation when grown on cheese curd agar. Lawns were incubated for 10 days. Photos were taken in natural light ∼20 cm above the surface of the cheese curd agar. Each square represents ∼2.25 cm2. (C) Color profiles of lawns of Brevibacterium after 10 days of rind development. Each dot represents a replicate cheese curd agar plate (n = 3). The a* and b* values were significantly different across strains , with the C3 and C7 strains having the highest levels of yellow and red pigmentation. (D) Color profiles of lawns of Staphylococcus after 10 days of rind development. Each dot represents a replicate cheese curd agar plate (n = 3). a* and b* values were significantly different across strains , with the C7 strain that contains the staphyloxanthin carotenoid operon having the highest levels of yellow pigmentation. Download FIG S3, TIF file, 2.0 MB.Differences in pigment production of individual bacterial strains. (A) Spots (10 μl) of concentrated experimental glycerol stocks were grown on PCAMS medium for 2 days in the dark to illustrate pigment differences across strains of Copyright © 2020 Niccum et al.2020Niccum et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Brevibacterium or Brachybacterium . The divergence was not due to stochastic community assembly across replicates, as we observed highly reproducible community structures across replicate experiments.Using synthetic communities with identical three-member communities isolated from nine distinct cheeses, our work demonstrated the significance of strain-level variation for microbiome assembly and function. Studies of plant and animal communities have demonstrated that intraspecific genetic and phenotypic diversity can impact community assembly and function –70. Here–Brachybacterium dominated, the levels of growth of a Brachybacterium strain alone and in interactions with community members were similar to those seen with other communities where Brevibacterium dominated . Future work exploring the roles of inhibitory and cooperative interactions will pinpoint specific mechanisms explaining the variable community assembly dynamics of cheese rind microbiomes.The goal of this work was to determine whether strain-level diversity can impact community dynamics and functions. The limitations imposed by the use of only nine communities made it difficult to pinpoint specific ecological or genetic mechanisms that might underlie the observed differences across the communities. One simple explanation for the dominance of different taxa across the nine rind communities is differences in growth of individual strains. Our experiments comparing growth of individual strains alone versus in the community demonstrated differing growth rates and interactions with the community for each of the three taxa. However, the results do not fully explain the community structures. For example, in C4, where The evolutionary processes that have generated the divergent species and community-level responses of our cheese microbiomes are currently unknown. It is possible that each microbiome has experienced different evolutionary histories in each cheese production environment. As new batches of cheese are introduced to a cave environment, communities may be repeatedly transferred to these new cheeses. This repeated colonization of the cheese substrate could allow rind microbiomes to evolve collectively as a community in the individual production environments . Each enOur model communities represent the widespread bacterial taxa found in cheese rinds. We acknowledge that these communities have limitations that might impact translation of our results to other systems. First, our communities harbored only three bacterial species. While some widespread microbiomes have low species diversity , 73, man15–Brevibacterium pigmentation across strains suggested that high intraspecific variation of functional traits within one species might be a driver of functional divergence across communities. Variation in levels of carotenoid production across strains of cheese Brevibacterium species has been previously reported  in the metagenomic sequence data from these samples. We initially plated out 50 cheese rinds and isolated putative colonies of Brachybacterium, Brevibacterium, and Staphylococcus from 19 of the samples. We collected isolates from only 19 samples because the remaining samples did not contain colonies with morphologies similar to those of the three target bacteria. A pure culture isolation on a fresh PCAMS plate was made from one colony from each unique morphotype identified within each rind community. Brachybacterium alimentarium colonies have medium growth rates, are large and flat, and are yellow-green in color. Brevibacterium auranticum colonies are usually slow-growing, medium-sized, and orange. Staphylococcus equorum colonies are usually fast-growing, smooth, medium-sized, flat, and either white or light golden in color. Initial identification of the isolates was done using the 16S rRNA region and primers 27f  was used to create a 16S rRNA gene maximum likelihood phylogeny. To identify phylogenomic relationships within each of the three species as well as between each of the nine communities, we used PanSeq  to identTo compare the presence and absence of genes across strains and species, core and accessory genes were identified by assigning protein-coding sequences to functionally orthologous groups using the MultiParanoid method of the Pan-Genome Analysis Pipeline (PGAP) . SpeciesTo measure community assembly of the distinct rind communities, approximately 20,000 CFU of each species was inoculated on the surface of 150 μl of cheese curd agar  distributed into replicate wells of a 96-well plate, as previously described , 43. ComPenicillium) perturbation experiments were conducted using the same community assembly assay but with 6% salt cheese curd agar or with the addition of Penicillium. Penicillium strain 12, isolated from a natural rind cheese in Vermont, was used in these experiments. We used this strain because it was isolated from a cheese where Staphylococcus, Brachybacterium, and Brevibacterium were also found and because it was used in previous experiments in our lab  and fungal  to allow a larger sampling area. To construct the rind communities, 600,000 CFU of each species was inoculated across the surface of the cheese curd agar. Synthetic communities were incubated for 10 days in the dark at 24°C followed by color and VOC analyses. For analysis of pigment production by individual strains of each species . This handheld colorimeter uses the CIELAB color space to quantify both lightness (L*) and two chromatic coordinates (a* and b*). Similar colorimeters have been used to quantify cheese rind color . Higher 10, an internal standard (obtained from RESTEK). The internal standard was used to determine the relative concentration of each compound. Organics were introduced into a gas chromatograph/mass spectrometer (GC/MS) by thermal desorption. In addition to the use of the Twister blanks, analysis of the cheese curd agar medium was performed to assess background interferences. Compounds present at equal or higher relative concentrations in the media compared to the samples were removed from the data.Volatiles were collected from synthetic rind communities by headspace sorptive extraction (HSSE) using polydimethylsiloxane (PDMS)-coated magnetic stir-bars. HSSE is an equilibrium-driven enrichment technique in which 10-mm-long-by-0.5-mm-thick stir-bars  are suspended 1 cm above the sample by placing a magnet on the top side of the collection vessel cover. Five replicates of each culture were sampled for 4 h. After collection, the stir bar was removed and spiked with 10 ppm ethylbenzene-dm/z 40 to m/z 350, with the electron ionization (EI) source at 70 eV. A standard mixture of C7 to C30 n-alkanes (Sigma-Aldrich) was used to calculate the retention index (RI) value for each compound in the sample.Analyses were performed using an Agilent 7890A/5975C GC/MS equipped with an automated multipurpose sampler (Gerstel). The thermal desorption unit  provided splitless transfer of the sample from the stir bar into a programmable temperature vaporization inlet . The TDU was heated from 40°C (0.70 min) to 275°C (3 min) at 600°C/min under 50 ml/min of helium. After 0.1 min, the CIS, operating in solvent vent mode, was heated from –100°C to 275°C (5 min) at 12°C/s. The GC column   was heated from 40°C (1 min) to 280°C at 5°C/min with 1.2 ml/min of constant helium flow. The MS was scanned from viz., RI and MS (positive identification), or in commercial libraries and literature (tentative identification). Once the data were assigned, the database was annotated to include compound name, CAS number, and RI. If neither positive nor tentative identification was possible (in cases in which the data represented an unknown compound), a numerical identifier was used to identify the compound. The database was annotated to include the GC/MS information described above. In contrast, if peak scans differed (representing unresolved peaks), the software searched for 3 to 5 invariant scans, averaged their spectra, and then subtracted the average spectrum value from the TIC signal. This process was repeated until the residual signal at each scan approximated background noise. If peak signals failed to meet the user-defined criteria described below, no additional information was obtained.Ion Analytics spectral deconvolution software (Gerstel) was used to analyze the GC/MS data , 87. A tt tests were used. Differences in a* and b* values in the pigmentation assay were determined using ANOVA. To determine differences in VOC composition across the nine communities, PERMANOVAs of Bray-Curtis dissimilarities of relative peak areas were used. A SIMPER analysis of relative peak areas of VOCs was used to identify the contributions of each VOC to Bray-Curtis dissimilarity.To determine differences in community composition, permutational multivariate analyses of variance (PERMANOVAs) with Bray-Curtis dissimilarity were used. ANOVA of log-transformed data was used to determine significant differences between total CFU levels across experiments. In cases of unequal variances , Kruskal-Wallis tests were used. To determine relationships between relative abundance and growth of individual strains, linear regressions were used. To compare total growth alone to growth in the community, GCA_002332225.1, GCA_002332445.1, GCA_002332365.1, GCA_003335435.1, GCA_003335365.1, GCA_002332335.1, GCA_003335395.1, GCA_002332395.1, and GCA_002332325.1 (for Brevibacterium auranticum); GCA_003335205.1, GCA_002332305.1, GCA_003335355.1, GCA_003335315.1, GCA_003335415.1, GCA_003335305.1, GCA_002332425.1, GCA_003335265.1, and GCA_003335295.1 ; and GCA_001747895.1, GCA_001747965.1, GCA_001747785.1, GCA_001747865.1, GCA_004143695.1, GCA_001747945.1, GCA_001747805.1, GCA_001747815.1, and GCA_001747825.1 (for Staphylococcus equorum) (the accession numbers are also listed in All genome assemblies have been deposited in NCBI under accession no.


