text:
Heterogeneity of proteome dynamics between connective tissue phases of adult tendon
Howard Choi,
Deborah Simpson,
Ding Wang,
Mark Prescott,
Andrew A Pitsillides,
Jayesh Dudhia,
Peter D Clegg,
Peipei Ping,
Chavaunne T Thorpe ,
Department of Physiology and Medicine, David Geffen School of Medicine, UCLA, United States;
Centre for Proteome Research, Biosciences Building, Institute of Integrative Biology, University of Liverpool, United Kingdom;
Department of Comparative Biomedical Sciences, Royal Veterinary College, United Kingdom;
Department of Clinical Sciences and Services, Royal Veterinary College, United Kingdom;
Department of Musculoskeletal Biology, Institute of Ageing and Chronic Disease, University of Liverpool, United Kingdom;
Howard Choi
Deborah Simpson
Ding Wang
Mark Prescott
Andrew A Pitsillides
Jayesh Dudhia
Peter D Clegg
Peipei Ping
Chavaunne T Thorpe
eLife digest
Maintenance of connective tissue integrity is fundamental to sustain function, requiring protein turnover to repair damaged tissue. However, connective tissue proteome dynamics remain largely undefined, as do differences in turnover rates of individual proteins in the collagen and glycoprotein phases of connective tissue extracellular matrix (ECM). Here, we investigate proteome dynamics in the collagen and glycoprotein phases of connective tissues by exploiting the spatially distinct fascicular (collagen-rich) and interfascicular (glycoprotein-rich) ECM phases of tendon. Using isotope labelling, mass spectrometry and bioinformatics, we calculate turnover rates of individual proteins within rat Achilles tendon and its ECM phases. Our results demonstrate complex proteome dynamics in tendon, with ~1000 fold differences in protein turnover rates, and overall faster protein turnover within the glycoprotein-rich interfascicular matrix compared to the collagen-rich fascicular matrix. These data provide insights into the complexity of proteome dynamics in tendon, likely required to maintain tissue homeostasis.
Muscles are anchored to bones through specialized tissues called tendons. Made of bundles of fibers (or fascicles) linked together by an 'interfascicular' matrix, healthy tendons are required for organisms to move properly. Yet, these structures are constantly exposed to damage: the interfascicular matrix, in particular, is highly susceptible to injury as it allows the fascicles to slide on each other.
One way to avoid damage could be for the body to continually replace proteins in tendons before they become too impaired. However, the way proteins are renewed in these structures is currently not well understood – indeed, it has long been assumed that almost no protein turnover occurs in tendons. In particular, it is unknown whether proteins in the interfascicular matrix have a higher turn over than those in the fascicles.
To investigate, Choi, Simpson et al. fed rats on water carrying a molecular label that becomes integrated into new proteins. Analysis of individual proteins from the rats' tendons showed great variation in protein turnover, with some replaced every few days and others only over several years. This suggests that protein turnover is actually an important part of tendon health. In particular, the results show that turnover is higher in the interfascicular matrix, where damage is expected to be more likely.
Protein turnover also plays a part in conditions such as cancer, heart disease and kidney disease. Using approaches like the one developed by Choi, Simpson et al. could help to understand how individual proteins are renewed in a range of diseases, and how to design new treatments.
Maintaining the structural and mechanical integrity of tissues in the musculoskeletal system, and other connective tissues, is fundamental to sustain tissue homeostasis and healthy function, requiring protein synthesis and degradation to repair and/or replace damaged tissue before damage accumulates and leads to injury (Humphrey et al., 2014). However, while the composition and structure of connective tissues is well defined (Scott, 1983), relatively little is known regarding proteome dynamics in connective tissues, particularly at the level of the individual constituent proteins.
Connective tissues consist of fibrous proteins (predominantly collagen) embedded in a glycoprotein-rich matrix (Scott, 1983), and variation in the organisation of both phases give rise to tissues with distinct structural and mechanical properties (Culav et al., 1999). Research in a variety of tissues including skin, tendon and cartilage indicates a relatively long half-life of collagens, with more rapid turnover of glycoproteins and other non-collagenous proteins (Thorpe et al., 2010; Maroudas et al., 1998; Sivan et al., 2006; Sivan et al., 2008; Verzijl et al., 2000). Indeed, several studies have reported negligible turnover of collagen, the major component of tendon and other connective tissues, within an individual's lifetime (Thorpe et al., 2010; Heinemeier et al., 2013; Heinemeier et al., 2016). However other studies have measured relatively rapid collagen synthesis in tendon, both at basal levels and in response to exercise (Miller et al., 2005), and also identified soluble collagen with a much shorter half-life compared to the majority of collagen in human articular cartilage (Hsueh et al., 2019); the location(s) of these more labile proteins remain to be identified. When taken together, these contradictory findings indicate that proteome dynamics in connective tissues is complex, and that the glycoprotein-rich phase may be replenished more rapidly than the collagen-rich phase. However, the turnover rate of individual proteins within the different phases of the extracellular matrix (ECM), and the potential contribution of differential regulation of protein turnover to maintenance of tissue homeostasis remain undefined.
Tendon provides an ideal model in which to separately interrogate protein turnover in these ECM phases, as it consists of highly aligned, collagen-rich fascicular matrix (FM), interspersed by a less dense glycoprotein-rich phase, termed the interfascicular matrix (IFM, also referred to as the endotenon) (Kastelic et al., 1978). While the FM is predominantly composed of type I collagen, glycoproteins are also present in this region at low abundance (Thorpe et al., 2016b). Similarly, the IFM contains small amounts of a variety of collagens (Södersten et al., 2013; Thorpe et al., 2016b). Due to the highly aligned structure of tendon tissue, it is possible to separate FM and IFM for individual analysis using laser capture microdissection (Thorpe et al., 2016b; Zamboulis et al., 2018). Indeed, these, and other studies, have shown greater expression of markers of ECM degradation, as well as increased neo-peptide levels, a proxy measure of protein turnover, within the IFM (Spiesz et al., 2015; Thorpe et al., 2015a; Thorpe et al., 2016b), suggesting this region is more prone to microdamage, likely due to the high shear environment that occurs due to interfascicular sliding (Thorpe et al., 2012; Thorpe et al., 2015b). Taken together, these findings suggest that there is greater turnover of ECM proteins localised to the IFM than within the FM to repair local microdamage and maintain tendon homeostasis.
Due to a lack of available methodology, it has not been possible until recently to study differential rate of turnover at the individual protein level. However, novel bioinformatics software developments, in combination with in vivo isotope labelling, and mass spectrometry technologies, now provide the capacity to determine the turnover rates of individual proteins within a sample (Kim et al., 2012; Lam et al., 2014; Lau et al., 2016). The aim of this study is therefore to apply this technique to tendon, firstly to establish the proteome-wide turnover rate of tendon, and secondly to combine this approach with laser capture microdissection to test the hypothesis that the proteome of the IFM is more dynamic than in the FM, with faster turnover of individual proteins. More rapid remodelling of the IFM would provide a mechanism by which shear-induced microdamage to this region could be repaired, preventing damage accumulation and subsequent injury.
2H enrichment in serum water
In rats labelled with deuterium over a period of 127 days (Figure 1a), 2H enrichment of serum occurred rapidly and in a similar manner to that reported previously in rodents (Kim et al., 2012), reaching a plateau of 5.6% by day 4, and remained constant throughout the study (Figure 1b). The enrichment curve, which was empirically derived from the GC-MS measurements at the sampled time points, defines two parameters: deuterium enrichment rate (kp) = 0.7913 and plateau (pss) = 0.0558; these values were used for subsequent kinetic curve fitting to calculate peptide turnover rate constants (k).
Metabolic labelling of rats using deuterium.
(A) Schematic showing 2H2O labelling of rats and details of sample collection, and interfascicular matrix isolation by laser capture microdissection (scale bar = 100 µm). (B) 2H enrichment in serum occurred rapidly, reaching a plateau of 5.6% by day 4 and remained constant throughout the study. Data are shown as mean ± SD and raw data provided in Figure 1—source data 1. (C) Example extracted ion chromatograms from tendon samples demonstrating increased abundance of higher mass isotopomer peaks over the course of the study (M1 – M5). (D) Example curve showing the relative abundance of the unlabelled monoisotopic peak (M0) for the cartilage oligomeric matrix protein (COMP) peptide QMEQTYWQANPFR, calculated by ProTurn software and plotted as a function of time.
Enrichment of serum water as measured by GC-MS.
190 proteins with ≥2 unique peptides were identified in whole tendon digests and protein interactions are shown in Figure 2. Of these proteins, 72 were classified as ECM or ECM-related proteins by MatrisomeDB (Hynes and Naba, 2012). In samples collected by laser capture microdissection of tendon cryosections, 266 proteins with ≥2 unique peptides were identified in the IFM, 79 of which were ECM or ECM-related proteins (Figure 3). In the FM, 116 proteins were identified, of which 71 were ECM or ECM-related proteins (Figure 4). Protein interactions for each tendon component demonstrate a complex and highly interconnected proteome in tendon and its ECM phases.
Protein-protein interaction map of proteins identified in whole tendon.
Unconnected nodes were removed to provide clarity of the interactome. The total cluster was built with STRING (Szklarczyk et al., 2019) allowing for experimentally verified and predicted protein-protein interactions at high confidence levels (0.700). Line thickness indicates the strength of supporting data. Source data are provided in Figure 2—source data 1.
Proteins identified in whole tendon samples used to create protein-protein interaction maps.
Protein-protein interaction map of proteins identified in the interfascicular matrix.
Proteins identified in the interfascicular matrix used to create protein-protein interaction maps.
Protein-protein interaction map of proteins identified in the fascicular matrix.
Proteins identified in the fascicular matrix used to create protein-protein interaction maps.
Protein turnover in whole tendon
Peptide turnover rates were calculated using ProTurn (v2.1.05; available at http://proturn.heartproteome.org; Lam et al., 2014; Lau et al., 2016; Lau et al., 2018; Wang et al., 2014), which automatically calculates turnover rate constants for all peptides that pass the selection criteria using non-steady state curve fitting (Figure 1). To assure data quality, we used a stringent cut-off, passing only peptides that are identified at 1% false discovery rate (FDR) and quantified at four or more time points. In total, 455 peptides, relating to 41 proteins, passed the ProTurn selection criteria and were used to calculate protein half-life in whole tendon samples.
The relative abundance of the unlabelled monoisotopic peak (M0), plotted as a function of time for selected decorin and collagen peptides are shown in Figure 5a&b. Non-steady state curve fitting was performed by ProTurn to calculate turnover rate constants (k) for each peptide, and resultant fractional synthesis curves in Figure 5c&d demonstrate much faster turnover of decorin peptides compared to collagen types 1 and 3. k values for all peptides identified, and corresponding protein half-lives are shown in Figure 6. As expected, the smallest k values related to collagenous proteins, with corresponding half-lives of 330 to 1086 days. By contrast, the protein identified with the fastest turnover was the glycoprotein clusterin, with a half-life of 1.4 days. The half-life of proteoglycans ranged from 21 days for decorin to 72 days for lumican. With the exception of collagens, which all exhibited low turnover rates, there was no clear relationship between protein class and rate of turnover (Figure 6c).
Calculation of protein turnover rate from mass isotopomer distribution over time.
Relative abundance of M0 in selected decorin (A) and collagen type 1, alpha one chain (B) peptides, and resulting k values calculated by non-steady state curve fitting using ProTurn software. Fractional synthesis rates (FSR), calculated from peptide k values, demonstrate more rapid turnover of decorin (C) compared to collagen type 1, alpha one chain (D). Source data, generated by ProTurn are available in Source data 1.
