Increasing anthelmintic resistance (AR) in livestock has stimulated growing efforts to monitor anthelmintic effectiveness (AE) on livestock farms. On-farm assessment of AE relies on measuring the reduction in faecal egg count (FEC) following treatment; and if conducted rigorously, qualifies as a formal FEC reduction test (FECRT) for AR. Substantial research effort has been devoted to designing robust protocols for the FECRT and its statistical interpretation; however, a wide range of factors other than AR can affect FEC reduction on farms. These are not always possible to control, and can affect the outcome and repeatability of AE measurements and confound the on-farm classification of AR using FECRT. This review considers confounders of FEC reduction, focusing on gastrointestinal nematodes of ruminants, including host and parasite physiology and demography; pharmacokinetic variation between drugs, parasites and hosts; and technical performance. Drug formulation and delivery, host condition and diet, and seasonal variation in parasite species composition, can all affect AE and hence observed FEC reduction. Causes of variation in FEC reduction should be attenuated, but this is not always possible. Regular monitoring of AE can indicate a need to improve anthelmintic administration practices, and detect AR early in its progression. Careful interpretation of FEC reduction, however, taking into account possible confounders, is essential before attributing reduced FEC reduction to AR. Understanding of confounders of FEC reduction will complement advances in FECRT design and interpretation to provide measures of anthelmintic efficacy that are both rigorous and accessible. Hence, in the case of AR, heritable resistance is present and likely to worsen if management is not changed, and recommendations could include switching to a different drug and altering treatment strategies to alleviate selection pressure ; whereasMonitoring of AE on farms is promoted as a way to identify drug failure at an early stage in the development of AR, when management can be altered in time to prolong the useful life of anthelmintics at farm and regional scales. Such monitoring often makes use of opportunities to assess AE alongside routine farm management activities, for example anthelmintic treatments to support productivity, which can introduce additional variability. Effectiveness and resistance are often conflated in practice, because monitoring of AE and classification of AR both use FECR as the key metric, and because the purpose of AE monitoring is usually to identify therapeutic failure as a result of AR. Although FECR < 95% does not necessarily denote AR, it is often taken to indicate that a given anthelmintic is failing due to parasite resistance, and to provoke a review of parasite control practices. Alternatively, AR might be present and yet the FECRT returns FECR > 95%, conferring a false sense of security and a missed opportunity for constructive management change. Simplifications of the FECRT, such as using pooled samples , 76 or dWhile advances in statistical analysis and improved FEC methods decrease the risk of AR misclassification due to technical error, biological processes can also confound FECR  and leadThe effectiveness of anthelmintics depends on exposing the parasite to an adequate drug concentration for sufficient time to achieve the target efficacy. If this is not achieved, effectiveness will be low even in the absence of AR. To guard against misinterpretation of poor AE, therefore, it is important to understand the pharmacology of the main anthelmintic chemical families, which depend on the pharmacokinetic and pharmacodynamic mechanisms of drug delivery and action , 55. PhaConsidering the complex factors that can affect parasite exposure to effective concentrations of anthelmintic, it follows that any adverse pharmacokinetic issue can decrease AE and cause therapeutic failure, and if not accounted for can lead to misclassification of AR using the FECRT.It is often assumed that, in the gut, it is the intraluminal concentration of anthelmintic that is critical for parasite exposure. However, anthelmintics are absorbed and metabolized in a variable fashion, and drug metabolites may or may not be active against the parasite. Some anthelmintics, notably many benzimidazoles (BZD), are not active in the form administered and require biotransformation, for example in the liver, gut and in other tissues, to then reach the target parasites. GIN may be exposed to the active form of anthelmintics while feeding on blood, tissue fluid or gut secretions, or by recirculation of the compound or active metabolites through the enterohepatic circulation . TranscuDissolution of drug particles in gastrointestinal fluids is particularly important for drugs administered as suspensions by the oral route . On the other hand, anthelmintics parenterally injected as drug