Centralized waiting lists (CWLs) for patient attachment to a primary care provider have been implemented across Canada, including Quebec. Little is known about the implementation of CWLs and the factors that influence implementation outcomes of such primary care innovations. The purpose of this paper is to explain variations in the outcomes of implementation by analyzing the characteristics of CWLs and contextual factors that influence their implementation.A multiple qualitative case study was conducted. Four contrasting CWLs were purposefully selected: two relatively high-performing and two relatively low-performing cases with regard to process indicators. Data collected between 2015 and 2016 drew on three sources: 26 semi-structured interviews with key stakeholders, 22 documents and field notes. The Consolidated Framework for Implementation Research was used to identify, through a cross-case comparison of ratings, constructs that distinguish high from low-performing cases.Five constructs distinguished high from low-performing cases: three related to the inner setting: network and communications; leadership engagement; available resources; one from innovation characteristics: adaptability with regard to registration, evaluation of priority and attachment to a family physician; and, one associated with process domain: engaging. Other constructs exerted influence on implementation , but did not distinguish high and low-performing cases.This is the first in-depth analysis of CWL implementation. Results suggest important factors that might be useful in efforts to continuously improve implementation performance of CWLs and similar innovations. Implementation of innovations is a key step in the diffusion–dissemination–implementation process in terms of maximizing the likelihood of achieving beneficial outcomes . HoweverDespite increasing recognition that context must be considered when looking at variations in the effectiveness of implementation, this research domain is still evolving. Some authors . Indeed,Innovations are introduced regularly in all countries to address problems that appear in healthcare systems. In Canada, the high number of patients without a regular primary healthcare provider has gained increasing attention in reform efforts . Canada’This study aims to explain and understand variations in the outcomes of implementation by analyzing the characteristics of CWLs and contextual factors that influence their implementation.In Canada, seven provinces have implemented CWLs to better manage supply and demand for attachment to a primary healthcare provider . AlthougThis study analyzes CWLs implemented in the province of Quebec, considered a pioneer in CWLs for unattached patients . This prThe CWLs in Quebec, called “Guichets d’accès aux clientèles orphelines” (GACOs) were implemented in 2008 by the Ministry of Health and Social Services (MSSS) in collaboration with the Quebec Federation of General Practitioners. They have a dual objective of: attaching patients to a family physician, and prioritizing vulnerable patients . GACOs wThe GACO in each HSSC is managed by a clerk who receives requests; a nurse who evaluates patient requests on a priority scale based on their clinical profile in collaboration with a local medical coordinator; a physician mandated to help attach patients with a family physician . People Almost a decade after launching the GACO model of CWLs, a research team in Quebec published a performance assessment focused on four outcomes of the implementation process: new requests for a family physician, change in the number of patients on the waiting list and numbers of patients and vulnerable patients attached to a family physician through the CWLs . The assThe Consolidated Framework for Implementation Research (CFIR) developed by the outer setting is defined by the larger socio-demographic, economic, political, geographic context surrounding the inner setting within which the GACO is implemented ;the inner setting refers to characteristics  of the organization where the GACO is implemented: the LHN. It includes both the HSSC in which the GACO is physically located, and all the primary care structures included in the LHN related to that HSSC refer to ;the characteristics of individuals involved in the implementation, including GACO staff members and the family physicians of the LHN, consider knowledge and beliefs, self-efficacy, stage of change and identification with the organization;the innovation characteristics describe characteristics of the GACO related to its adaptability to a given setting, complexity, trialability, evidential support and its relative advantages (or disadvantages); andthe process of implementation refers to planning, engaging, executing, reflecting and evaluating the GACO process as interrelated sub-processes known to have a key role in implementation.Taking a whole system approach, the five domains were operationalized and adapted in the present study as follows:A formative evaluation using qualitative multiple case study design was chosen to gain an in-depth understanding of phenomena in “real world settings [where] the researcher does not attempt to manipulate the phenomenon of interest” .Our study builds on results of the recent performance assessment of Quebec’s CWLs for unattached patients, which emphasized the need to better understand variations arising both from within the GACOs and from external local or regional influences . Cases wTo compare performance across the province, indicators for each GACO were transformed into rates per 10,000 population, and were classified into tertiles of relative performance: low, average or high. Contrasting cases were selected (lower vs higher performing GACOs) to understand and explain contextual conditions that led to different process outcomes  and suppData collection took place between May 2015 and June 2016 and drew on three main sources in order to triangulate data: semi-structured interviews with a range of GACOs stakeholders, documentation from the GACOs and field notes taken during and after interviews .For every case, we conducted semi-structured interviews with between five and eight stakeholders with different perspectives. All staff involved in the GACO implementation  and a few family physicians, with experience in attaching patients from the CWLs, were asked to take part in the study. A total of 26 key stakeholders were interviewed, using an interview guide based on the main domains of the CFIR. Interviews lasted between 45 and 90 min. It should be noted that in one case (Case 2), the local medical coordinator position was vacant at the time of data collection and the nurse declined our invitation to participate in the study.Field notes were taken during and after each interview by the interviewers to summarize the main elements discussed with participants, to capture factors emphasized by participants, to describe observations made by interviewers, to reflect on potential explanatory factors and to note modifications to make to the interview guide or clarifications to seek in upcoming interviews. In total, 22 documents  about the GACO structure and process were collected and reviewed to understand each component of the GACOs, and key stakeholder roles at strategic and operational levels.All three forms of data  were analyzed. Interviews were transcribed and coded using NVivo software. Documents and field notes were coded manually. Analysis followed a three-stage process. First, we applied a deductive approach using a codebook based on the CFIR to guide data coding and analysis. The codification was controlled by the technic of double coding: a research assistant and a researcher, both blinded to the implementation performance status of the four GACOs, coded all interviews independently, meeting periodically to compare and revise codes. When disagreements were observed, the codebook was adjusted and codes inspired from the CFIR were redefined to better fit the context of GACO implementation. Finally, persistent discrepancies were resolved through discussion with the larger research team. A narrative summary, based on the CFIR constructs, was created for each interview within each case to provide a rich description of each GACO’s story. A