Peptide turnover rate constants and corresponding protein half-lives in whole tendon.
(A) The peptide rate constants (k) for individual proteins are plotted in descending order on a logarithmic scale, with the median value represented by a red line. (B) The median k values for each protein were used to calculate protein half-life, assuming a first order reaction. (C) Protein turnover rates plotted against MatrisomeDB and PANTHER categories, with the median value represented by a red line. Due to space constraints, gene, rather than protein, names are displayed in parts A and B. Source data, generated by ProTurn are available in Source data 1.
Protein turnover in tendon compartments
246 peptides, relating to 20 proteins, and 121 peptides, relating to 12 proteins, passed the ProTurn selection criteria in the FM and IFM respectively. 55 peptides were present both in the IFM and FM, and k values were significantly greater for these peptides in the IFM compared to the FM (median: 0.018 vs. 0.010; p<0.0001), demonstrating an overall faster rate of protein turnover in the IFM. 39 peptides relating to collagen type I were identified in both the IFM and FM, with significantly greater k values in the IFM (p<0.0001; Figure 7a). The turnover rate constants and resultant half-lives for proteins identified in each tendon phase are shown in Figure 4b. Turnover rate constants for Col1a1 and Col1a2 were significantly higher in the IFM compared to the FM, but there were no significant differences in rate constants for Col3a1 between tendon phases.
Peptide turnover rate constants and corresponding protein half-lives in tendon phases.
(A) Rate constants (k) for collagen type I peptides identified in both tendon phases were significantly greater in the IFM compared to the FM (n = 39). (B) Median peptide decay curves for Col1a1 and Col1a2 in the FM (▲; solid line) and IFM (●; dashed line), showing goodness of fit (r2) and standard error (S.E.). (C) Turnover rate constants (k) and corresponding half-lives for proteins identified in each phase. *** indicates p<0.0001. Source data, generated by ProTurn are available in Figure 7—source data 1 for the FM and Figure 7—source data 2 for the IFM.
ProTurn output for FM.
hl tab contains protein half-life information organized by peptide sequence and hl-data tab contains isotopomer relative abundance at each time point.
ProTurn output for IFM.
Due to ProTurn software identifying a low number of proteins in laser captured samples, particularly in the IFM, turnover rate constants in tendon phases were calculated manually for a number of proteins of interest (Figure 8). Rate constants of turnover of decorin peptides were significantly higher in the IFM compared to the FM (Figure 8). It was not possible to assess differences in turnover of other proteins due to a low number of peptides identified at sufficient time points to allow accurate curve fitting (Figure 8c).
Manual calculation of turnover rate of selected proteins.
(A) Relative abundance of M0 as a function of time, and resultant non-linear curve fitting for peptides corresponding to decorin identified in the FM (▲; solid line) and IFM (●; dashed line). (B) Resultant k values for decorin peptides were significantly greater in the IFM than in the FM (p=0.042). (C) Manually calculated turnover rate constants (k) and corresponding half-lives for tendon proteins of interest. Source data are available in Figure 8—source data 1.
GraphPad Prism output showing the manually calculated relative abundance of M0 at different time points for decorin, fibromodulin, biglycan and Col3a1 peptides, and resultant K values.
This is the first study to investigate the proteome-wide turnover rate of tendon and its constituent ECM phases, demonstrating complex proteome dynamics in whole tendon. Protein turnover rate in tendon varied almost 1000-fold, with half-lives ranging from 1.5 days to over 1000 days. The results also support our hypothesis, demonstrating significantly faster turnover of proteins in the glycoprotein-rich IFM compared to the collagen-rich FM, both overall and when directly comparing individual proteins.
In agreement with previous studies (Thorpe et al., 2016b), the IFM had a more complex proteome than the FM, with a greater number of proteins identified in this region. Indeed, more proteins were identified within the IFM than in whole tendon samples; this is likely due to the predominance of collagen in whole tendon samples which precludes detection of proteins present at lower abundance in these samples. As many of the proteins present in the IFM were identified in less than four samples, it was not possible to perform curve fitting to calculate the turnover rate for these proteins. Due to the small size of the rat Achilles tendon, it was not possible to laser-capture a larger volume of IFM, and pooling samples from each time point for LC-MS/MS analysis did not result in a greater number of protein hits than when each sample was analysed separately. In addition, it was necessary to calculate turnover rates of some proteins manually as automated identification of isotopomer peaks was unsuccessful, particularly in IFM samples. While turnover rates calculated manually cannot be compared directly to those calculated automatically by ProTurn, due to differences in curve fitting, it is possible to compare turnover rates of IFM and FM proteins, where both are calculated manually. Additionally, only tendons from female rats were used, such that any differences in turnover rate between sexes could not assessed. While no previous studies have directly compared tendon turnover rates in males and females, it has been demonstrated that there are very small differences in the transcriptome and proteome of Achilles tendons from male and female mice (Sarver et al., 2017). Despite these limitations, results reveal a diverse rate of protein turnover in both the FM and IFM, with values for individual proteins similar to those observed when the tendon matrix was analysed as a whole.
Collagenous proteins had the slowest turnover of all tendon matrix proteins identified, in the whole tendon, and in the FM and IFM, with a half-life from approximately 1.5 years in the IFM to 3 years in the FM for type I collagen. Collagen type III appeared to have a somewhat faster turnover rate than type I in both the FM and IFM, with no significant difference between phases. The IFM is known to be enriched in collagen type III, whereas the majority of collagen type I localises to the FM (Södersten et al., 2013; Thorpe et al., 2016b). While the collagen turnover rates measured are relatively slow, they contrast with previous studies which have reported that collagen is essentially inert in the adult rat patellar tendon (Bechshøft et al., 2017) and in tendons from larger animals (Heinemeier et al., 2013; Thorpe et al., 2010), as well as in cartilage and intervertebral disc (Heinemeier et al., 2016; Sivan et al., 2008). However, these previous studies have either analysed tissues as a whole, or separated the tissue into collagenous and non-collagenous fractions, rather than calculating turnover of individual proteins, such that they lack the specificity achieved by using deuterium labelling combined with mass spectrometry analysis. Other studies that have measured collagen synthesis in human tendon using stable isotope labelling and performing tendon biopsies, report contradictory findings, calculating a FSR of 0.04–0.06% hour−1, which equates to a half-life ranging from 48 to 64 days (Miller et al., 2005; Babraj et al., 2005; Smeets et al., 2019). However, these were very short term studies, and it is unlikely that all newly synthesised protein would be incorporated into the matrix, such that FSR is over-estimated (Heinemeier et al., 2013). The results we present here help to explain these apparent contradictory findings, suggesting that the pool of more labile collagen identified previously in human tendon and cartilage (Miller et al., 2005; Hsueh et al., 2019) may be located in the glycoprotein-rich ECM phase rather than with the bulk of the fascicular collagen.
Another factor that is likely to influence tendon remodelling is the regulation of post-translational modifications. ECM proteins, particularly collagens, undergo a wide range of post-translational modifications during synthesis and assembly which occur both intra- and extra-cellularly, either through regulated pathways or spontaneously (Yamauchi and Sricholpech, 2012; Avery and Bailey, 2005). Post-translational modifications are essential for determining protein structure and function, and are also likely to influence susceptibility to degradation by limiting enzyme access to cleavage sites (Jaisson et al., 2007). Indeed, a previous study has shown that, with ageing, partially degraded collagen accumulates within the equine superficial digital flexor tendon (Thorpe et al., 2010). This is accompanied by increased advanced glycation end products which may render the ECM more resistant to degradation and subsequent remodelling, reducing the ability to repair damage within the tendon (Thorpe et al., 2010). However, the effects of alterations in post-translations modifications on rates of protein turnover remain to be determined.
In addition, it is likely that overall protein turnover rate is more rapid in small compared to large animals. While few studies have directly compared protein turnover kinetics between species, a recent study of protein turnover in dermal fibroblasts from different rodent species demonstrated a negative correlation between median k values and lifespan (Swovick et al., 2018). In support of this, the half-life of serum albumin is approximately 10 fold greater in the human compared to the rat (Chaudhury et al., 2003; Jeffay, 1960). Taken together, it can be presumed that turnover of tendon ECM proteins is also likely slower in larger animals than those we report here for the rat, but the relative differences observed between individual proteins and ECM phases are likely to persist in humans and other large animals.
It is also important to recognise that specific proteins that are not core components of the tendon ECM will not be metabolised within the tendon, but instead will be synthesised elsewhere and transported to the tendon via the circulation. Specifically, serum albumin and serotransferrin are predominantly synthesised and secreted by the liver (Tavill, 1972), whereas haemoglobin is synthesised in the bone marrow and its turnover provides a measure of erythrocyte lifespan (Koury, 2016). In addition, anionic trypsin-1, which had a particularly long half-life of nearly 2000 days in the FM, is synthesised in the pancreas. The long half-life measured for this protein is rather surprising, as generally enzymes have a much shorter half-life than ECM proteins (Lake-Bakaar et al., 1980) Proteases have a role in tissue repair (Shah and Mital, 2018); it is possible that there is a pool of trypsin within tendon that remains inactive until required for repair, which would explain the long half-life measured.
The measured rate constants of protein turnover will also be dependent on the protein extraction methods used. We chose GuHCl extraction for whole tendon as this has previously been shown to result in best proteome coverage for tendon (Ashraf Kharaz et al., 2017). It was not possible to use the same extraction method for laser-captured samples due to the small amount of starting material, therefore we used a method previously optimised for this sample type (Thorpe et al., 2016b). Despite differences in extraction techniques used, protein half-lives were similar in the whole tendon and the fascicular matrix, suggesting minimal effects from using different extraction techniques. Indeed, previous studies, in which the rat Achilles was acid-solubilised post deuterium labelling and FSR measured by GC-MS, reported a FSR of 0.66 % day−1 (Katsma et al., 2017) which equates to a half-life of 105 days, supporting the results we present here, in which average protein half-life in whole tendon is 131 days.
As well as providing the first data on individual protein turnover rates in tendon, this work builds on our understanding of the role of the IFM in tendon function. Previous work has shown that the IFM has specialised composition and function, facilitating sliding between tendon fascicles to provide extensibility to the tendon (Thorpe et al., 2012; Thorpe et al., 2015b; Thorpe et al., 2016b; Thorpe et al., 2016c; Thorpe et al., 2016a). Cyclic sliding and recoil increases the risk of microdamage to this region (Spiesz et al., 2015), and therefore greater turnover of IFM proteins may be a protective mechanism to repair this damage. We have previously demonstrated that elastin and lubricin are localised to the IFM and likely contribute to its mechanical function (Godinho et al., 2017; Thorpe et al., 2016a). However, these proteins are difficult to identify using LC-MS/MS (Thorpe et al., 2016b) so we were unable to assess their rate of turnover.