solutions, including some formulations of macrocyclic lactones (ML) or levamisole (LEV), do not require dissolution before systemic absorption, and the digestive secretion process is an important step to assure drug-nematode contact in the gut. Drug absorption is a main limiting factor that determines the amount of drug reaching the systemic circulation. The reversible exchange between the bloodstream and tissues then allows the drug and/or its metabolites to achieve concentrations that are anthelmintically active in the tissues occupied by parasites .Some orally administered anthelmintics, notably the benzimidazole (BZD) methylcarbamates  have limited water solubility and small differences in drug solubility may have a major influence on their absorption, systemic availability and resultant clinical efficacy . While sMacrocyclic lactones (ML) can be administered by oral or parenteral routes, or trans-cutaneously via pour-on formulations. Oral administration of ML facilitates the achievement of higher drug concentrations and enhanced efficacy against nematodes located in the gastrointestinal tract compared to transcutaneous treatment in sheep, cattle and horses , 58, 85.Following administration of ML pour-on formulations to cattle, anthelmintic exchange between cattle through natural grooming behavior, i.e., self- or allo-licking, can significantly impact on AE . Higher Cooperia in cattle was not found to be affected by rainfall [Human factors can cause variation in the dose of anthelmintic administered, for example through poor calibration of dosing equipment, inaccurate estimation of animal weight, or failure to part the coat sufficiently well to ensure contact of topically applied anthelmintic with the skin. Once applied, absorption of topical anthelmintic can be affected by other factors, such as bathing or heavy rainfall – although ivermectin pour-on efficacy against rainfall . Given tDifferences between batches in the quantity of the active ingredient, its bioavailability, or degradation during storage and transport may result in variable efficacy. For luminal-acting anthelmintics, particle size is very important, with fine particulate size far superior to coarse; this is affected further by passage along the intestine . ErraticCombinations of two or more anthelmintics are increasingly used to manage AR . The occPharmacokinetic and pharmacodynamic confounders of AE are carefully excluded from therapeutic trials needed for drug registration , with goMany host-related factors may affect the kinetic behaviour and resultant clinical efficacy of anthelmintic compounds, through the processes of dissolution, absorption, and biotransformation.Differences in anthelmintic metabolism occur between host species and also within them according to breed and age. Pronounced pharmacokinetic differences among animal species have been documented for different anthelmintic molecules, with lower systemic availability in goats compared to sheep treated at the same dose rates, being among the most established. The rate of liver cytosolic production of reduced flubendazole metabolites can differ nearly 100-fold between sheep and pigs . Within Haemonchus contortus [In young ruminants, orally administered anthelmintics may partially bypass the rumen and reticulum to enter the omasum and abomasum following closure of the oesophageal-reticular groove, a reflex especially developed in the nursing ruminant but inconsistently active in the adult . Thus, vontortus , althougIndividual variation in drug pharmacokinetics will further generate inter-individual differences in therapeutic effectiveness , adding Temporary feed restriction prior to anthelmintic treatment has been shown to increase the absorption and bioavailability of BZD in ruminants. Thus, fasting cattle prior to intra-ruminal albendazole treatment modified the absorption and disposition kinetics of albendazole metabolites, increasing plasma and tissue availability . StarvatFeed type also influences gut transit and drug absorption. Binding of BZD compounds to dietary fibre can substantially modify the duration of the so-called rumen reservoir effect , alterinGIN infection, and a change to a high-grass diet, can both reduce gut transit time and cause diarrhoea in grazing ruminants, especially at times when they are most likely to be treated under farm conditions. Under these circumstances, AE could be compromised regardless of AR. Moreover, high water content dilutes egg density in faeces and decreases observed FEC , 90. FECProlonged feed restriction leading to poor nutritional status can negatively affect anthelmintic bioavailability through reduced BZD biotransformation in the liver and resultant pharmacokinetics , while bipso facto, effectiveness in the presence of infection, accumulated gut damage in chronic infection could lower expected efficacy.Parasitic infection can itself induce important changes to