case-specific matrix with illustrative quotes for each GACO summarized information related to how each CFIR construct influenced implementation. This process led to four narrative summaries and four case-specific matrices organized according to the CFIR constructs.Second, the first author applied ratings, based on the summaries and matrices, to the coded constructs at the GACO level, using the rating rules described by Application of the ratings was checked by two co-authors  for a random subset of constructs within each case; and a double rating was done by (M-AS) for another random subset of coded constructs.In the third stage of analysis, we compared ratings for each construct across all four GACOs using a cross-case analytic matrix that was developed to identify patterns of variation by construct across cases. Finally, a detailed matrix of the specific CFIR constructs with a special focus on categories that distinguished between high and low implementation performance allowed us to draw conclusions on patterns of variation in factors that influenced GACO implementation outcomes. As per In our study, we examined the difference in positive or negative ratings between high and low-performing cases to determine if the construct was an important distinguishing factor and also relied on the supporting qualitative summary. If a difference was of at least two points, the construct was considered to make a strong distinction.To ensure scientific rigor, double blinded coding was performed on all transcripts and double rating and checks were also conducted on a random subset of constructs within each case. Also, an audit trail was kept of all changes made to the codebook as coding and analysis progressed throughout the study . PrelimiIn this section, we discuss the five main constructs that helped identify relevant and rich explanations of variation between high and lower performance level cases. These constructs were: network and communications; leadership engagement; available resources; adaptability; and engaging.Outer setting constructs ; inner setting constructs ; innovation characteristics , and individual characteristics  exerted influence on GACO’s implementation, but did not distinguish cases by level of performance.Two levels were considered: within services of the HSSC where the GACO is implemented and between the services of the LHN.So when patients call me back, they might tell me between noon and two o’clock. We’ve created codes so that I write that the patient called back, and can be reached between noon and two, and I put a code, for example PM. In prioritizing, the nurse will see that it’s between noon and two, with the PM code to call that person.(Case 4 Clerk)There was evidence in all cases of good communication and network relationships sustained through informal and regular formal meetings of GACO staff (e.g. clerk and nurse). However, among GACO staff, collaborative practice to address common work issues and rapidly match a patient to a family physician appeared more developed in Cases 3 and 4 (rated high). In Cases 1 and 2 (rated low), staff were still trying to improve work procedures. Moreover, differences were seen between high and low performers in the extent of communication among GACO staff around the goal of minimizing delays to prioritize patients. In Cases 3 and 4 (rated high), staff had developed innovative communication strategies to solve problems arising from the complexity of the GACO process. While all sites were aware of this problem, only GACOs 3 and 4 had smoothed workflow procedures by instituting a communication system involving formal or informal procedures. In Case 4, staff recorded their initial comments and nurses and clerks developed symbol systems and codes that helped the nurse identify priority patients quickly, ensured work continuity, and enhanced patient access to a family physician:The most innovative practice was a formal communication strategy developed in Case 3 (rated high) using an online computer-based system to perform a quick pre-prioritization of patients with special needs and reduce delays for urgent patients (e.g. vulnerable patients) to avoid compromising their health status.We’re really waiting for the computer program that will help us prioritize them, which will help. We’d certainly like to do more to prioritize them, but we still have to take the time to assess them.(Case 1 Manager)It is noteworthy that in Case 1 (rated low), despite attempts by the clerk to help the nurse prioritize participants, both manager and clerk admitted that problems persist, and said they were eager to put in place a computer communication system to facilitate pre-prioritization of patients:In Cases 3 and 4 (rated high), participants described good working relationships between GACO staff and health professionals working in collaborating clinics. In Case 3, for instance, good communication between the GACO clerk and some of the local clinics facilitated patient care in case of emergency. In Cases 3 and 4, transmission of medical information and health assessments to family physicians via the medical record of patients referred from the GACO was seen to speed follow up and increase family physician willingness to participate in the GACO.Some participants  in Case 4 (rated high) mentioned that communication and collaboration between GACO staff and physician clinics was sometimes difficult (e.g. GACO staff have to follow up with clinics to know if a patient has been refused by the physician). Nevertheless, GACO staff made efforts to organize discussion with clinics to overcome breaks in communication and minimize their negative impact on patients.Here, we have many clinics, 30 or so clinics that work in silos and don’t speak to each other. There’s no real spirit of belonging to a HSSC. Now it’s starting to improve between certain clinics, but there are some who don’t participate in anything. There are some we never see. These people will almost never take on patients from the GACO.In contrast, in Case 1 (rated low), most physician clinics on the territory operated in isolation, which hampered their participation in the GACO. Poor communication between clinics limited collaborative practice and hindered family physician participation in GACO:Regarding patients, no, (physicians have not received information from the GACO). The […] information we received, and it’s been a while, it’s our physician lead (of the clinic) who went to get it.(Case 2 Family Physician 1)In Case 2 (rated low), some participants reported that the GACO nurse facilitated referral of certain cases through personal contact with family physicians, and helped with patient transfers following retirement of a family physician. However, local family physicians in clinics had different perceptions and complained of poor communication from the GACO about patients on the waiting list and changes in GACO procedures. They even mentioned that the medical director of the clinic had to seek out this information himself:In Case 2 (rated low) and Cases 3 and 4 (rated high), participants emphasized the leadership of formally appointed leaders  or emergent leaders (Case 4) to achieve GACO goals.In Cases 2 and 3, medical coordinators played a role in solving problems, encouraging family physicians to attach patients, managing patient requests and attaching many vulnerable patients. In Case 4, the medical coordinator’s lack of engagement was compensated by the leadership executed by two professionals who emerged as champions and played an active role in implementation efforts: the GACO nurse, who was enthusiastic, proactive and keen on reducing long waiting times for patients, and created adaptive strategies to leverage resources; and a young family physician who was highly engaged and attached a large number of babies and vulnerable patients (400) from the GACO during his first year of practice.I went to see several clinics because I knew a few physicians who had worked here in the past. But with doctors, it takes a doctor to talk to them. I may have the best intentions, make action plans, say “I’ll go see this or that clinic.” Little things get done, but the bulk of it has to go through physicians.Despite, similarities across Cases  regarding leadership, some