We extend previous observations in tendon and other musculoskeletal tissues (Thorpe et al., 2010; Sivan et al., 2006; Maroudas et al., 1998; Hsueh et al., 2019), measuring considerably faster turnover of proteoglycans compared to collagens in tendons, FM and IFM. Interestingly, there were variations in rates of small leucine-rich proteoglycan turnover between tendon phases. Significantly faster turnover of decorin was observed in the IFM compared to the FM, whereas the results suggest that faster fibromodulin turnover may occur within the FM. Indeed, previous studies have shown differential abundance of specific proteoglycans in the IFM and FM, with enrichment of fibromodulin in the FM, but no significant differences in amounts of decorin, biglycan or lumican between tendon phases, as determined by proteomic analysis and immunohistochemistry (Thorpe et al., 2016b; Thorpe et al., 2016a). While use of mouse knock out models have established the roles of a variety of proteins during tendon development, with collagen type V, decorin, biglycan, fibromodulin, lumican and thrombospondins all serving roles in modulating collagen fibrillogenesis (Connizzo et al., 2013; Delgado Caceres et al., 2018), the contributions of these proteins to the function of mature tendon are less well characterised. Recent work using decorin and biglycan inducible knock-out mice demonstrated alterations in collagen fibril diameter and decreased tendon mechanical properties in the adult, indicating an important role for these proteoglycans in FM and subsequently, tendon homeostasis (Robinson et al., 2017). It is likely that proteoglycans also contribute to homeostasis within the IFM, but the roles of specific proteins in the IFM during development and in the adult remain to be defined.
Our results show that proteome dynamics in tendon is highly complex, with differences in protein-selective turnover rates observed in distinct IFM and FM ECM phases. Regulation of protein turnover is multifactorial, involving pathways controlling protein synthesis, folding, transport, assembly, disassembly, and degradation (Powers et al., 2009). Recent studies have identified the important role of transforming growth factor-β (TGF-β) in regulating ECM remodelling in tendon and other tissues (Gumucio et al., 2015; Rys et al., 2016). Indeed, it has been shown that TGF-β localises to the IFM in developing and adult tendon (Kuo et al., 2008; Russo et al., 2015), which is consistent with the notion that increased TGF-β signalling could have a role in this faster protein turnover identified in the IFM.
This is the first study to determine the turnover rate of individual proteins in different connective tissue phases, demonstrating complex proteome dynamics in tendon, with an overall faster turnover of proteins in the glycoprotein-rich IFM phase. Greater capacity for turnover of this phase of the tendon matrix may be indicative of a mechanism that reduces the accumulation of damage caused by the high shear environment within this region, although this remains to be directly determined. The techniques used here provide a powerful approach to interrogate alterations in connective tissue protein turnover with ageing and/or disease which are likely to influence injury risk.
Key resources table
Reagent type
(species) or resource
Source or reference
Strain, strain background (Rattus Norvegicus, Female) Wistar Charles River RRID:RGD_13508588 Female
Commercial assay or kit Pierce protein assay ThermoFisher 22660
Chemical compound, drug [2H]2O CK isotopes LtD DLM-2259
Chemical compound, drug Acetonitrile with 0.1% formic acid LCMS grade Fisher Scientific 10723857
Chemical compound, drug Ammonium bicarbonate Sigma 09830
Chemical compound, drug Chondroitinase ABC Sigma C3667
Chemical compound, drug Dithiothreitol Melford laboratories MB1015
Chemical compound, drug Formic acid (0.1% v/v) LCMS grade Fisher Scientific 10188164
Chemical compound, drug Guanidine hydrocholoride Sigma G3272
Chemical compound, drug Iodoacetamide Sigma Aldrich I1149
Chemical compound, drug RapiGest SF Waters 186001861
Chemical compound, drug Trifluoroacetic acid Optima Fisher Scientific 10723857
Chemical compound, drug Trifluoroacetic acid (0.1% v/v) LCMS grade Fisher Scientific 10516625
Chemical compound, drug Trypsin Gold MS grade Promega V5280
Software, algorithm Peaks Studio v8.5 Bioinformatics Solutions www.bioinfor.com/peaks-studio
Software, algorithm STRING v11.0 PMID:30476243 string-db.org
Software, algorithm MatrisomeDB PMID:21937732 matrisomeproject.mit.edu
Software, algorithm PANTHER PMID:23868073 www.pantherdb.org
Software, algorithm ProLuCID PMID:26171723 fields.scripps.edu/yates/wp/?page_id = 821
Software, algorithm ProteoWizard v3 PMID:28188540 proteowizard.sourceforge.net/index.html
Software, algorithm ProTurn PMID:24614109 proturn.heartproteome.org
Software, algorithm Prism v8.2 GraphPad www.graphpad.com
Other Filter units, Vivacon 500 10000 MWCO Sartorius VN01H02
- Female Wistar rats (n = 24, 12 week old, weight: 270 ± 18 g, range: 228–299 g, specific pathogen free, Charles River Company, UK) were randomly housed in polypropylene cages in groups of 3, subjected to 12 hr light/dark cycle with room temperature at 21 ± 2°C and fed ad libitum with a maintenance diet (Special Diet Services, Witham UK). All procedures complied with the Animals (Scientific Procedures) Act 1986, were approved by the local ethics committee at the Royal Veterinary College, were performed under project licence PB78F43EE and are reported according to the ARRIVE guidelines (Kilkenny et al., 2010).
In vivo isotope labelling
All rats, with the exception of the controls (n = 3) received two intraperitoneal injections of 99% [2H]2O (10 ml/kg; CK isotopes Ltd) spaced 4 hr apart, and were then allowed free access to 8% [2H]2O (v/v) in drinking water for the duration of the study to maintain steady-state labelling (Kim et al., 2012). All rats were acclimatized for 1 week prior to commencement of the study, and monitored daily and weighed weekly throughout the study; no adverse effects were observed. Rats in the control group were sacrificed on day 0. Rats in the isotope labelled groups were sacrificed on day 1, 3, 6, 15, 31, 63 and 127 (n = 3 per time point). Rats were culled at the same time of day (10 am) at each time point, to minimise any effect from diurnal variations. Blood was collected from each rat immediately post-mortem, and allowed to clot at room temperature. Blood samples were centrifuged at 1500 g for 10 min, and serum collected and stored at −20°C prior to analysis. Both Achilles tendons were harvested within 2 hr of death. The left Achilles was snap frozen in n-hexane cooled on dry ice for proteomic analysis of the whole tissue, and the right Achilles was embedded in optimal cutting temperature compound and snap frozen in n-hexane cooled on dry ice for isolation of fascicular and interfascicular matrices (Figure 1a).
GC-MS analysis of serum water
2H labelling in serum was measured via gas chromatography-mass spectrometry (GC-MS) after exchange with acetone and extraction with chloroform as described previously (McCabe et al., 2006). 30 µl extract was analysed using GC-MS (Waters GCT mass spectrometer, Agilent J and W DB-17MS column (30m × 0.25 mm x 0.25 µm)). The carrier gas was helium (flow rate: 0.8 ml/min). The column temperature gradient was as follows: 60°C initial, 20 °C/min increase to 100°C, 1 min hold, then 50 °C/min increase to 220°C. The injection volume was 1 µl and injector temperature was 220°C. The mass spectrometer operated in positive ion electron ionisation mode, the source temperature was 180°C and the range scanned was m/z 40–600 (scan time: 0.9 s). Mass spectral intensities for m/z values 58 and 59 were produced by combining the mass spectra in the acetone peak to create an averaged spectrum (Waters MassLynx). Serum 2H enrichment was calculated by comparison to a standard curve, and first order curve fitting (GraphPad Prism 8) used to calculate the rate constant (kp) and plateau (pss) of deuterium enrichment (Figure 1b).
Protein extraction from whole tendon
Left Achilles tendons were thawed, and their surface scraped with a scalpel, followed by PBS washes, to remove the epitenon and any residual non-tendinous tissue. Tendons were finely chopped, flash frozen in liquid nitrogen and pulverised in a dismembrator (Sartorius, mikro-dismembrator U, 1800rpm, 2 mins.). Proteins were extracted using previously optimised methodologies (Ashraf Kharaz et al., 2017). Briefly, following deglycosylation in chondroitinase ABC, proteins were extracted in guanidine hydrocholoride (GuHCl) as described previously (Kharaz et al., 2016). Protein content was determined using a Pierce protein assay according to the manufacturer's instructions. Samples were stored at −80°C prior to preparation for LC-MS/MS by centrifugal filtration. Filter units (Vivacon 500; 10 000 MWCO) were rinsed with 1%(v/v) formic acid and a balance volume of buffer (4M GuHCl/50 mM ammonium bicarbonate (ambic)) was added. A volume equivalent to 50 µg protein was added to each filter and the samples vortexed gently. Filters were centrifuged (15 min, 12500 rpm, 20°C), washed with GuHCl buffer and centrifuged again. Proteins were reduced by DTT incubation (100 µl of 8 mM in 4M GuHCl, 15 min, 56°C) and then centrifuged (10 min, 12500 rpm). DTT was removed by washing twice with buffer as described above. Proteins were alkylated with 100 μl 50 mM iodoacetamide in 4M GuHCl solution, vortexed and incubated in the dark (20 min., room temperature). Iodoacetamide was removed by washing twice with GuHCl buffer as described above. Buffer was exchanged to ambic by 3 washes with 50 mM ambic, centrifuging after each wash. 1 µg trypsin in 50 mM ambic (40 µl) was added and proteins digested overnight at 37°C with mixing. Flow through was collected after centrifugation and addition of 40 µl 50 mM ambic. The flow though was combined and acidified using trifluoroacetic acid (10%(v/v)), and diluted 20-fold in 0.1%(v/v) TFA/3%(v/v) acetonitrile for LC-MS/MS analysis.
Isolation and extraction of fascicular and interfascicular matrix proteins
Longitudinal cryosections (15 µm) from the right Achilles tendons (five time points, n = 2–3/time point) were adhered to membrane slides (PEN, Leica) and stored at −80°C. Sections were prepared for laser capture as described previously (Thorpe et al., 2016b) and approximately 1 mm2 of FM and IFM from each sample were collected into 50 µl molecular biology grade H2O (Leica LMD7000; Figure 1a). Samples were immediately frozen and stored at −80°C. Prior to digestion, samples were centrifuged immediately after removal from −80°C storage. Volume was adjusted to 80 µl by adding 50 mM ambic, and Rapigest SF (1%(v/v) in 25 mM ambic, Waters, UK) was added to a final volume of 0.1%(v/v). Samples were mixed (room temperature, 30 min) then incubated at 60°C (1 hr, 450 rpm). Samples were centrifuged (17200 g, 10 min), vortexed, then incubated at 80°C (10 min). Proteins were reduced by incubating with DTT (5 μL, 60 mM in 25 mM ambic, 10 min, 60°C) then alkylated by adding 5 μl 178 mM iodoacetamide in 25 mM ambic (30 min. incubation, room temperature, in dark). Trypsin (Promega Gold sequencing grade) was diluted in 25 mM ambic and added at an enzyme to protein ratio of 1:50 (based on estimated protein amount from tissue volume collected). Digests were incubated overnight at 37°C with an enzyme top-up after 3.5 hr. Rapigest was hydrolysed with TFA (0.5 μl, 37°C, 45 min.). Digests were centrifuged (17200 g, 30 min.), aspirated into low-bind tubes and de-salted on stage-tips as previously described (Thorpe et al., 2016b).
LC-MS/MS analyses
Data-dependent LC-MS/MS analyses were conducted on a QExactive quadrupole-Orbitrap mass spectrometer (Williamson et al., 2016; Michalski et al., 2011) coupled to a Dionex Ultimate 3000 RSLC nano-liquid chromatograph (Thermo Fisher, UK). Digest was loaded onto a trapping column (Acclaim PepMap 100 C18, 75 µm x 2 cm, 3 µm packing material, 100 Å) using a loading buffer of 0.1%(v/v) TFA, 2%(v/v) acetonitrile in water for 7 min (flow rate: 9 µl/min). The trapping column was set in-line with an analytical column (EASY-Spray PepMap RSLC C18, 75 µm x 50 cm, 2 µm packing material, 100 Å) and peptides eluted using a linear gradient of 96.2% A (0.1%(v/v) formic acid):3.8% B (0.1%(v/v) formic acid in water:acetonitrile [80:20](v/v)) to 50% A:50% B over 30 min (flow rate: 300 nl/min), followed by 1% A:99% B for 5 min and re-equilibration of the column to starting conditions. The column was maintained at 40°C, and the effluent introduced directly into the integrated nano-electrospray ionisation source operating in positive ion mode. The mass spectrometer was operated in DDA mode with survey scans between m/z 300–2000 acquired at a mass resolution of 70,000 (FWHM) at m/z 200. The maximum injection time was 250 ms, and the automatic gain control was set to 1e6. The 10 most intense precursor ions with charges states of between 2+ and 4+ were selected for MS/MS with an isolation window of 2 m/z units. The maximum injection time was 100 ms, and the automatic gain control was set to 1e5. Peptides were fragmented by higher-energy collisional dissociation using a normalised collision energy of 30%. Dynamic exclusion of m/z values to prevent repeated fragmentation of the same peptide was used (exclusion time: 20 s).
Peptide and protein identification