the pharmacokinetics, side-effects and effectiveness of anthelmintics. For example, parasite-mediated inflammatory reactions in the gut change mucosal permeability, luminal pH and gut transit time, altering BZD bioavailability. Elevation of abomasal pH, a characteristic outcome of abomasal GIN infection , decreasFasciola hepatica [Liver function is important for the pharmacokinetics of BZD , 97, andhepatica , also pohepatica . The effTeladorsagia circumcincta for this reason [Many biocidal drugs, such as antimicrobials, require a competent immune system to achieve high efficacy. The extent to which immature immune systems impede AE in ruminants, as they do in mice , is unkns reason . Includis reason  allows ns reason . Similars reason , but is Different GIN species respond differently to anthelmintics through species-specific processes influencing bioavailability and lethal dose, irrespective of AR, and product licences and data sheets reflect these differences. The AE achieved against a mixed population of GIN in natural infections is therefore likely to vary with species composition, and such variation accentuated by differing levels of AR among species. Furthermore, the responses of parasite communities to treatment include physiological and evolutionary adaptations, and biotic interactions, which affect outcomes including FECR.Nematodirus) versus trichostrongylid nematodes in ruminants. Concealed species variation, however, fundamentally affects FECR results. In mixed-species GIN infections in sheep in Scotland, FECR indicated AE of 65–77%, but Teladorsagia circumcincta dominated post-treatment FEC and was more highly resistant to both BZD and ML than undifferentiated FECR would suggest [Haemonchus contortus was found in cattle despite FECR exceeding 95% because of their low abundance relative to other species such as H. placei [Cooperia-Ostertagia infection in cattle [Calculation of overall FECR for GINs generally uses total FEC, separating only morphologically distinct taxa, e.g., moleinid (e.g.,  suggest . In catt. placei . High le. placei . When ARn cattle .Studies repeating FECR with species identification on individual farms over time are rare and difficult to achieve given requirements for adequate starting FEC levels , but seaThe egg output of different GIN species varies widely , 94, 95,H. contortus L3 decreases survival but increases subsequent fecundity [Climatic conditions also affect fecundity, for example desiccation of ecundity . Seasonaecundity  and lifeecundity , 51. If The stringent requirements of a formal FECRT for AR places it out of the practical reach of routine farm management, hence various more pragmatic approaches have been proposed to provide actionable information on AE against GIN. These, however, entail compromises that can affect the reliability of FECR under farm conditions. Statistical interpretation of the FECRT is not discussed in detail here. Although different statistical methods and the assumptions on which they are based affect the degree to which technical errors and other confounders alter AR classification, the underlying true FECR that they seek to interpret is also open to technical bias.Meticulous treatment is necessary for a defensible FECRT, including administration of the correct dose of anthelmintic for the weight of each individual animal included. When AE monitoring is aligned with normal farm management, however, such accuracy depends on having adequate facilities and sufficient time to weigh and dose individuals. A large proportion of farmers estimate anthelmintic dose by visual inspection or using the average weight of the group, risking under-dosing, while calibration of dosing equipment is infrequent . Sub-theper rectum is prone to allow egg development or contamination with free-living nematodes, while labelling errors can misattribute samples to treatment groups and cause serious misclassification. Storage of faecal samples can introduce inaccuracies through egg development or altered egg flotation after fixation [Pre-analytic factors such as collection, labelling, and storage of faecal samples prior to analysis can increase variability in FECRT results. Sampling faeces from the ground rather than fixation , 70, henfixation , 86, andAggregated distribution of helminths between individual animals in a group, and clumping of eggs within the faecal mass, require that adequate numbers of animals and faeces from each be included if FECR is to be representative of the group treated. The error structure of FEC and implications for the FECRT have been widely discussed and explored through simulation studies , 53, 54.The diagnostic portfolio for FEC includes firmly established methods, e.g. McMaster and Mini-FLOTAC , althougIncreasingly variable weather conditions confound attempts to measure AR using FECRT, which require adequate starting egg density. In grazing livestock, delayed rise in