key differences between low and high-performing cases were highlighted. Participants in Case 2 mentioned the difficulty of mobilizing family physicians to attach patients during a period when the medical coordinator was absent and there was a void in leadership. The manager who temporarily filled his role admitted having limited capacity to recruit family physicians and emphasized that implementation had to be physician driven to incite family physicians to attach patients from the GACO:Also, despite the Case 2 (rated low) medical coordinator’s support for staff in resolving problems encountered with family physicians, he did not, despite intending to do so, implement a strategy to offer patients alternative health services; a more proactive approach was evident in Cases 3 and 4 (rated high). Moreover, staff expressed their concerns about lack of continuity in the medical coordinator role and difficulties sustaining improvements to the GACO after the coordinator’s departure.He  tried to do a little public relations with the clinics. […] He doesn’t have a very aggressive management style and, as well, these are his colleagues. We can’t forget that this is a doctor talking to other doctors. It’s a delicate balance. He can’t impose on them: he’s a doctor himself. He practices in a clinic, he has his clientele, he knows what he’s talking about because he lives it every day; he can’t impose upon them […]. I don’t know what his relation is, but he’s a very nice man, not aggressive, very forthcoming, but […] that’s that.(Case 1 Nurse)In Case 1 (rated low), no specific champion was mentioned. Participants noted that the medical coordinator fulfills a traditional role, providing support when needed and dealing with problematic family physicians. Compared to other GACOs, he was insufficiently committed to enhance family physician participation. The tie with family physicians working in clinics was weak:The nurse’s statement was endorsed by the medical coordinator himself, who admitted “not twisting the arms of family physicians” to attach patients from the GACO. “No, I didn’t twist their arms like that” . He was not known by one of the family physician interviewed practising in one clinic. “The medical coordinator at the GACO? No I don’t know who that is” (Case 1 Family Physician 2).That’s it? They start cutting: the nurse who was supposed to be there five days a week is now on three days. Then she leaves on retirement, and there’s a chance she won’t be replaced. I may end up on my own and then I’ll really lose my mind.(Case 1 Clerk)Lack of adequate staffing, inadequate technology, high staff turnover and financial constraints were highlighted in all cases as barriers to implementing the GACOs. Inadequate human resources caused problems in both processing patients’ registration on the GACO, and in attaching them to a family physician. For example, a lack of family physician capacity on the territory had a negative impact on GACO ability to handle requests for registration. Lack of stability in the clerk’s role led to delays as new clerks had to be trained. As well, the lack of additional funds allocated for implementation efforts constrained recruitment of additional human resources. No additional staff was made available for GACO implementation efforts and no extra support was provided to the HSSC within which the GACO operated. The HSSC had to make do with existing staff, who were assigned additional tasks to implement the GACO:The process works well and the fact that we’ve – I’ve talked to others about it when I find myself with other managers – trained a good number of nurses, here and there; we’re open on weekends, and sometimes people don’t show up for their Saturday or Sunday appointments. It’s a good time to catch people at home. The weekend nurses, everyone knows, turn on the computer and sign into the SIGACO (database). All that contributes to reducing the need to bring in pregnant nurses and all that. When I mention this elsewhere, nobody’s doing it […] “That’s a great idea,” they say. Because no one has a budget for this. We have a 0.8 clerk and I believe the salary of one nurse, and in some places that can be a very part-time nurse, so […] in that way, we manage to make the machine roll along smoothly.(Case 4 Nurse)Along with human resource challenges, GACOs had difficulties accessing the medical insurance database to verify patients’ attachment status . All cases faced these challenges, but Cases 3 and 4 (rated high) found creative ways to deal with them. Case 4 developed an adaptive strategy to optimize GACO resources by making full use of the local HSSC nursing staff. For example, a full-time clerk and several nurses who were not initially assigned to patient prioritization worked on GACO efforts whenever their schedule allowed. Training many nurses to conduct patient evaluations expanded the potential resource pool and reduced reliance on a single person:I would say that she [the nurse in the other department] has a lot on her plate. She helps us depending on the number of patients she has. There are evenings when she can’t do anything for the GACO, and evenings when she can help us out […] She does some prioritization, telephone evaluations for prioritization. It depends. Sometimes we ask her to do the RAMQ [Régie d’Assurance Maladie du Quebec-Quebec Health Insurance Board] checks. Sometimes, in researching on the RAMQ site, you find that they (the patients) have found a family physician by themselves. So there’s an elimination in that time.(Case 3 Nurse)Along the same lines, Case 3 (rate high) managed to overcome human resource constraints through their innovative pre-prioritization system. They recruited a nurse clinician working in another department who agreed to help GACO staff prioritize patients, and check their status in the medical insurance database:In all four cases, the GACO was adapted to meet community needs. Adaptations were related to one of three GACO activities: registration, evaluation of priority and attachment.If they don’t come to the GACO, the GACO will come to them […]. So I tell them “I’m the system that comes to see you.” I’m the HSSC nurse who goes out to present them the services offered by the CLSC, and register them for the GACO as well. So then, personally giving them the leaflet. “So now you’ll get your life on track, call us back. Your wait to get a family doctor has already begun” […] I prioritize them on the spot. You check if they have their RAMQ [Quebec Health Insurance Board] number […] I look, because I have access, to see if they have a doctor. With some, I’ve even called RAMQ with them because even I get lost in the system when I call there, so you can imagine what it’s like for this poor man.(Case 4 Nurse)In response to an overwhelming volume of calls, all four cases decided to replace telephone registration by an online registration form. What differentiated high-rated and low-rated GACOs were the procedures designed to support registration of specific population groups such as newborns and homeless patients. Cases 3 and 4 (rated high) implemented procedures to facilitate registration, which was not done in low-performance cases. In Case 3, GACO staff developed an internal procedure to register newborns on the waiting list even though the provincial GACO software did not permit registration until the baby had received a health insurance number. Case 4 used an outreach strategy whereby the GACO nurse visited homeless shelters monthly to help patients sort out their eligibility for the GACO and register:A major difference between the two groups of GACOs was that Cases 3 and 4 (rated high) put in place creative initiatives to offer alternative health services while patients waited on the GACO. This was not done in lower-rated Cases 1 and 2. For instance, Case 3 offered the possibility of receiving transitional care while waiting for a regular physician from an ambulatory unit for patients with high needs. Case 4 developed a center for disease prevention for patients in good health, which helped to evaluate non-urgent patients and offer them a check-up by a nurse, based on the guidelines, while they were on the waiting list. If a change in their health status or needs was noted, the process of being attached to a family physician could be accelerated.In general, we try to provide a mix of cases. If he just wants diabetic patients, we’ll try to maybe send him four out of five, with the fifth being a patient with diabetes and mental health issues. We put some effort into accommodating them, but also have a clear view of our reality, which is far from easy.