Proteins were identified from RAW data files, with trypsin specified as the protease, with one missed cleavage allowed and with fixed modifications of carbamidomethyl cysteine and variable modifications of oxidation of methionine and proline (Peaks Studio v8.5, Bioinformatics Solutions, Waterloo, Canada). Searches were performed against the UniProt Rattus Norvegicus database (www.uniprot.org/proteomes), with an FDR of 1%,≥2 unique peptides per protein and a confidence score >20. Protein network analysis was performed using the Search Tool for Retrieval of Interacting Genes/Proteins (STRING), v11.0 (Szklarczyk et al., 2019), and proteins were further classified using MatrisomeDB (Hynes and Naba, 2012) and the Protein ANalysis THrough Evolutionary Relationships (PANTHER) Classification System (Mi et al., 2013). The proteomic data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (Perez-Riverol et al., 2019) with the data set identifier PXD015928 and 10.6019/PXD015928.
Calculation of protein turnover rates
Peptides at each time point were identified using ProLuCID (Xu et al., 2015) searching against a reverse-decoyed protein sequence database (UniProt Rattus Norvigicus, reviewed, accessed 23/04/2018). Fixed modifications of carbamidomethyl cysteine and ≤3 variable modifications of oxidation of methionine and proline were allowed. Tryptic, semi-tryptic, and non-tryptic peptides within a 15-ppm mass window surrounding the candidate precursor mass were searched. Protein identification was performed using DTASelect (v.2.1.4) (Cociorva et al., 2006), with ≤1% global peptide FDR and ≥2 unique peptides per protein. Protein turnover rates and resulting half-lives were calculated using custom software (ProTurn v2.1.05; available at: http://proturn.heartproteome.org) as previously described and validated (Lam et al., 2014; Lau et al., 2016; Lau et al., 2018; Wang et al., 2014). Briefly, RAW files were converted into mzML format (ProteoWizard, v3.0.11781)(Adusumilli and Mallick, 2017) for input into ProTurn, along with DTAselect-filter text files for protein identification. ProTurn parameters were as follows: area-under-curve integration width: 60 p.p.m., extracted ion chromatograph smoothing: Savitzky-Golay filter (Savitzky and Golay, 1964) over seven data points. Non-steady state curve fitting was used to account for the initial delay in uptake of 2H, using a first-order kinetic curve to approximate the equilibration of 2H2O in body water. Values of kp and pss were inputted from the resultant curve fitting of 2H enrichment in serum samples. To control against false positive identifications, only peptides that were explicitly identified in ≥4 data points were accepted for the calculation of protein turnover. The 'Allow Peptide Modification' option was selected to include any identified post-translationally modified peptides in kinetic curve-fitting, and peptide isotopomer time series were included if R2 ≥0.8 or standard error of estimate (S.E)≤0.05 (Lam et al., 2014). Protein-level turnover rate is reported as the median and median absolute deviation of the turnover rate constants of each accepted unique constituent peptide, from which protein half-life can be calculated assuming a first order reaction (Lau et al., 2018).
For some proteins of interest, particularly in the IFM, automated calculation of turnover rates was unsuccessful, due to unsuccessful automated identification of isotopomer peaks. In these cases, turnover rate constants were calculated manually, using isotopomer peak height to calculate the relative abundance of M0 as a function of time (Thermo Xcalibur v2.2), and fitting first order kinetic curves to estimate k (GraphPad Prism v8.0.2). As the labelling pattern of each peptide is dependent on the number of available labelling sites (N), unlabelled relative abundance of the 0th isotopomer (a), as well as the plateau level of enrichment of deuterium (pss); these values were used to calculate the plateau values of the 0th isotopomer (A0) which occurs when the peptide is fully labelled. N was calculated from the literature (Lam et al., 2014; Commerford et al., 1983). a was calculated from the peptide sequence and natural abundance of heavy isotopes of carbon, nitrogen, oxygen and sulphur (Lam et al., 2014). pss was calculated from the serum enrichment as detailed above. The following equation was then used to calculate the plateau of A0:
A0,PLATEA=a(1−pss)N
Previous work demonstrates this accurately predicts the plateau measured experimentally (Lam et al., 2014), therefore, for curve fitting calculations the plateau was constrained to the calculated value for each peptide. To assess the degree of differences in curve fitting performed by automated and manual approaches, k values were calculated using both approaches for 10 peptides. Resulting k values differed by an average of 23%. This method does not allow for direct comparison between manual and automated calculations of k, due to differences in curve fitting method, but does allow differences in peptide k values between IFM and FM to be assessed, when both are calculated manually.
When ≥ 3 peptides were identified corresponding to a particular protein, statistical differences in turnover rate constants in tendon phases were assessed using paired t-tests, Wilcoxon matched pairs tests, or Mann-Whitney tests, with p<0.05 (GraphPad Prism, v8.2). The statistical test chosen was dependent on whether data were normally distributed, which was assessed with Kolmogorov-Smirnov tests, and whether the same peptides were identified in tendon phases, which allowed for paired statistical tests.
The proteomic data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD015928 and 10.6019/PXD015928. Source files for 2H enrichment data, ProTurn output files and manually calculated turnover rates are provided with the manuscript.
Choi H
Simpson D
Wang D
Prescott M
Pitsillides AA
Dudhia J
Clegg PD
Ping P
Thorpe CT
(2020) ProteomeXchange
Heterogeneity of proteome dynamics between connective tissue phases of adult tendon.
https://doi.org/10.6019/PXD015928
Adusumilli R
Mallick P
(2017) Data conversion with ProteoWizard msConvert
Ashraf Kharaz Y
Zamboulis D
Sanders K
Comerford E
Clegg P
Peffers M
(2017) Comparison between chaotropic and detergent-based sample preparation workflow in tendon for mass spectrometry analysis
Proteomics 17:1700018.
https://doi.org/10.1002/pmic.201700018
Avery NC
Bailey AJ
(2005) Enzymic and non-enzymic cross-linking mechanisms in relation to turnover of collagen: relevance to aging and exercise
Scandinavian Journal of Medicine and Science in Sports 15:231–240.
Babraj JA
Cuthbertson DJ
Smith K
Langberg H
Miller B
Krogsgaard MR
Kjaer M
Rennie MJ
(2005) Collagen synthesis in human musculoskeletal tissues and skin
American Journal of Physiology-Endocrinology and Metabolism 289:E864–E869.
https://doi.org/10.1152/ajpendo.00243.2005
Bechshøft CL
Schjerling P
Bornø A
Holm L
(2017) Existence of life-time stable proteins in mature rats-Dating of proteins' age by repeated short-term exposure to labeled amino acids throughout age
Chaudhury C
Mehnaz S
Robinson JM
Hayton WL
Pearl DK
Roopenian DC
Anderson CL
(2003) The major histocompatibility complex-related fc receptor for IgG (FcRn) binds albumin and prolongs its lifespan
Journal of Experimental Medicine 197:315–322.
Cociorva D
L. Tabb D
(2006) Validation of tandem mass spectrometry database search results using DTASelect
Current Protocols in Bioinformatics 16:4.
https://doi.org/10.1002/0471250953.bi1304s16
Commerford SL
Carsten AL
Cronkite EP
(1983) The distribution of tritium among the amino acids of proteins obtained from mice exposed to tritiated water
Radiation Research 94:151–155.
Connizzo BK
Yannascoli SM
Soslowsky LJ
(2013) Structure-function relationships of postnatal tendon development: a parallel to healing
Matrix Biology 32:106–116.
https://doi.org/10.1016/j.matbio.2013.01.007
Culav EM
Clark CH
Merrilees MJ
(1999) Connective tissues: matrix composition and its relevance to physical therapy
Physical Therapy 79:308–319.
https://doi.org/10.1093/ptj/79.3.308
Delgado Caceres M
Pfeifer CG
Docheva D
(2018) Understanding tendons: lessons from transgenic mouse models
Godinho MSC
Greenwald SE
Screen HRC
(2017) Elastin is localised to the interfascicular matrix of energy storing tendons and becomes increasingly disorganised with ageing
Scientific Reports 7:9713.
Gumucio JP
Sugg KB
Mendias CL
(2015) TGF-β superfamily signaling in muscle and tendon adaptation to resistance exercise
Exercise and Sport Sciences Reviews 43:93–99.
https://doi.org/10.1249/JES.0000000000000041
Heinemeier KM
Heinemeier J
Magnusson SP
(2013) Lack of tissue renewal in human adult achilles tendon is revealed by nuclear bomb (14)C
FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology 27:2074–2079.
https://doi.org/10.1096/fj.12-225599
Møller MB
Grum-Schwensen T
Petersen MM
(2016) Radiocarbon dating reveals minimal collagen turnover in both healthy and osteoarthritic human cartilage
Science Translational Medicine 8:346ra90.
https://doi.org/10.1126/scitranslmed.aad8335
Hsueh MF
Önnerfjord P
Bolognesi MP
Easley ME
Kraus VB
(2019) Analysis of "old" proteins unmasks dynamic gradient of cartilage turnover in human limbs
Science Advances 5:eaax3203.
https://doi.org/10.1126/sciadv.aax3203
Humphrey JD
Dufresne ER
Schwartz MA
(2014) Mechanotransduction and extracellular matrix homeostasis
Hynes RO
Naba A
(2012) Overview of the matrisome--an inventory of extracellular matrix constituents and functions
Cold Spring Harbor Perspectives in Biology 4:a004903.
https://doi.org/10.1101/cshperspect.a004903
Jaisson S
Larreta-Garde V
Bellon G
Hornebeck W
Garnotel R
Gillery P
(2007) Carbamylation differentially alters type I collagen sensitivity to various collagenases
Jeffay H
The Metabolism of Serum Proteins: III. Kinetics of serum protein metabolism during growth
Kastelic J
Galeski A
Baer E
(1978) The Multicomposite Structure of Tendon
Connective Tissue Research 6:11–23.
Katsma MS
Patel SH
Eldon E
Corbell KA
Shimkus KL
Fluckey JD
Carroll CC
(2017) The influence of chronic IL-6 exposure, in vivo , on rat Achilles tendon extracellular matrix
Cytokine 93:10–14.
https://doi.org/10.1016/j.cyto.2017.04.011
Kharaz YA
Tew SR
Canty-Laird EG
(2016) Proteomic differences between native and tissue-engineered tendon and ligament
PROTEOMICS 16:1547–1556.
Kilkenny C
Browne WJ
Cuthill IC
Emerson M
(2010) Improving Bioscience Research Reporting: The ARRIVE Guidelines for Reporting Animal Research
PLOS Biology 8:e1000412.
Kim TY
Kim AK
Lau E
Lin AJ
Liem DA
Zhang J
Zong NC
Lam MP
(2012) Metabolic labeling reveals proteome dynamics of mouse mitochondria
Molecular & Cellular Proteomics 11:1586–1594.
https://doi.org/10.1074/mcp.M112.021162
Koury MJ
(2016) Red Blood Cell Production and Kinetics Rossi's Principles of Transfusion Medicine
Wiley Online Library.
https://doi.org/10.1002/9781119013020.ch08
Kuo CK
Petersen BC
Tuan RS
(2008) Spatiotemporal protein distribution of TGF-betas, their receptors, and extracellular matrix molecules during embryonic tendon development
Developmental Dynamics 237:1477–1489.
https://doi.org/10.1002/dvdy.21547
Lake-Bakaar G
Rubio CE
McKavanagh S
Potter BJ
Summerfield JA
(1980) Metabolism of 125I-labelled trypsin in man: evidence of recirculation
Gut 21:580–586.
https://doi.org/10.1136/gut.21.7.580
Ng DC
Liang X
Bleakley BJ
Liu C
Tabaraki JD
Cadeiras M
Deng MC
(2014) Protein kinetic signatures of the remodeling heart following isoproterenol stimulation
The Journal of Clinical Investigation 124:1734–1744.
https://doi.org/10.1172/JCI73787
Cao Q
Dincer TU
Bot BM
Ge J
(2016) A large dataset of protein dynamics in the mammalian heart proteome
Lam MPY
Ng DCM
Lee JM
Hermjakob H
(2018) Integrated omics dissection of proteome dynamics during cardiac remodeling
Maroudas A
Bayliss MT
Uchitel-Kaushansky N
Schneiderman R
Gilav E
(1998) Aggrecan turnover in human articular cartilage: use of aspartic acid racemization as a marker of molecular age
Archives of Biochemistry and Biophysics 350:61–71.
https://doi.org/10.1006/abbi.1997.0492
McCabe BJ
Bederman IR
Croniger C
Millward C
Norment C
Previs SF
(2006) Reproducibility of gas chromatography-mass spectrometry measurements of 2H labeling of water: application for measuring body composition in mice
Analytical Biochemistry 350:171–176.
https://doi.org/10.1016/j.ab.2006.01.020
Mi H
Muruganujan A
Casagrande JT
Thomas PD
(2013) Large-scale gene function analysis with the PANTHER classification system
Nature Protocols 8:1551–1566.
Michalski A
Damoc E
Hauschild JP
Lange O
Wieghaus A
Makarov A
Nagaraj N
Cox J
Horning S
(2011) Mass spectrometry-based proteomics using Q exactive, a high-performance benchtop quadrupole orbitrap mass spectrometer
Molecular & Cellular Proteomics 10:M111.011015.
Miller BF
Olesen JL
Hansen M
Døssing S
Crameri RM
Welling RJ
Flyvbjerg A
(2005) Coordinated collagen and muscle protein synthesis in human Patella tendon and quadriceps muscle after exercise
https://doi.org/10.1113/jphysiol.2005.093690
Perez-Riverol Y
Csordas A
Bai J
Bernal-Llinares M
Hewapathirana S
Kundu DJ
Inuganti A
Griss J
Mayer G
Eisenacher M
Pérez E
Uszkoreit J
Pfeuffer J
Sachsenberg T
Yilmaz S
Tiwary S
Audain E
Walzer M
Jarnuczak AF
Ternent T
Brazma A
Vizcaíno JA
(2019) The PRIDE database and related tools and resources in 2019: improving support for quantification data