GIN FEC in dry conditions will postpone the earliest date at which FECR might sensibly be measured; while follow-up of apparently low AE using a FECRT will also rely on adequate reinfection and hence weather and grazing conditions. Low observed FEC in summer could pressurize farmers and advisors to conduct FECRT at egg densities too low to produce accurate AR classification, or to defer the test to later in the year, increasing confounding by species composition and host immunity. Technical improvements in FEC methods that permit valid FECRT at lower starting FEC  could hepost mortem worm counts in animals following treatment, which is rarely feasible on farms, leaving the FECRT as the only appropriate test for AR that is widely applicable between GIN species and anthelmintic classes. The failure of the livestock industry to adequately monitor anthelmintic efficacy has much to do with the limitations of this test, which is onerous and expensive if done thoroughly, and often leaves considerable uncertainty around the results of a particular test and its repeatability at farm level. Simplification of the FECRT in an effort to align its application with routine parasite treatments and so increase uptake, however, adds to variability in results due to the real-world confounders discussed here. The same dilemma applies to epidemiological studies of AR, which must trade sample size against effort per farm, limiting the strength of conclusions that apply rigorous FECRT but on a small number of farms [The detection and management of AR is a major and growing challenge for sustained parasite management on farms, and hence for the sustainability of grass-based livestock production. Confirmation of AR requires of farms , 78.Although the factors listed above are highly likely to be common on farms and hence to influence FECR results, the extent to which they actually confound estimates of AE and AR is not well known. There are good reasons for this. Following up therapeutic failure with formal FECRT requires resources, commitment and competent advisors, and is not always feasible. The achievement of high efficacy in drug licensing trials despite natural variation in animal and parasite physiology might also suggest that many of the factors listed are only potential confounders, whose impact in reality is not large enough to materially affect FECR results and AR classification. As AR takes hold, however, efficacy becomes increasingly marginal and confounders become more important to outcomes. Furthermore, therapeutic failure will accentuate the impact of parasites on the health and physiology of livestock and reinforce parasite-dependent confounders. As AR takes hold and testing rates remain low , the sitWhile hard evidence for the role of confounders in AE and AR classification is hard to come by, there are strong grounds to be vigilant. Refinements such as improved FEC methods and statistical interpretation of FECRT results are helpful, but they do not address the full range of potential confounders of FECR results. It is important that veterinary professionals and other advisors, as well as parasitologists and animal scientists, are aware of these, and that expectations of the FECRT as a standard test are appropriately managed, especially when modified to improve practicality. False expectations of a clear and lasting result from a single FECRT can lead to inappropriate management decisions, which have important implications for sustained parasite control, and can be avoided if potential confounders of test results are appreciated. Ultimately, a wider array of approaches to monitoring AE on farms is needed to more fully track trajectories and to adjust parasite management strategies in an evidence-based and timely manner.


PIM1 is a serine/threonine kinase over-expressed in prostate cancer. We have previously shown that PIM1 phosphorylates the androgen receptor (AR), the primary therapeutic target in prostate cancer, at serine 213 (pS213), which alters expression of select AR target genes. Therefore, we sought to investigate the mechanism whereby PIM1 phosphorylation of AR alters its transcriptional activity. We previously identified the AR co-activator, 14-3-3 ζ, as an endogenous PIM1 substrate in LNCaP cells. Here, we show that PIM1 phosphorylation of AR and 14-3-3 ζ coordinates their interaction, and that they extensively occupy the same sites on chromatin in an AR-dependent manner. Their occupancy at a number of genes involved in cell migration and invasion results in a PIM1-dependent increase in the expression of these genes. We also use rapid immunoprecipitation and mass spectrometry of endogenous proteins on chromatin (RIME), to find that select AR co-regulators, such as hnRNPK and TRIM28, interact with both AR and 14-3-3 ζ in PIM1 over-expressing cells. We conclude that PIM1 phosphorylation of AR and 14-3-3 ζ coordinates their interaction, which