(Case 4 Clerk)Cases 3 and 4 (rated high) developed strategies to deal with family physician preferences, whereas Cases 1 and 2 (rated low) complied fully with physician demands. Case 3 implemented a restrictive approach, and did not allow physician preferences to influence patient referrals. Case 4 implemented a more flexible strategy, with GACO staff adjusting referrals to physician preferences while also taking into consideration the patient’s health profile and the importance of their needs. In Case 3, GACO staff urged family physicians to attach patients referred to them, keeping an inventory of the characteristics of each family physician’s practice to minimize the chance they would refuse a patient; GACO staff would also reduce the number of referrals as needed to a given family physician to ensure that patients would be seen by them within appropriate timelines. In Case 4, despite trying to accommodate family physicians, participants decided to refer patients with mental health problems even when family physicians did not want to take them:In Cases 3 and 4 (rated high), adaptation according to distance and region was a noteworthy theme, whereas this was not done in Cases 1 and 2 (rated low). Case 3 used postal codes to refer patients to the closest GACO; in Case 4, this was not always possible due to limited family physician capacity on the territory covered by the GACO, which did not allow patients to choose a region for attachment.My own family doctor didn’t know about the GACO, and she’s on the territory. Not all small clinics attend the regional department of family physicians meetings, which keeps them up to date, so there is a need for, like in [one region], they do a lot of PR [public relations], they even have a little pamphlet, I kept it, I found it cute that the coordinator himself went around to the clinics and handed them out. Definitely, new doctors who arrive, if they’re looking […].(Case 1 Clerk)The promotion of the GACO to patients and family physicians in Cases 1 and 2 (rated low) was slower and less developed than in the high-rated cases. Participants from lower performing Cases 1 and 2 acknowledged that promoting the GACO, attracting family physicians to attach patients from the GACO and encouraging patients to use GACO services, were major concerns. In interviews, participants stressed the enormous efforts required to publicize the GACO. For example, in Case 1 (rated low), they suggested promoting the GACO by distributing pamphlets to patients in clinics. In Case 2, the manager admitted that there is still a lot of work to be done with family physicians to inform them about the GACO in order to obtain results:We promoted on-line registration at that point. We made little bookmarks that we distributed to the clinics on our territory.(Case 3 Manager)Compared to the other sites, Case 3 (rated high) was keen on marketing and driving use of the GACO, providing clinics with updates on adjustments made during implementation (e.g. distributing bookmarks when launching the online registration) to increase GACO use by family physicians and patients:We left them in pharmacies and clinics. Little pads like this. They were all over the area.(Case 4 Clerk)Case 4 (rated high) limited promotion of the GACO at first, because they were afraid of having too many patient requests, but later distributed flyers in French and English through clinics, pharmacies, and community organizations :Our study succeeded in identifying factors that enhance implementation effectiveness and may be used to address performance shortcomings in CWLs. Five main influencing factors were seen to operate at different levels, interact synergistically and work together in mutually reinforcing ways to produce implementation process outcomes. These factors were also seen in four other similar studies  put in place to overcome implementation challenges. Similar strategies have been adopted when implementing advanced access, an innovation to improve timely access to primary care (With regard to the innovation (GACO) characteristics, our results indicate that high-performing GACOs were more innovative in embracing complexity than low-performing GACOs, and adapted the innovation to their local settings and the needs of patients on the waiting list. GACOs required continuous adaptation by staff. Indeed, adaptation is likely to occur when complex innovations unfold in real-world context  directives and the tacit knowledge of local stakeholders , who contribute empirically grounded knowledge of the local context and their own lived experience  and management at provincial level; and prioritization according to five new categories reflecting urgency and health needs . The chaDespite the numerous studies examining models that similarly aim to improve timely access to healthcare in different countries , very few have identified and explained factors that differentiate high from lower-performing sites in the implementation of such innovations. Studies that have attempted to address this relationship , and interviews with family physicians, who are knowledgeable informants directly involved in GACOs implementation and/or impacted by the GACOs. Second, the researchers who coded and analyzed data were blinded to the status of implementation in the four GACOs, which helped reduce bias in qualitative findings and ensure trustworthiness. Double coding was performed using an iterative process, which also helped to increase credibility. Analyzing the results through the CFIR helped synthetize results, will facilitate future comparison of findings across other similar studies adopting the same methodology.A few limitations to the current study should be mentioned. First, the number of participants at Site 2 was less than the number of the other three sites and may have yielded a less rich picture. Additional results may have been captured if the GACO nurse had agreed to be interviewed.Second, we selected four sites among 94, given our objective was not to produce statistically generalizable results, but rather a rich contextualized understanding of each GACO. The final limitation of this study relates to the absence of patients’ experience with the GACO. Future research should include interviews with patients who were attached with a family physician through these CWLs, and with those who remain on the waiting list.We also point out that we have presented in detail only 5 among a possible 39 distinguishing constructs. We do not consider this problematic given that use of a limited number of constructs has been recommended by the designers of the framework  and otheThis study provides the first in-depth analysis of CWLs implementation. Findings can be used to develop strategies to overcome barriers to implementation, better manage wait lists, and improve performance. Ultimately, they could contribute to reducing inequities in access to a family physician, and in health outcomes, notably for vulnerable populations and those with complex physical and/or mental healthcare needs. Findings are also relevant for decision makers responsible for designing complex innovations whose decisions shape the development, implementation and scale-up of CWLs. They may also more generally inform the dissemination efforts of similar complex organizational models in different contexts. When implementing this innovation in similar real-world healthcare delivery contexts, and when redesigning implementation strategies, greater consideration should be given to the combination of organization-level factors , intervention characteristics (adaptability) and the process domain (engagement) identified as factors important to achieving implementation outcomes. Moreover, a mandated innovation that is simultaneously top-down and less prescriptive creates a good opportunity for stakeholders in the field to identify practical ways to bring about change.