Powers ET
Morimoto RI
Dillin A
Kelly JW
Balch WE
(2009) Biological and chemical approaches to diseases of proteostasis deficiency
Annual Review of Biochemistry 78:959–991.
https://doi.org/10.1146/annurev.biochem.052308.114844
Robinson KA
Sun M
Barnum CE
Weiss SN
Huegel J
Shetye SS
Lin L
Saez D
Adams SM
Birk DE
(2017) Decorin and biglycan are necessary for maintaining collagen fibril structure, fiber realignment, and mechanical properties of mature tendons
Matrix Biology 64:81–93.
Russo V
Mauro A
Martelli A
Di Giacinto O
Di Marcantonio L
Nardinocchi D
Berardinelli P
Barboni B
(2015) Cellular and molecular maturation in fetal and adult ovine calcaneal tendons
Journal of Anatomy 226:126–142.
https://doi.org/10.1111/joa.12269
Rys JP
Monteiro DA
Alliston T
(2016) Mechanobiology of tgfβ signaling in the skeleton
Matrix Biology: Journal of the International Society for Matrix Biology 54:413–425.
Sarver DC
(2017) Sex differences in tendon structure and function
Journal of Orthopaedic Research 35:2117–2126.
https://doi.org/10.1002/jor.23516
Savitzky A
Golay MJE
(1964) Smoothing and differentiation of data by simplified least squares procedures
Analytical Chemistry 36:1627–1639.
https://doi.org/10.1021/ac60214a047
(1983) Connective tissues: the natural fibre-reinforced composite material
Journal of the Royal Society of Medicine 76:993–994.
Shah D
Mital K
(2018) The role of trypsin:chymotrypsin in tissue repair
Advances in Therapy 35:31–42.
Sivan SS
Tsitron E
Wachtel E
Roughley PJ
Sakkee N
van der Ham F
DeGroot J
Roberts S
(2006) Aggrecan turnover in human intervertebral disc as determined by the racemization of aspartic acid
(2008) Collagen turnover in normal and degenerate human intervertebral discs as determined by the racemization of aspartic acid
Smeets JSJ
Horstman AMH
Vles GF
Emans PJ
Goessens JPB
Gijsen AP
van Kranenburg JMX
van Loon LJC
(2019) Protein synthesis rates of muscle, tendon, ligament, cartilage, and bone tissue in vivo in humans
Södersten F
Hultenby K
Heinegård D
Johnston C
Ekman S
(2013) Immunolocalization of collagens (I and III) and cartilage oligomeric matrix protein in the normal and injured equine superficial digital flexor tendon
Connective Tissue Research 54:62–69.
Spiesz EM
Chaudhry S
Riley GP
Birch HL
Screen HR
(2015) Tendon extracellular matrix damage, degradation and inflammation in response to in vitro overload exercise
Journal of Orthopaedic Research 33:889–897.
Swovick K
Welle KA
Hryhorenko JR
Seluanov A
Gorbunova V
Ghaemmaghami S
(2018) Cross-species comparison of proteome turnover kinetics
Molecular & Cellular Proteomics 17:580–591.
https://doi.org/10.1074/mcp.RA117.000574
Szklarczyk D
Gable AL
Lyon D
Junge A
Wyder S
Huerta-Cepas J
Simonovic M
Doncheva NT
Morris JH
Jensen LJ
Mering CV
(2019) STRING v11: protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets
Tavill AS
(1972) The synthesis and degradation of liver-produced proteins
Streeter I
Pinchbeck GL
Goodship AE
(2010) Aspartic acid racemization and collagen degradation markers reveal an accumulation of damage in tendon collagen that is enhanced with aging
Udeze CP
(2012) Specialization of tendon mechanical properties results from interfascicular differences
Journal of the Royal Society Interface 9:3108–3117.
https://doi.org/10.1098/rsif.2012.0362
Lei II
Varone A
(2015a) Tendon overload results in alterations in cell shape and increased markers of inflammation and matrix degradation
Scandinavian Journal of Medicine & Science in Sports 25:e381–e391.
https://doi.org/10.1111/sms.12333
(2015b) The interfascicular matrix enables fascicle sliding and recovery in Tendon, and behaves more elastically in energy storing tendons
Journal of the Mechanical Behavior of Biomedical Materials 52:85–94.
https://doi.org/10.1016/j.jmbbm.2015.04.009
Karunaseelan KJ
Ng Chieng Hin J
(2016a) Distribution of proteins within different compartments of tendon varies according to tendon type
Peffers MJ
Halliwell E
(2016b) Anatomical heterogeneity of tendon: fascicular and interfascicular tendon compartments have distinct proteomic composition
(2016c) Fascicles and the interfascicular matrix show adaptation for fatigue resistance in energy storing tendons
Acta Biomaterialia 42:308–315.
https://doi.org/10.1016/j.actbio.2016.06.012
Verzijl N
Thorpe SR
Bank RA
Shaw JN
Lyons TJ
Bijlsma JW
Lafeber FP
Baynes JW
TeKoppele JM
(2000) Effect of collagen turnover on the accumulation of advanced glycation end products
(2014) Characterization of human plasma proteome dynamics using deuterium oxide
PROTEOMICS - Clinical Applications 8:610–619.
https://doi.org/10.1002/prca.201400038
Williamson JC
Edwards AV
Verano-Braga T
Schwämmle V
Kjeldsen F
Jensen ON
Larsen MR
(2016) High-performance hybrid orbitrap mass spectrometers for quantitative proteome analysis: observations and implications
Proteomics 16:907–914.
Xu T
Park SK
Venable JD
Wohlschlegel JA
Diedrich JK
Lu B
Liao L
Hewel J
Han X
Wong CCL
Fonslow B
Delahunty C
Shah H
(2015) ProLuCID: an improved SEQUEST-like algorithm with enhanced sensitivity and specificity
Journal of Proteomics 129:16–24.
https://doi.org/10.1016/j.jprot.2015.07.001
Yamauchi M
Sricholpech M
(2012) Lysine post-translational modifications of collagen
Essays in Biochemistry 52:113–133.
https://doi.org/10.1042/bse0520113
Zamboulis DE
(2018) Pre- and post-natal development of the tendon inter-fascicular matrix and fascicles in the equine superficial digital flexor tendon and common digital extensor tendon
Osteoarthritis and Cartilage 26:S403–S404.
https://doi.org/10.1016/j.joca.2018.02.783
Clifford J Rosen
Senior Editor; Maine Medical Center Research Institute, United States
Calvin Vary
Reviewing Editor; University of Maine, United States
Reviewer; University of Maine, United States
This is an interesting study that uses mass spectrometry combined with deuterium isotope labeling (via initial injection and D2O water ad lib) to gain an unbiased look at the protein composition and turnover in the interfascicular and fascicular (IFM and FM, respectively) matrix of the rat Achilles tendon. The results support the hypothesis that the proteome of the IFM is more dynamic than the FM, and point out that these data in this model system have relevance to tendinous tissue remodeling and repair.
Thank you for submitting your work entitled "Heterogeneity of proteome dynamics between connective tissue phases of adult tendon" for consideration by eLife. Your article has been evaluated by Clifford Rosen as the Senior Editor, a Reviewing Editor and one peer reviewer.
There are some issues that need to be addressed before acceptance, as outlined below:
Overall, the results were convincing that there is a difference in IFM and FM composition and turnover. However, some concerns were noted regarding the validation of some aspects of this model system. Addressing these concerns would strengthen the paper overall.
First, there were no corroborating data for the mass spectrometric results. For example, western blotting for IFM and IF proteins would be useful not only to corroborate the MS data but additionally, to gauge the efficiency of the separation of IFM and FM tissue sections and their relative levels of enrichment for at least 1 or a few selected proteins. An alternative approach (given the low amounts of IFM material) could include immunofluorescence or immunohistochemical staining of tendon sections for representative proteins.
Second, regarding the equal representation of sex in the experiments, justification for using only female rats should be offered.
The Discussion should address whether turnover of protein post translational modifications (e.g., glycosylation) is an important feature of tendinous remodeling. Also, the Discussion would be enhanced by a concise discussion of any relevant knockout mouse phenotypes that support the importance of IFM- or FM-expressed proteins to tendinous or other tissue function.
Finally, if this was not included, provision for submission of these MS/proteomic data should be made to an appropriate public-accessible data base (e.g., PRIDE, PRoteomics IDEntifications database), consistent with Journal policies.
This paper investigates the proteome dynamics in tendon by comparing calculated protein turnover rates in the interfascicular matrix (IFM) vs. the fascicular matrix (FM).
This is a very interesting study illustrating the different protein turnover rates in different regions of the tendon, isolated by laser capture microdissection. The methods used are highly adequate and the data interpretation is sound. The main conclusions drawn are supported by the data presented and the study is well presented in the manuscript.
In the database search (subsection "Peptide and Protein Identification") – it seems like you did not use oxidation of proline as a variable modification, please comment. The number of collagen peptides identified would be significantly increased by using that modification and I would highly recommend a new search using this modification to see how the collagen data is affected.
[…] Overall, the results were convincing that there is a difference in IFM and FM composition and turnover. However, some concerns were noted regarding the validation of some aspects of this model system. Addressing these concerns would strengthen the paper overall.
Thank you for this useful suggestion. We were planning to immunolabel some tendon sections for key ECM proteins (collagen types 1 and 3, decorin, biglycan, fibromodulin) but unfortunately have not been able to do so due to lab closures as a result of COVID-19. However, distribution of these proteins within tendon is well described in the literature, and we have discussed our findings in light of these (Discussion, third and ninth paragraphs).
Thank you for raising this point. We used female rats to maintain consistency with a related study, for which female rats were chosen to reduce the possibility of having to singly house animals after surgery due to animals fighting (which is common in males). We have added use of a single sex as a limitation of the study (Discussion, second paragraph).
Thank you for raising this point, which we had not considered. We have added a section to the Discussion considering the effect of PTMs on tendon remodelling (Discussion, fourth paragraph). We have also expanded on the discussion of relevant KO mice phenotypes (Discussion, ninth paragraph).
The data have been uploaded to the PRIDE repository, as described in the subsection "Peptide and Protein Identification".
[…] In the database search (subsection "Peptide and Protein Identification") – it seems like you did not use oxidation of proline as a variable modification, please comment. The number of collagen peptides identified would be significantly increased by using that modification and I would highly recommend a new search using this modification to see how the collagen data is affected.
Thank you for this suggestion, we have now included oxidation of proline as a variable modification in the searches, which has resulted in a large increase number of collagen peptides identified in whole tendon, FM and IFM (Results). We have re-calculated protein half-life using this data, which has allowed us to calculate the turnover rates of a number of additional minor collagens (see Figure 6). It has also increased the number of collagen type I peptides identified, increasing the significance of our results (see Figure 7).
Department of Physiology and Medicine, David Geffen School of Medicine, UCLA, Los Angeles, United States
Software, Formal analysis, Writing - review and editing
Centre for Proteome Research, Biosciences Building, Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom
Investigation, Methodology, Writing - review and editing
Software, Writing - review and editing
Investigation, Methodology
Department of Comparative Biomedical Sciences, Royal Veterinary College, London, United Kingdom
Conceptualization, Writing - review and editing
Department of Clinical Sciences and Services, Royal Veterinary College, Hatfield, United Kingdom
Department of Musculoskeletal Biology, Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, United Kingdom
Resources, Software
Conceptualization, Funding acquisition, Investigation, Methodology, Writing - original draft, Project administration, Writing - review and editing
cthorpe@rvc.ac.uk
Arthritis Research UK (21216)
National Heart, Lung, and Blood Institute (R35 HL135772)
National Heart, Lung, and Blood Institute (T32 HL139450)
The authors would like to thank Professor Rob Beynon for his advice regarding study design and when preparing the manuscript.
Animal experimentation: All procedures complied with the Animals (Scientific Procedures) Act 1986, were approved by the local ethics committee at the Royal Veterinary College, were performed under project licence PB78F43EE and are reported according to the ARRIVE guidelines. All of the animals were handled according to National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) guidelines.
Clifford J Rosen, Maine Medical Center Research Institute, United States
Calvin Vary, University of Maine, United States
Accepted: April 16, 2020
Version of Record published: May 12, 2020 (version 1)
© 2020, Choi et al.
proteome dynamics
interfascicular matrix
proteostasis