in turn recruits additional co-regulatory proteins to alter AR transcriptional activity. Ruff et al. demonstrate that phosphorylation of the androgen receptor and 14-3-3 ζ by PIM1 kinase coordinates their interaction and chromatin occupancy at genes involved in cell migration and invasion. The authors conclude that PIM1 phosphorylation of these substrates regulates the AR transcriptome in prostate cancer cells. PIM1 has also been linked to a variety of oncogenic processes in both prostate cancer and other cancers, including cell-cycle progression, genomic instability, resistance to chemotherapeutics, and increased tumorigenicity in mouse xenograft models6. We and others have previously shown that PIM1 phosphorylates AR, the primary therapeutic target in prostate cancer, at S213, affecting the expression of select AR target genes8. This phosphorylation of AR at S213 is associated with higher-grade tumors, castration-resistant disease, and altered AR transcriptional activity9. However, the mechanism by which AR phosphorylation at S213 affects AR transcriptional activity is not fully understood.PIM1 is a serine/threonine kinase upregulated in prostate cancer. While PIM1 protein level is very low in benign prostate tissue, PIM1 expression is high in about half of prostate cancer samples10. Among the substrates identified was 14-3-3 ζ at serine 64. 14-3-3 ζ has been previously identified as one of the two most amplified genes in metastatic prostate cancer relative to paired local tumors11. In addition to gene amplification in over 50% of castration-resistant prostate cancer samples, 14-3-3 ζ protein expression is indicative of poor prognosis in patients12. Previous studies also show an interaction between the AR and 14-3-3 ζ, and co-activation of AR by 14-3-3 ζ13. However, the mechanism by which 14-3-3 ζ co-activates AR, and the resulting transcriptional outcome, have not been studied.We have previously identified a set of endogenous PIM1 substrates in LNCaP cells to elucidate the mechanisms by which PIM1 exerts its oncogenic effects14. Noting that PIM1 has been shown to foster interactions between the 14-3-3 proteins and its substrates, we hypothesized that PIM1 phosphorylation of AR mediates its interaction with the co-activator 14-3-3 ζ17. Finding this to be the case, we used RNA seq and ChIP seq to further characterize the transcriptional changes and chromatin occupancy resulting from PIM1 phosphorylation and 14-3-3 ζ co-regulation of AR. The results indicate a coordinated response in which PIM1 phosphorylation of AR and 14-3-3 ζ alters AR-dependent gene expression.Here, we sought to characterize how PIM1 phosphorylation of AR and the AR co-activator, 14-3-3 ζ, alters AR transcriptional activity in prostate cancer. 14-3-3 ζ is a regulatory protein that interacts with phosphorylated serine and threonine residues, and its optimal interaction motif closely resembles the PIM1 consensus phosphorylation motif8. To study the effect of this phosphorylation on AR transcriptional activity, we generated a doxycycline-inducible cell line expressing PIM1 in LNCaP cells. We confirmed that in the presence of doxycycline, the cells over-express PIM1. This resulted in an increase in phosphorylation of AR at S213, as detected by the AR pS213 antibody generated in our laboratory as described previously , we found a substantial reduction in luciferase activity, consistent with previous reports of 14-3-3 ζ acting as an AR co-activator . Metascape pathway analysis indicates that the upregulated genes are involved in extracellular matrix organization, cell adhesion, and cytokine signaling, among other pathways -inducible PIM1 over-expressing LNCaP cells Fig. , compariays Fig. 22. The gily Fig. .Fig. 2PIUltimately, we wanted to identify which of these transcriptional changes could be attributed to PIM1 phosphorylation of AR and 14-3-3 ζ and their co-regulatory interaction. To do this, we performed ChIP-seq on AR, pS213 AR, and 14-3-3 ζ in the same control and PIM1 over-expressing LNCaP cells. We observed that in PIM1 over-expressing cells, there is a shift in overall AR chromatin occupancy, with about 7% new occupancy sites and a loss of about 25% of occupied sites Fig. . When weWhen next looked at 14-3-3 ζ chromatin occupancy. We find again that PIM1 over-expression results in a shift in 14-3-3 ζ occupancy, with some sites lost and other sites gained Fig. . However22. Although the categories of cellular processes identified in the ChIP-seq analysis appear to be broader than those identified in the RNA-seq analysis, this is likely due to the sheer number of genes showing the AR and 14-3-3 ζ occupancy, compared with the relatively few genes with changes in expression at the RNA level. While there was not complete overlap, we chose to focus on genes in these similar classes to determine whether their expression is dependent on 14-3-3 ζ and AR binding.When next examined