The isolated SCs were then treated in vitro with a control ) or a high substrate growth media (HSM) . Cells were assessed on their ability to proliferate, differentiate, and fuel substrate oxidation after differentiation. The effect of HSM was measured as the percentage difference between SCs exposed to HSM compared to control media. In vitro SC function was not affected by donor age. OVF reduced SC proliferation rates (–19% p < 0.05) but did not influence differentiation. Cellular proliferation in response to HSM was correlated to the donor’s body mass index (BMI) . When exposed to HSM, SCs from normal weight (BMI 18–25 kg/m2) participants exhibited reduced proliferation and fusion rates with increased fatty-acid oxidation (p < 0.05), while SCs from participants with higher BMIs (BMI 25–32 kg/m2) demonstrated enhanced proliferation in HSM. HSM reduced proliferation and fusion (p < 0.05) in SCs isolated from subjects before OVF, whereas HSM exposure accelerated proliferation and fusion in SCs collected following OVF. These results indicated that diet has a greater influence on SC function than age and BMI. Though age and BMI do not influence in vitro SC function when grown in controlled conditions, both factors influenced the response of SCs to substrate challenges, indicating age and BMI may mediate responses to diet. Satellite cells (SC) aid skeletal muscle growth and regeneration. SC-mediated skeletal muscle repair can both be influenced by and exacerbate several diseases linked to a fatty diet, obesity, and aging. The purpose of this study was to evaluate the effects of different lifestyle factors on SC function, including body mass index (BMI), age, and high-fat overfeeding. For this study, SCs were isolated from the vastus lateralis of sedentary young (18–30 years) and sedentary older (60–80 years) men with varying BMIs (18–32 kg/m Muscle mass correlates with increased metabolic rate and lower rates of obesity, as well an improved overall quality of life ,2,3,4. ITo explain the loss in muscle mass associated with age and disease, many researchers have assessed the mechanisms of tissue repair, specifically those mediated by the satellite cell (SC) ,12,13. SSCs commonly exist as quiescent cells located between the sarcolemma and basement membrane of skeletal muscle  and can SC function can be altered by disease and negative lifestyle factors, such as a sedentary lifestyle  and obesDiet-induced obesity (DIO) in rodents is associated with altered SC function, specifically, blunted activation and proliferation and diminished insulin receptor signaling ,32,33. SThis study sought to determine the influence of donor diet, age, and body mass index (BMI) on SC function as defined by their ability to proliferate, and fuse into multinucleated fibers. SCs were collected from: young sedentary lean, young sedentary overweight/obese, aged sedentary lean, and high-fat/hypercaloric fed young sedentary lean donors. 2, and body fat was between 11–33%. All participants were male and sedentary . Participants were grouped as sedentary normal weight , sedentary overweight or obese , or sedentary aged . The SedOld group had an average appendicular skeletal muscle index  of 9.70 ± 0.13 kg/m2  [Human research participants: Skeletal muscle biopsies were obtained under Virginia Polytechnic and State University, Internal Review Board-approved protocols , indica al. [2) ,40.n = 4) from skeletal muscle biopsies collected following the lead-in diet (before OVF) and following OVF diet and used for the studies described herein.Dietary intervention: Using a controlled feeding approach, participants were provided with a 10–14 day lead-in diet immediately followed by a high-fat hypercaloric diet (OVF) for four weeks as described by Osterberg et al. . The leaTM, Lonza, Rockland, ME, USA) for 5 days until they reached 60–80% confluency. After proliferation, cells were differentiated for seven days in a low-serum media (DMEM with 5 mM glucose and 2% horse serum) with differentiation media replaced every 2 days. Amphotericin-B (0.5 ug/mL), antimitotic (for penicillin 100 U/mL and streptomycin 100 ug/mL), and gentamycin (0.01 mg/mL) were added for protection against bacterial and fungal contamination.SCs were collected from tissue biopsies of the vastus lateralis muscle of participants as described in Muoio et al. . SCs werHigh substrate media (HSM) was applied during the proliferation stage of SCs. HSM contained the same ingredients as the control growth media with additional supplementation of 400 mM, 2:1, oleate–palmitate complexed with 5% BSA , and the glucose concentration increased from 5 mM to 25 mM  . GLUT1, ® NF Cell Proliferation Assay Kit . Doubling times were calculated using the PRISM exponential growth equation software [Cells were counted with a hemocytometer and each cell line was seeded in triplicate at 2500 cell/well in 96-well plates. Cells used for proliferation measures were generated after their third passage (P3). Plates were collected on days 1 through 6, and nuclear material was counted using the CyQUANTsoftware   and DNase I treatment , according to the manufacturer’s instructions. qRT-PCR was performed using a ViiA 7 Real-Time PCR System  and TaqMan Universal PCR Master Mix, used according to the manufacturer’s specifications . Target gene expression in human skeletal muscle primary-cell culture was normalized to peptidyl-prolyl cis-trans isomerase B (PPIB) RNA levels. TaqMan probes were purchased as pre-validated assays for Pax7(Hs00242962_m1), MyoD (Hs00159528_m1), Myogenin (Hs01072232_m1), and PPIB (Hs00168719_m1) . Relative quantification of target genes was calculated using the ΔCT method. Derivation of the ΔCT equation has been described in Applied Biosystems User Bulletin no. 2 (P/N 4303859).t-test. Differentiation and the myogenic index, as well as RNA expression, were analyzed using repeated-measures ANOVA and a Tukey’s post hoc analysis to compare daily differences. Pearson’s correlations were used to explore relationships between SC function and age, bodyfat percentage, and BMI. The HSM effect is reported as percent change from control values. Data is presented as mean ± SEM and statistical significance was set at a p-value of ≤0.05. The samples were taken during two different Virginia Polytechnic Institute and State University, Internal Review Board-approved studies (#12–652 and #11–077). SC-culture approval was further granted under #14–1234. Written consent was obtained for both studies.One-way ANOVA was used to compare proliferation rates between the SedLn vs. SedOvOb and SedLn vs. SedOld with a Tukey post hoc test for multiple comparisons. SCs isolated before vs. following OVF and control vs. HSM samples were compared using a paired 2 (average (avg) 24.2 ± 0.6 kg/m2, avg bodyfat 21.3 ± 1.5%)  vs. overweight/obese , there were no differences in proliferation (p = 0.59) (p = 0.04), which was primarily driven by reduced spontaneous fusion (fusion prior to the addition of differentiation media) in the SedOvOb group (Influence of bodyweight/adiposity on SC function: SC cultures were obtained from 23 young (23.6 ± 0.7 years) participants with BMI ranging from 20–33 kg/m = 0.59) A. SedLn Ob group C. There Ob group D. A negaOb group E.2, p < 0.01) and body fat levels  compared to the younger SedLn group, but both groups participated in minimal physical activity    B. Withinp = 0.03, p = 0.05), and, thus, had higher rates of oxidative efficiency (p = 0.03) (The SedLn and SedOld groups achieved similar simple fusion (2+ nuclei) rates throughout differentiation C. Both g = 0.03) D. p = 0.03). There was no change in fasting blood glucose  or triglycerides . HDL , LDL , and total cholesterol  trended on changes but were not statistically significant. SCs collected after 4 weeks of OVF [p = 0.03)  A. Howeve = 0.03) B,C.p < 0.01) (p < 0.05) (p = 0.03). The effects of HSM exposure during the proliferative phase suppressed fusion after eight days of differentiation reducing simple and robust fiber nucleation by 15.7% (p = 0.02) and 16.5% (p = 0.03), respectively. Though there is a reported reduction in both proliferation and fusion prior to differentiation treatment it is possible that cells which attempted to spontaneously differentiate perished. Due to the proliferation assay only counting live cells, those that did not survive spontaneous differentiation result in a reduced measured proliferation.Effects of HSM on SedLn SC Function: SedLn (BMI 18–25) SCs treated with HSM demonstrated reduced proliferation (4.83 ± 1.95%) compared to control