text:
The Ever-Changing Pushkinskaya 10 Art Center in St. Petersburg, Revisited
Text: Nikolai Sadik-Ogli 14.8.2017
Photographs: Ali Sadik-Ogli
The Pushkinskaya 10 Art Center, a self-described Free Cultural Fellowship (http://www.p-10.ru/), has been a central cultural and artistic hub in St. Petersburg since it was founded in an abandoned building by a squatting group of independent artists, musicians, and writers in 1989 known as the "Free Culture" Society in direct opposition to Soviet-enforced cultural conformity. This author first visited the complex in 1996, primarily to attend one of the weekly improvisation performance salons hosted by composer Yuri Kassyanik. That particular evening's concert was dedicated to the memory of the recently deceased American jazz singer, Ella Fitzgerald. At that time, the center was a loose confederation of independent musicians and artists working together outside of the official system. Its underground status was reflected by the nearby shops that primarily specialized in bootleg cassettes and CDs of American and European rock and pop music. The dilapidated building's courtyard seemed to house many bums and alcoholics, which sometimes made it feel almost scary to visit.
Today, the facility is a clean, well-organized, and officially recognized cultural destination. The entrance is well marked and has been moved to the opposite side of the block at 53 Ligovsky Prospekt. The complex consists of four buildings that house a variety of galleries and concert spaces that host changing exhibitions and programs amongst the many artist's apartments and studios that are still there. Art is everywhere – drawn on the walls, filling the stairwells, hallways, and empty spaces inside and out. There is a reception area that charges an entrance fee of 500 RUB for foreign residents but only 200 RUB for locals(1). Even the surrounding shops have become legitimate businesses that mainly cater to occult, New Age, and other exotic items and subject matter.
The centerpiece of the complex is the Museum of Non-Conformist Art, which is housed in two separate Big and Small Halls in Buildings C and D, respectively. The exhibition "#Rejected" was on display in the Big Hall through 23 July 2017 and featured pieces that had been either 1) rejected by Soviet censors or other juries when they were submitted for exhibitions and competitions, or by collectors who never purchased them; 2) rejected by the artists themselves for various reasons (without being destroyed, however); or 3) reflected the idea of rejection as the subject matter of the work. The pieces included Alexander Goncharuk's four-panel homage to Malevich's "Black Square," each of which served as the background to a forest of dead flies pinned to the paintings in steady rows like zoological display. Recalling the way in which the "Black Square" had originally been displayed in the upper corner of the exhibition hall, the paintings on the right in the picture above had been inspired by Russian Orthodox icons in a more traditional manner. Another piece consisted of the board that had been set beneath the easel and happened to randomly accumulate dense abstract patterns created by paint dripping during the creation of other works.
The Small Hall housed the more historically interesting exhibition, "Leningrad Non-Conformist Art from the 1950-1960s: Paintings and Graphics from the MoNa Collection," which is on display through 10 September 2017. Today in retrospect, it is difficult to imagine how these mostly impressionistic or humoristic pieces could ever have been considered controversial. The exhibition included a historically important map of the many clandestine apartment exhibitions that had dared to display these contested works throughout the city. Ultimately, however, the true story behind what the exhibition's title alludes to is obviously much larger than what can be displayed in its three rooms.
Among the other venues, the Pons Per Styx Gallery in Building C featured an exhibition of humorously subversive "function-collages" by Vadim Voinov. They included "Start," a simple commemoration of Yuri Gagarin's first space flight on a blue and black background painted on a board to which a silver toy rocket was attached. Many of Voinov's other pieces featured memorabilia depicting life in the former Soviet Union and its leaders, like Joseph Stalin, in unflattering ways.
The stairwell between the fourth and fifth floors in Building C houses The Door Gallery, which displayed the "All Seeing Eye," an installation by Janno Bergman and the Estonian "Art Container" Group, through 23 July. The piece consists of a three-dimensional model of the pyramid seen on the back the American one-dollar bill inside a large plastic case. The case's lights turn off as people pass by, and in the dimness, the eye at the top of the pyramid rises up from within.
The Museum of The New Academy of Fine Arts in Building B featured an exhibition dedicated to "Xenia," the deceased wife of artist Timur Novikov. Also in Building B, the Art-Liga Gallery displayed "Games of Reality: An Exhibition in the Framework of the 'New Names' Project" with paintings by Lo Si, Sasha Sandler, and others. Additionally, the first floor Corridor Gallery between Buildings B and C houses the permanent exhibition, "Samizdat A4," dedicated to literary, artistic, and musical activities conducted in opposition to Soviet repression since 1956. In conjunction with Voinov's pieces and the retrospective on display in the Small Hall, these exhibitions effectively demonstrate the original, rebellious spirit behind the founding of the Pushkinskaya experiment during Soviet times while also demonstrating its contemporary historical importance as a living, central repository for that independent tradition.
A visit to Pushkinskaya 10 can be capped off by refreshments from either the trendy Fish Fabrique café and concert venue on the ground floor near the entrance or in the Clipping Room (Montazhnaya) café on the fourth floor of Building D, which features video art, media exhibitions, and a wide mix of interesting background music. There is also a small Hotel Pushkinskaya 10 with eight individually decorated rooms.
Several of the other facilities were not open at the time of this visit. The Sound Museum in Building D features changing exhibitions, such as Vladislav Makarov's recent video installation "Inner Applause," as well as occasional concerts. The Lampa Studio in Building B is described as an experimental space for young artists and curators. The complex also houses the office of Nikolai Vasin's Temple of Love, Peace, and Music named after John Lennon, which displays Vasin's fifty-year-old collection of Beatles memorabilia. A street sign identifying the unofficial "Ulitsa John Lennon" has also been posted in the courtyard. In a similar but more domestic spirit, Building C contains a Museum of the Reality of Russian Rock.
The history of the center has been thoroughly documented in the extensive book: Art Center "Pushkinskaya-10" – Parallelosphere: 1989-2009 (St. Petersburg: Pushkinskaya-10 Art League, 2010) by Sergey Kovalsky, Evgeny Orlov, and Yuri Ribakov.
(1) The Pushkinskaya 10 logo, as seen above the entryway and in all of their advertising materials, immediately recalls The Resident's trademark image of an eyeball in a top hat, as seen in the poster for this recent documentary: http://residentsmovie.com/.
Edellinen artikkeliEdellinen Mediaalinen läpivalaisu: ajatuksia transmedialesta
Seuraava artikkeliSeuraava Rikkaharjoja ja yt-neuvottelusankareita – Tuntematon sotilas 2017