the sites occupied by 14-3-3 ζ and pS213 AR. We observed in PIM1 over-expressing cells that the majority of pS213 AR sites overlap with 14-3-3 ζ occupancy Fig. 25. Numerous studies have linked MMP7 to prostate cancer progression and metastasis, as well as specifically to promoting bone metastases29. Although less studied, MMP10 has also been found to be increased at the protein level in metastatic prostate cancer relative to benign tissue30. TLL2, also a zinc-dependent metalloproteinase, is a member of the bone morphogenic protein (BMP) family. It has been shown to breakdown laminin-5, and is upregulated in invasive breast cancer32. While not linked to prostate cancer as of yet, the cytoplasmic dynein protein DYNC1I1 is associated with increased cell migration in gastric cancer, as well as shortened progression-free survival in breast cancer34. Finally, we included COL4A6, a type IV collagen protein. Collagen proteins are understood as playing both the pro- and anti-tumorigenic roles, depending on the context. They can provide not only a barrier to metastatic cells but also a scaffold which dynamically alters extracellular matrix remodeling and can promote tumor progression, depending on the stage of the tumor35. Altogether, we aimed to examine a panel of genes which show AR and 14-3-3 ζ chromatin occupancy, as well as PIM1-dependent expression, to explore the mechanism by which PIM1 may mediate AR-dependent gene expression.To validate the gene expression changes in our RNA seq data, we selected a panel of genes involved in extracellular matrix organization, cell adhesion, cytokine signaling, and cell migration. Altogether, these pathways are key for cell migration and invasion, which is necessary for tumor metastasis. We chose genes which showed chromatin occupancy for either AR or pS213 AR and 14-3-3 ζ, hypothesizing that AR and/or 14-3-3 ζ may play a role in the changes in expression of these genes , and treated for 24 h with 10 nM of the synthetic androgen R1881. We hypothesized that genes for which the expression increased with AR knockdown would decrease with R1881 treatment, and vice versa. In fact, we saw this was the case, with DYNC1I1, MMP7 (ns), and COL4A6 showing increased expression with R1881 treatment, and MMP10 and TLL2 showing reduced expression , MMP7, and COL4A6) and, unexpectedly, an increase in expression for other genes [MMP10 and TLL2 (ns)] Fig. . Althougion Fig. . This inion Fig. . TherefoOur ChIP seq data shows that 14-3-3 ζ and AR or pS213 AR occupy chromatin at a variety of PIM1 upregulated genes  on AR and 14-3-3 ζ in LNCaP cells in the presence of PIM122. For AR, we identified many known AR co-regulators, as well as proteins previously identified in RIME experiments for AR. For instance, Stelloo et al. performed RIME analysis on AR, showing validated interactions with known interactors ARID1A (a component of SWI/SNF chromatin remodeling complex), HOXB13 (a homeodomain family transcription factor responsible for androgenic response), HSP90 (a chaperone that promotes the maturation of the AR LBD), FOXA1 (a member of the forkhead class of DNA-binding proteins), and PARP1 (modifies various nuclear proteins by poly(ADP-ribosylation), as well as their novel findings of AR interaction with TLE3  and TRIM28 (corepressor with E3 SUMO-protein ligase activity)48. Among these, we identified AR interacting in at least two of the three replicates with each of these proteins , and they both also significantly co-occur with hnRNPK amplification  , PIM1, and one or more of these co-factors. Using available studies of metastatic castration-resistant prostate cancer from cBioPortal, we found that PIM1 and YWHAZ amplification significantly co-occur . 293 cells were cultured in DMEM, LAPC4 cells were cultured in IMDM, and 22RV1 and LNCaP cells were cultured in RPMI (containing phenol red and L-glutamine), all with 10% FBS and 1% penicillin/streptomycin. All cell lines were regularly assessed for mycoplasma contamination. For chemical treatments, the following were used: doxycycline (Sigma), SGI-1776 , and Enzalutamide (MedKoo).7 using HiFi kit (NEB) , pcDNA3-HA-AR . For stable 14-3-3 ζ lentiviral infection in LNCaP cells, 14-3-3 ζ was cloned into pCW57-MCS1-P2A-MCS2 (Neo) using HiFi kit (NEB) . S64A mutation was introduced to 14-3-3 ζ plasmid using QuikChange Lightning Multi Site-Directed Mutagenesis Kit (Agilent), with the single primer CTTCCGTCTTTTGTTCAATAGCTGAGACGACCCTCCAAGATG. All plasmids were sequenced to confirm the mutations and to ensure no additional changes in the sequence were introduced.Constructs used were as follows: pCW57-MCS1-P2A-MCS2 (Neo) PIM1, backbone a gift from Adam Karpf, Addgene plasmid #89180, insert cloned from plasmid previously described in Ha et alCommercial antibodies used were as follows: PIM1 , 14-3-3 ζ , AR , hnRNPK , TRIM28 , HSP90 , Tubulin , Myc-tag , SP1 , and BRG1 . Immunoprecipitations for