conditions ( < 0.01) A. HSM tr < 0.05) A,B. HSM 2, total FAO, and the ratio of complete to incomplete FAO (CO2:ASM) all improved (p < 0.05)  A,C,D. HS < 0.05) E. A meta < 0.05) E,F. Thisp < 0.05) of Pax7 expression (p < 0.01) and increased  expression of myogenin (p < 0.01), but with no change in MyoD compared to control SCs. After day one, HSM exposure had no effect on Pax7, MyoD, or myogenin expression.Pax-7, MyoD, and MyoG RNA-expression varied throughout proliferation and differentiation A–D. At dp < 0.05). In pre-OVF SCs, HSM treatment reduced proliferation by −5.35 ± 1.14%. Conversely, for SCs collected from the same individuals post-OVF, HSM exposure caused an increased proliferation rate of 7.17 ± 3.65% (p < 0.05) (p < 0.05), yet HSM treatment increased simple and robust fusion of SCs collected post-OVF  ( < 0.05) B. The sapost-OVF B,C.The primary findings of these studies are: (1) a high fat/hypercaloric feeding intervention in humans reduced cultured SC proliferation; (2) in sedentary participants age did influence in vitro SC function under control conditions, and (3) elevated concentration of fatty acids and glucose during SC proliferation have differential effects on SC function that may be influenced by the BMI, age, and diet of the SC donor. The effects of overfeeding on SCs may be linked to substrate availability as in vivo OVF and in vitro HSM treatments, resulting in similar changes to SC function.SCs may adapt to the substrate levels of their environment as demonstrated by the opposing responses to the HSM treatment of lean vs. overweight/obese SCs and SCs collected before and following overfeeding. Both BMI and bodyfat percentage showed some influence of SC function with overweight/obese SCs presenting with reduced fusion and bodyfat, negatively correlating with proliferation. These data, when taken with the OVF and HSM results from this study and previously demonstrated effects of DIO models ,32, showDonor age appeared to have no influence on in vitro function of SCs in humans after physical maturity. Donor age caused no differences in an SCs ability to proliferate, fuse, or differentiate into metabolically functional tissue. These results are contrary to findings from previous human and rodent models that suggest aging alters the function of SCs at their core, as aging has been shown to disrupt SC function in vivo ,50,51,52Previous studies have demonstrated that in vivo phenotypes of metabolic disease, such as insulin resistance ,34,55 anSC myogenesis is transcriptionally regulated through the sequential expression of key transcription factors (TFs): Pax-7   make it difficult for any human study to isolate the true effects of chronological aging. Large differences in adiposity between young and aged counterparts is a common complication seen in aging studies, as it is difficult to recruit a lean aged population. Even in the subject demographics for this work, there was a significant difference between the adiposity of the young and aged group. With the high prevalence of body fat differences in aging studies, we must ask if the differences currently attributed to age are truly due to the chronological age of the participants or could the variation be more accurately ascribed to the adiposity of the aged population.Perhaps one of the most intensive studies designed to elicit the mechanistic difference between young and old SCs is that of the work done by Roberts et al., published in 2011. This work found that Cyclin D1 and MyoD were determined to have different expressional patterns in young vs. aged SCs. In response to exercise, MyoD increased in the young group as expected, but there was no increase in the aged group.  While thThese results highlight the resiliency and adaptability of SCs, as age did not influence SC function. SCs were negatively affected by BMI, bodyfat, and overfeeding with reduced proliferation and fusion. While overfeeding causes a variety of systemic effects, such as inflammation and altered hormonal signaling, the results of the high substrate in vitro treatment indicate that changes to substrate levels alone are enough to alter SC function. This data suggests a reasonable viability for the use of dietary interventions to alter SC function. Age and BMI were influential in the SCs’ response to the substrate challenge, with SCs from both older and heavier donors preferring higher substrate. This is an indication that older and more obese populations may respond differently to dietary interventions designed to influence muscle function.


The broad product characterization included high-resolution transmission electron microscopy, X-ray diffraction, X-ray fluorescence, scanning electron microscopy and energy dispersive spectroscopy analyses. The macrostructural parameters, pore size distribution, mechanical resistance, stiffness, hygric and thermal parameters of the composites matured for 28-days were also the subject of investigation. The combination of diatomite and graphene nanoplatelets greatly reduced the porosity and average pore size in comparison with the reference material composed of MOC and silica sand. In the developed composites, well stable and mechanically resistant phase 5 was the only precipitated compound. Therefore, the developed composite shows high compactness, strength, and low water imbibition which ensure high application potential of this novel type of material in the construction industry.A high-performance magnesium oxychloride cement (MOC) composite composed of silica sand, diatomite powder, and doped with graphene nanoplatelets was prepared and characterized. Diatomite was used as a 10 vol.% replacement for silica sand. The dosage of graphene was 0.5 wt.% of the sum of the MgO and MgCl During the sintering itself, a large amount of CO2 is released because of the decomposition of limestone [The overall amount of greenhouse gas (GHG) emissions released during the production of Portland cement (PC) accounts for 5–7% of all global emissions . Predictimestone ,4. This imestone ,7,8. Ano2-H2O and there are four known phases of this sort of material which differ depending on the stoichiometric ratio between magnesium oxide, magnesium chloride and water. At ambient temperature, Phase 3 (3Mg(OH)2·MgCl2·8H2O) and Phase 5 (5Mg(OH)2·MgCl2·8H2O) are formed. At temperatures above 100 °C, Phase 2 (2Mg(OH)2·MgCl2·4H2O and 2Mg(OH)2·MgCl2·5H2O) and Phase 9 (9Mg(OH)2·MgCl2·4H2O) are present [Magnesium oxychloride cement (MOC), discovered in 1867 , is an a present ,13,14,15 present ,20,21,22Graphene and other graphene derivatives belong to the group of carbon-based nanomaterials ,24,25. GAnother distinct advantage in comparison to PC is the bonding ability to the wide range of fillers, due to its unique microstructure. It is known, that fillers such as silica glass , fuel as2·nH2O, so it can be described as a silica-bearing material [Diatomite can be described as a mineral formed in the process of sedimentation of the fragments of the carapace of diatom algae. It is a pale-colored, lightweight material mainly composed of the phase SiOmaterial ,45,46. Tmaterial  or as a material ,49,50. Tmaterial ,52,53,54In this paper, a composite material based on MOC and silica sand with graphene additive and diatomite powder was prepared and characterized using various analytical methods. Such material composition is unique and based on our analysis no similar material has been reported in the literature up to now. Diatomite was used as a partial sand substitute. A reference sample containing only MOC and silica sand was also prepared and used for comparison. The samples were analyzed in terms of their phase and chemical analysis using X-ray diffraction and energy dispersive spectroscopy. Optical microscopy was used to analyze the microstructures of all the samples. All of the samples were subjected to mechanical tests to show their compressive strength, flexural strength and elastic moduli. Ability to transport and accumulate water was characterized by the measurement of hygric parameters, porosity and pore size analysis. The thermal parameters of composites were the subject of the investigation as well.2·6H2O (p.a. purity) delivered by Lachner s.r.o.  had density of 26 Bé°. The 0–2 mm fraction of silica sand  was used as only filler in reference samples. The loose bulk density of sand used was 1678 kg·m−3. In modified composite mix, fine-grained diatomite (Blaine fineness 2087 m2·kg−1) produced in LB MINERALS s.r.o.,  was used as 10% volumetric replacement of silica sand. The grain size analysis of quartz sand was done by sieve method in accordance with the EN 933-1 [The light burnt magnesia (MgO) was a product of Styromagnesit Steirische Magnesitindustrie Ltd. . A hydrous solution of MgClEN 933-1 . The par2·g−1 were provided by Alfa Aesar . The purity of graphene used was 99.9 wt.