redpajama_set_name: RedPajamaCommonCrawl


glamorous humming bird bird bath hummingbird bird bath oil pan r9097688.
new humming bird bird bath hummingbird bird bath mister o0866620.
liveable humming bird bird bath hummingbird bird bath video p5609032.
liveable humming bird bird bath hummingbird bird bath stained glass s7218095.
qualified humming bird bird bath hummingbird bird bath diy r1232627.
likeable humming bird bird bath hummingbird bird bath a2232199.
good humming bird bird bath how to make a hummingbird bath fountain q9157132.
awesome humming bird bird bath hummingbird bird bath at kmart g8165357.
fantastic humming bird bird bath hummingbird bird bath fountain y1380117.
newest humming bird bird bath hummingbird bird bath canada g5028595.
favorite humming bird bird bath hummingbird bird bath mister z6510533.
favorite humming bird bird bath how to make a hummingbird bath fountain n2319822.
superb humming bird bird bath hummingbird bird bath stained glass j4754715.
artistic humming bird bird bath how to make your own hummingbird bath l5370809.
qualified humming bird bird bath hummingbird bird bath fountain v6037857.
glamorous humming bird bird bath how do you make a hummingbird bath t4456060.
amazing humming bird bird bath how to make a homemade hummingbird bath x1937600.
unusual humming bird bird bath how to make a hummingbird bath fountain u0824813.
minimalist humming bird bird bath hummingbird bird bath mister s3517467.
valuable humming bird bird bath hummingbird bird bath stained glass m5217610.
staggering humming bird bird bath how to make your own hummingbird bath r3572372.
delightful humming bird bird bath hummingbird bird bath instructions f6791337.
wondeful humming bird bird bath hummingbird bird bath diy f9934333.
delightful humming bird bird bath how to make a hummingbird bath u9243795.


text:
MONTANA DEPARTMENT OF COMMERCE
Funded Projects Human Resources and Payroll Leadership Legislative Information Mission, Vision, Values Welcome Message
Boards Boards
Board of Horse Racing Board of Housing Board of Investments Coal Board Hard Rock Mining Impact Board Montana Heritage Commission Montana Facility Finance Authority Small Business Development Center Advisory Council State Tribal Economic Development Commission Tourism Advisory Council
Connect Employee Directory Red Tape Relief
Divisions Divisions
Brand MT Business MT Community Development Montana Housing
Legal Affairs Legal Affairs
Administrative Rule Information Public Information Requests
ARPA Public Comment Media Contacts Public Meetings
Research and Information Services Research and Information Services
Census and Economic Information Center
Industry Development Program Industry Development Program
Seminar Series Data Centers Malting
Broadwater County Receives $400,000 of Grant Funding to Support Three Forks Brewery Expansion
HELENA – The Montana Department of Commerce announced today that $400,000 of federal economic development grant funding has been allocated to Broadwater County to provide a loan to Jackrabbit Pub, LLC for the purchase of new commercial kitchen equipment. The grant funding comes from Commerce's Community Development Block Grant (CDBG) Economic Development Grant program.
"At Commerce, we're always proud to support Montana's small businesses, which help our rural communities grow their economies and prosper. Projects like the Jackrabbit Pub will help create a true boost for the Three Forks area," said Montana Department of Commerce Director Scott Osterman. "The revenue generated by this expansion will provide integral financial benefits to Three Forks and the surrounding communities through job creation, tourism promotion, and an increased tax base."
The Jackrabbit Pub, located near Three Forks, is a new expansion of Bozeman's Bridger Brewing. The pub plans to use locally grown ingredients for its restaurant, as well as a local workforce to staff the pub. Seventeen new jobs will be created as part of the expansion.
"Jackrabbit Pub and Bridger Brewing are excited to work with Commerce's CDBG program on this economic development project. These funds are critical in allowing us to create jobs and spur the economy in a rural county like Broadwater," said David Sigler, owner of Bridger Brewing. "We also love the fact that as we pay these funds back, other businesses in the community can take advantage of this capital in their future endeavors to further support not only job growth, but small businesses, which are the heart and soul of every community."
The CDBG Economic Development Grant program supports businesses by awarding grants to local governments and making fixed-rate financing available to those businesses at low interest rates. Businesses work with local governments to apply to the program, and the local government then loans to an eligible business or grants to a non-profit.
CDBG funding originates from the U.S. Department of Housing and Urban Development (HUD).
For more information about CDBG Economic Development grants, visit COMDEV.MT.GOV.
Tags: Community MT
Want more updates from the Department of Commerce? Subscribe to receive email updates here.
301 S. Park Ave.
Helena, MT 59620-0501
Choose Montana
Consolidated Plan
Made in Montana
Visit Montana
Department of Environmental Quality
Department of Natural Resources and Conservation
Governor's Office of Economic Development
We use cookies. We want to make our website more user-friendly and continuously improve it. If you continue to use the website, you agree to the use of cookies.


text:
New Item

        <form class="form-horizontal" data-ng-submit="create()" novalidate>
            <fieldset>

                    Name

                        <input type="text" data-ng-model="name" id="name" class="form-control" placeholder="Name" required>

                    Price

                        <input type="number" data-ng-model="price" id="price" class="form-control" placeholder="0" required>

                        <textarea rows="10" data-ng-model="description" class="form-control" placeholder="a description">
                        </textarea>

                    <input type="submit" class="btn btn-default">

            </fieldset>
        </form>

redpajama_set_name: RedPajamaGithub


text:
Q: How to force Flutter to use new developer certificate? I am currently developing an application with Flutter. I used to be able to run the code directly using VS code or the command flutter run. Ever since I revoked my developer certificate and generated a new one, the above options when executed, the app crashes on my iPhone. If, however, I run it on the simulator, it works as expected.
I suspect that this is due to the change in certificates. Xcode says my app is signed with the certificate B39****** where as flutter run says it is signing with 578*******
I have even tried flutter config --clear-ios-signing-cert as well as flutter upgrade and it still signs with 578.
How can I force flutter to sign my app with the new certificate? Currently, this means I cannot use the hot reload feature on my physical devices.

A: Run flutter config to see your current settings and a certificate choice applied.
To clear the choice according to the documentation you can run:
flutter config --clear-ios-signing-cert.

--clear-ios-signing-cert                     Clear the saved development certificate choice used to sign apps for iOS device deployment.

Flutter 2.0.5.

A: I think check this file vim ~/.flutter_settings will be well.
{
  "ios-signing-cert": "Apple Development: XXXX@gmail.com (XXXXXXXX)",
  "enable-macos-desktop": true
}

A: I had a very similar issue a couple months ago.  It took me several hours and basically I had to sign out of and then sign back into XCode:
XCode->Preferences->Accounts
Completely sign out of any accounts and then sign back in with the newest one.

A: Use

flutter clean

in the terminal. When will clear the selected certificate and will present a choice to you next time you run you code

A: In code sign page of Xcode.
There is sub pages, with tab: All, Debug, Release and Profile.
It takes me several days to figure out I have selected the wrong develop team.
And then takes me few hours to find out that I have somehow clicked the Profile tab, instead of All.
I guess you have the same problem.

A: I just fixed it by going into Xcode and setting the correct team. I think perhaps Flutter does have a saved file somewhere with these old default c

A: I was trying to change the default one that gets used as I create a new project, here's what worked for me:
1-Removing old certificate from keychain access
2-Adding new one
3-Restarting terminal session and creating a new project
ps: other steps that might be needed as well:
Sign out of old account on Xcode and sign in with the new one

redpajama_set_name: RedPajamaStackExchange


Robin Williams- when I hear that name I think of my childhood. Watching Robin portray amazing characters like in the film Jack, a young soul trapped in an old man's body or being stuck in a board game in the movie Jumanji.
When you watched him on the screen, it did not feel like you were watching a comedy legend, it felt like you were watching a kid, just like yourself, going through these extraordinary obstacles. He was never an old man trying to act young in a family comedy film, like most of the actors you see nowadays. He owned every character he played.
Then I went to college and learnt the art of improvisational comedy in a course. It was the most satisfying form of performance art for me, so I decided to learn more about it and start performing.
During my years of performing Urdu and English improvisation on television and theatre, I realised that my current comedy heroes like Will Ferell and Conan O' Brian were both great improvisers. But not only that, I saw Robin Williams star in the American television show Whose Line Is It Anyway? as a guest improviser and he owned it! He dominated the entire episode with great laughter from his performance. It was amazing!
The point I am trying to make here is that as a performer, I grew with Robin Williams by my side the entire time. Every stage of life I went into, Robin was there greeting me and showing me how versatile an artist can be. He was not just the best stand up, improviser and comedian, but he could also do an Oscar winning performance in a drama film like Good Will Hunting just to show you how vast his widen his scope.
The last hallmark of my journey with Robin Williams was when I saw him in New York, where he performed and spoke about his life. It was unreal to see him up-close; I remember being so nervous that it was embarrassing.
I couldn't help it – that is the affect a legend has on you; someone who brought happiness to so many people for so long.
I liked him in 'Mork & Mindy' …and as the T.V. Frog prince with actress Teri Garr from the 80's..
Just watched 'Old Dogs' last weekend with Robin Williams & John Travolta.


acid rich oils of Evening Primrose, Avocado, Olive and Raspberry Seed provide instant hydration and softness, while Chamomile helps calm and soothe irritated skin for a healthier, more radiant complexion. Fantastic option for those not wanting sunscreen included in their moisturiser. Suitable for all skin types, especially dry and mature.


text:
Radixan Religious Knowledge
Understand Christianity
Home > Understand Christianity > Self Supporting Seventh Day Adventist Churches
Self Supporting Seventh Day Adventist Churches
The 2013 budget of the Seventh-day Adventist Church's world headquarters funds mission work and administrative support outside of North America. to continue until the work is established and.
What Does The Bible Say About Dying For Your Faith What does the Bible really say? Recently I asked 25 friends. Here's my best effort to condense their responses into the 5 most important teachings of the Bible. 1. A loving God created our world. They are also a profound betrayal of the most fundamental traditions of the faith their university is meant to preserve.
Shortly after, officials from the Seventh Day Adventist and Alliance Church joined in on. and we appreciate every little gift." Those interested in supporting the church can make an online donation.
Berkley Center For Religion Peace And World Affairs What Is First Eucharist In The Catholic Church Basic Texts for the Roman Catholic Eucharist THE ORDER OF MASS. This webpage contains the English translation of the Mass used up until 2011. As of Nov. 27, 2011, English-speaking Catholics are using a new English Translation of the Third Edition of The Roman Missal. Second Edition
The Seventh-day Adventist Church acknowledges literature evangelism as a self- supporting missionary in harmony with the church's teachings and principles.
Adventist-laymen's Services & Industries (ASI) is a membership-based. who are enthusiastic about actively participating in the church's worldwide mission. In 1947 these self-supporting entities formed the Association of Seventh-day.
New York United Sabbath-Day Adventist Church. The International Association of Free Seventh-day Adventists is a network of self-supporting SDA churches.
On Sunday evening, April 15, suspected terrorists opened fire in front of the Quetta Seventh-day Adventist Church in Pakistan. During this difficult time, we are humbled by the care and support of.
A truck drove into a crowd during Bastille Day ceremonies. seven Adventist members have been injured during the attack. Blood donations are more than welcome, according to local authorities. "We.
Heritage is a self-supporting coeducational boarding academy placed amid. of education described in the Encyclopedia of the Seventh-day Adventist Bible. and their church, choosing professions that enable them to serve others and to.
View Larger Citing financial reasons, board members of Weimar Institute of Health & Education in Northern California announced in a statement last month that the self-supporting. advocated by the.
Ted N.C. Wilson, president of the Seventh-day Adventist. and the supporting ministries, many of which belong to that wonderful organization called ASI, that work side-by-side with the official.
Hartland Day Academy. null. Hartland administration is committed to working closely with the worldwide Seventh-day Adventist Church. Read More.
Seventh-day Adventists are not fundamentalists," said Andrews University Church History professor Nicholas Miller. She herself did not support it, said Miller. After Ellen G. White's death in 1915,
Aug 22, 2018. The SDA Church still operates a handful of food production facilities. self- supporting Seventh-day Adventist medical missionary, founded.
the headquarters of the Seventh-day Adventist Church in Wash-. The Far Eastern Division of Seventh-day Adventists granted. Self-supporting missionaries.
Thus, the Seventh-day Adventist Church's position on tithe has been based upon the. Ellen White believed in loyal self-supporting institutional work, too.
Prayer To St Rita Patron Saint Of The Impossible A thematic bibliography of Christian heresy, heterodoxy, dissent, and witchcraft, and their repression by various means (inquisition etc A small portion of an ankle bone belonging to Saint Toribio Romo — the patron saint of. it is not impossible. If we have his intercession and his protection, we can do anything. Let us then commend.
and the public at large that the Seventh-day Adventist church in Jamaica does not support any political party." Harvey, who sits on the controversial board of the National Housing Trust, was quoted as.
The station is a self-supporting subsidiary of WAU. Adventist Media Center– Brazil, an entity of the Seventh-day Adventist Church's South American Division,
A network of historic self-supporting Seventh-day Adventist churches and ministries that promotes the Bible and the Spirit of Prophecy. Founding date.
When Judith Miranda and her husband visited Malibu, California over 10 years ago, there weren't any Seventh-day Adventist churches in city limits. Adventist radio and television support, health.
In the midst of a hurting community, local Seventh-day Adventist. church members joined the long lines of blood donors at Red Cross blood drives. Members are also coming to the church and.
Jun 9, 2010. More than 100 physicians and dentists operated self-supporting clinics as officials of the Seventh-day Adventist Medical and Dental Association.
Internet outreach of the Seventh-day Adventist Church and its supporting ministries will be coordinated by a new Global Internet Evangelism Committee, church leaders have voted. The initiative was.
Sunday's mass lootings in the capital of the Central African Republic have not affected Seventh-day Adventist Church operations. More than a dozen South African troops supporting the president were.
Tucker-Norcross Free SDA Church is a self-supporting Seventh-day Adventist. of 1996, and later merged with Berean Church of Free Seventh-day Adventists,
On November 26, 2016, Ontario Conference of Seventh-day Adventists inaugurated what is. impaired at Christian Record Services, to see a church for the visually impaired in Ontario. With the support.
ASI — the organization of Seventh-day Adventist lay persons involved in. as well as support the global mission of the Seventh-day Adventist Church. about the role the college was playing in establishing a self-supporting work that would.
Editor's note: This story is part of a series of historical articles published this year marking the 150th anniversary of the Seventh-day Adventist Church. got to find a way to support their.
Gift Ideas For First Time Visitors At Church Make-A-Wish needs local residents to make it happen by volunteering their time and compassion to assist with fulfilling. The Ladies of Mary Society of Exaltation of the Holy Cross Church, 420 Main. If you have a friend who loves sweets, but hates to cook, why not help them by mixing the ingredients together ahead of
For the past 30 years, we have had a number of churches and parsonages. We are endeavoring to do this through placing self-supporting couples in villages.
Dear Brothers and Sisters in Christ, about three weeks ago on October 10, I appealed to you, the nearly 21 million Seventh-day Adventist members around the. As you may know, during Annual Council,
Seventh-day Adventists affirm the traditional view of marriage as being between a man and a woman and call on others to support legislation that so defines marriage, according to statements from.
List of Seventh-day Adventist Secondary Schools (Academies/High Schools) of. The list includes Conference, OCI, and self-supporting schools, and notes.
The Seventh-day Adventist Church announced a new website and. place where they can search for all the service opportunities offered by the Adventist Church and other supporting ministries.
McEdward shared his appreciation for Sudan, a predominately Muslim nation, to provide official recognition to the Seventh-day Adventist Church. "It is a great pleasure to be able to have our church.
Charge of Apostasy Against the Seventh-day Adventist Church. of administration and a parallel self-supporting ministry independent of the organized system.
A difficult saga for the Seventh-day Adventist. a tremendous sense of support from our worldwide church family who've been praying, along with us, that God's purpose will prevail." Bertil Wiklander.
It will also help the local Adventist churches to realize their mission, through the. to the SDA church, especially in the developing countries, to be self-reliant,
Category: Understand Christianity - Comments: Comments Off on Self Supporting Seventh Day Adventist Churches
Discover God


I have made up mind to close this website down. Since I no longer have the time to keep it updated.
I would like to say a very big thank-you to everyone who has looked at since website, since it first went on-line back in 1998.
Web design Gordon S Valentine.


text:
Home » Coronavirus , Facebook , Google , Pandemic , Technology , Twitter » Twitter Staff Can Now Work From Home Forever
Twitter Staff Can Now Work From Home Forever
In what has been described as "an era-defining moment" by one digital innovation expert, Twitter has given its staff the leeway to work from home 'forever' if they wish, as the company looks towards the future after the coronavirus pandemic - BBC reports.
Twitter cited the success recorded while working from home during the lockdown as the reason for its decision.
The social media giant however admitted it would allow workers to return to the office if they choose when it reopens.
This comes after Google and Facebook announced that their staff can work from home until the end of the pandemic and the year.
Twitter issued a statement which read thus:
"The past few months have proven we can make that work. So if our employees are in a role and situation that enables them to work from home and they want to continue to do so forever, we will make that happen."
Twitter added that for staff who are keen to return to the company's office, 'they will be welcomed and with some additional precautions'.


text:
Q: PHP Chatbox : Cannot Send to MySQL Database Code :
        $user = $_POST["user"];
        $message = $_POST["message"];
        $message = htmlspecialchars($message);

        $db=mysqli_connect("host","username","pwd","db");

        if (mysqli_connect_errno()){
            echo "Failed to connect to MySQL: " . mysql_connect_error();
        }

        #Connected

        if(isset($message) == true && $message != "" && $message != " "){
            if(isset($user) == false){
                $user = "visitor";
            }

            $sql="
            INSERT INTO ChatBox (DateTime, User, Content)
            VALUES
            (now(),'$_POST[User]','$_POST[Content]')
            ";

            if (!mysqli_query($db,$sql))
            {
            die('Error: ' . mysqli_error($db));
            }
            }

            mysqli_close($db);

This is my code in a chatbox.
However, I use the line if(isset($message) == true && $message != "" && $message != " "){ to find out whether $_POST['message']  is null or not.
Finally, I cannot insert anything in my chat box.
This is my chatbox link : http://jamie-is-cool.comeze.com/chat.php
The problem is that : After clicking Send! nothing happened. It just reload and didn't send.
What should I do?
A complete list of code is available here.

A: Insert code block will on fire if the statement if( isset( message) ... returns a true.
But in your HTML form, there is no such element with name message but a user and content.
<form action="chat.php" style="margin:0px;padding:1px;" method="POST">
  User: <input type="text" style="width:15%;" name="User" /> |
  <input type="text" style="width:65%;" name="Content" />
  <input type="submit" value="Send!" style="width:10%;" />
</form>

Add an input type element named message to the form with some value and then submit to get succeed.
<form action="chat.php" style="margin:0px;padding:1px;" method="POST">
  User: <input type="text" style="width:15%;" name="User" /> |
  <input type="text" style="width:65%;" name="Content" />
  <input type="text" style="width:65%;" name="message" />
  <input type="submit" value="Send!" style="width:10%;" />
</form>

For better display, adjust the form table display width accordingly.


text:
package com.google.samples.apps.topeka.activity.quiz;

public class HistoryQuizTest extends BaseQuizActivityTest {

    @Override
    int getCategory() {
        return 2;
    }
}


text:
Q: vsTest targets wrong .NET version My Azure DevOps build pipeline has a vsTest task in it. The test is specified in azure-pipelines.yml like so:
- task: VSTest@2
  inputs:
    testSelector: 'testAssemblies'
    arguments: '/Framework:.NETCoreApp,Version=3.0'
    testAssemblyVer2: |
      **\*test*.dll
      !**\*TestAdapter.dll
      !**\obj\**
    searchFolder: '$(System.DefaultWorkingDirectory)'

When running the build pipeline, no tests get run and when looking in the log I find this line: Test run will use DLL(s) built for framework .NETFramework,Version=v4.0 and platform X86.
Why does it look for a version different than the one specified in the YAML-file and how can I fix this?

A: One of the users suggests the following approach in the comments section of this page:

You can for example add otherConsoleOptions:/Framework:.NETCoreApp,Version=v3.1 in the task to set command line
  options for VSTest.exe