% and negligible traces of other elements such as S, Si, and Fe were identified. The microstructure of the graphene was studied by HR-TEM . The TEM data presented in The graphene nanoplatelets having declared surface area 500 m2·6H2O and tap water. Part of the solution was used for the dispersion of graphene nanoplatelets. For this purpose, the magnesium chloride solution with graphene was first sonicated in ultrasonic bath for 15 min and then dispersed for 5 min using a T18 UltraTurrax  operating at 7000 rpm. The resulting suspension was added to MgO powder and mixed for 90 s. After that the sand or sand/diatomite mix were added and the mixture was stirred for another 90 s.A magnesium chloride solution of required concentration was prepared using MgCl2·6H2O mass.The fresh composite was then poured in two layers into prismatic iron molds having dimensions of 160 mm × 40 mm × 40 mm and compacted on a vibrating table. The specimens were demolded after 24 h, and then they were cured for 27 days in laboratory at temperature of (23 ± 2) °C and relative humidity of (50 ± 5)%. D The composition of investigated composites is presented in The graphene nanoplatelets were subjected to the HR-TEM. For that purpose, an EFTEM 2200 FS microscope  was applied.U = 30 kV, I = 10 mA).XRD data were collected at room temperature on a D8 Phaser powder diffractometer  with parafocusing Bragg–Brentano geometry using CuKα radiation  and AZtecEnergy software (v. 3.0).The morphology was investigated using SEM with a FEG electron source . EDS was measured by an X-MaxOptical microscopy of composite samples was performed by a Navitar macro-optics microscope  with optical zoom up to 110× and recorded with 2/3” digital camera  having a resolution of 5 Mpix. The sample was illuminated by a white LED ring light source with individually addressable segments and intensity. NIS-Elements BR 5.21.02 software with an Extended Depth of Focus Module (EDF) was used for imagining and analysis of the samples.For the 28-days matured composites, structural, micro-structural, mechanical, hygric, and thermal parameters were determined. Except for the MIP test, five samples of each material were tested. The presented data represents mean value calculated based on the results obtained for the particular samples. Where applicable, the expanded combined uncertainty of the presented data was given.Basic characterization of the hardened composites was done using the bulk density, specific density and total open porosity assessment. The bulk density test was conducted in compliance with the standard EN 1015-10 . For theff (MPa), compressive strength fc (MPa), and dynamic modulus of elasticity Ed (GPa). The strength tests were realized as prescribed in the EN 1015-11 [Mechanical resistance and stiffness were characterized by flexural strength  1015-11 . A VikasWa (%) and 24-h water absorption Wa24 (%) were measured according to the EN 13755 [Aw (kg·m−2·s−1/2) and apparent moisture diffusivity κapp (m2·s−1) were evaluated based on the free water intake experiment. This test was conducted as introduced in the EN 1015-18 [The poor resistance of MOC-based materials against moisture damage is reported in the literature ,60. TherEN 13755 . The wat 1015-18 , and the 1015-18 .Among thermophysical parameters, thermal conductivity, thermal diffusivity, and volumetric heat capacity were tested. They were examined using a Hot Disk TPS 1500 thermal constants analyzer  operating on a transient plane source technique . The tesIn this study, the impact of graphene and diatomite addition to the magnesium oxychloride matrix was investigated. Prepared high-performance composites are shown in 3(OH)5Cl·4H2O (ICDD 00-007-0420) and MgO (ICDD 00-001-1169). The diatomite as well as graphene are not visible in MOC-DG sample due to amorphous nature of diatomite and very low graphene content.The phase composition of both samples was studied using X-ray Diffraction. The XRD analysis was conducted on paste samples in order to avoid very strong reflections of quartz. Usually, when sand is present in the samples, the quartz is the only visible phase. Both diffraction patterns can be seen in The morphology of the composites was studied by SEM see . Highly To determine the chemical composition of the composites, EDS was used. The qualitative analysis showed the presence of the following major elements: magnesium, oxygen, carbon, chlorine, calcium, silicon and aluminum, whose elemental maps can be seen in 2. The presence of calcium and iron is caused by the low amount of impurities present in the raw materials, namely the caustic MgO.The quantitative analysis results are shown in The microstructure of both samples was analyzed using optical microscopy see . Both MOPHg), were almost similar. The use of diatomite and graphene nanoplatelets led to the high drop in porosity which was due to the low dimension of diatomite particles and graphene agglomerates.The basic structural properties of the analyzed composites are presented together with the mechanical parameters in Because of the lower specific density of diatomite than that of silica sand, both the bulk density and specific density of MOC-DG material were slightly reduced. The drop of the investigated macrostructural parameters is well apparent from Consistent with the decrease in porosity, the mechanical strength of MOC-DG composite was greatly improved. It was the result of the three mutually acted effects: (i) low porosity, (ii) activation of two-dimensional graphene nanoplatelets that bridged the gaps between the Phase 5 needles, unreacted MgO, and diatomite particles, (iii) high hardness, compressive strength, and flexural of graphene nanoplatelets ,66,67. T3·g−1 (MOC-DG) to 0.03721 cm3·g−1 (MOC-REF). Accordingly, the average pore size decreased from 0.0412 to 0.0174 μm.The pore size distribution measured by mercury porosimetry is shown in Aw value of about two orders of magnitude higher [Aw values should be 0.4 and 0.2 kg·m−2·s−1/2 respectively [−1·K−1). Of course, it is lower value than obtained in our case, but their material had much higher porosity.The effect of the use of graphene and diatomite on the hygrothermal performance of the newly developed composite is evident from e higher ,69. For e higher  introducectively . It meanectively  who anal3(OH)5Cl·4H2O) and unreacted MgO residue;the identified crystalline phases in both composites were well stable and durable phase 5 (Mgthe highly compacted structure of graphene-doped composite was identified, where possible defects were inter-grown by phase 5 needle like crystals;diatomite and graphene were well distributed and fixed in MOC matrix;the porosity, bulk density, and specific density were reduced by the use of graphene nanoplatelets and diatomite;the compressive strength of MOC-DG composite was greatly improved due to the high hardness and mechanical strength of graphene, lowered porosity, and activation of two-dimensional graphene-based reinforcement;as the average pore size was significantly reduced by the mutual action of diatomite and graphene in composite mixture, the water transport and accumulation was highly limited in MOC-DG materials which is very promising finding for its improved durability in the sense of moisture damage;the use of diatomite slightly reduced the thermal conductivity of the newly developed composite, but its heat transport properties remained high.The impact of graphene and diatomite admixing on the performance and properties of MOC composites was studied. A broad test campaign which involved sophisticated and effective characterization analyses such as HR-TEM, XRD, XRF, SEM, and EDS were applied to investigate the raw materials and composites matured for 28-days. The data acquired from the conducted tests allows us to point out the following most substantial results:Summarizing the main findings of the study above listed, the diatomite/graphene enriched MOC composite represents an interesting alternative material that meets the current technical criteria and functional standards imposed on novel high-performance materials for construction industry. The acquired data will be used in the near future for the design of new construction composites designed for specific construction purposes and applications.