Western blot were carried out using Protein A/G PLUS-agarose beads (SCBT).Transient plasmid transfections were done using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. siRNA transfections  were done using Lipofectamine RNAi Max (Invitrogen) according to the manufacturer’s protocol. Viral transductions were carried out in 293 T cells using 5 μg lentiviral construct, 2 μg pMD2.G , and 3 μg psPAX2  using Lipofectamine reagent (Invitrogen). Target cells were treated with viral supernatant and polybrene  overnight, and media replaced. Pools selected using Geneticin .Luciferase assays were conducted using stably transfected ARR3-luciferase LNCaP cells. Cells transfected with siRNA were seeded into 384-well plates at 5000 cells/well, followed by 24 h treatment with R1881. Both were measured using the ONE-Glo detection kit (Promega) and data are standardized to CellTiter-Fluor (Promega) viability readings to account for potential variations in well to well cell number. Four biological replicates were used for the experiment.4HCO3, 5 mM DTT, 8 M urea. For cysteine alkylation, samples were mixed with 0.5 μl 0.15 M iodoacetamide followed by 30-min incubation at room temperature in dark. Alkylation reactions were then mixed with 50 μl of the solution containing: 50 mM NH4HCO3, 5 mM DTT, and 10 ng/μl trypsin/Lys-C mix (Promega), followed by overnight incubation to complete proteins digestion. Digestion reactions were mixed with 5 μl 20% heptafluorobutyric acid and peptides were desalted using C18 spin tips according to manufacturer protocol (Thermo Scientific). Desalted peptides were dried under vacuum and reconstituted in 12 μl of 0.1% formic acid before LC-MS analysis on Orbitrap Fusion Lumos mass spectrometer. Peptides concentration was measured spectrophotometrically at 205 nm using NanoDrop One (Thermo Scientific).Three microgram of recombinant 14-3-3 ζ  were combined with 250 ng of recombinant PIM1 kinase  in a buffer containing 20 mM MOPS pH 7.2, 5 mM EDTA, 25 mM glycerol-2-phosphate, 1 mM DTT, 500 μM ATP, and 75 mM MgCl2 for 15 min at 30 °C. The experiment was performed in duplicate. Proteins (~3 μg) from phosphorylation reactions were precipitated using 4 vol. of acetone at −20 °C for 1 h. Acetone pellets, collected by 10-min centrifugation at 16,000 g, were rinsed with 80% acetone and dried on air. Dried pellets were dissolved in 5 μl of denaturing buffer: 50 mM NH66.Peptides were analyzed by LC-MS on Orbitrap Fusion Lumos mass spectrometer coupled with Dionex Ultimate 3000 UHPLC. During each run, 0.5-2 μg of peptides from individual samples were injected and resolved on 50-cm long EASY-Spray column (Thermo Scientific) by 90-min long linear gradient of 4–40% acetonitrile in 0.1% formic acids at flowrate 0.25 μl/min. Method for the data-dependent acquisition was based on published protocol with exception of each cycle was set to last for 2 s instead of 3 swww.uniprot.org) combined with a set of known protein contaminants (supplied with MaxQuant). Parameters were set to search for peptides of at least five amino acids long, containing at most 2 missed trypsin cleavages, and, depending on the experiment, having dynamic modifications, including carbamidomethylation of cysteine, oxidation of methionine, phosphorylation of serine, threonine, or tyrosine, acetylation of lysine and protein N-terminus, mono- and dimethylation of lysine and arginine. In MaxQuant, the intensity determination option was set to “Total sum” and “Match between runs” option was enabled. All the other parameters were left at their defaults. For analysis, each sample was normalized to protein A/G abundance. IgG-bound peptides were subtracted from antibody-bound peptides, and then samples were normalized to the amount of AR or 14-3-3 ζ pulled down in each replicate.Peptides identification and label-free quantitation were done in Proteome Discoverer 2.1 or MaxQuant 1.6. Search engines, Sequest and Andromeda respectively, were supplied with a protein database consisting of human or mouse proteome downloaded from UniProt  at 1 unit per microgram of protein for 30 min at 37 °C for 30 min before adding loading buffer.Phos-tag gels were prepared with gel containing 40 μM of Phos-tag Acrylamide (Wako) and 80 μM MnClhttps://rosalind.onramp.bio/), with a HyperScale architecture developed by OnRamp BioInformatics, Inc. . Reads were trimmed using cutadapt67. Quality scores were assessed using FastQC68. Reads were aligned to the Homo sapiens genome build hg19 using STAR369. Individual sample reads were quantified using HTseq and normalized via Relative Log Expression (RLE) using DESeq2 R library71. Read Distribution percentages, violin plots, identity heatmaps, and sample MDS plots were generated as part of the QC step using RSeQC72. DEseq2 was also used to calculate fold changes and p values and perform optional covariate correction. Clustering of genes for the final heatmap of differentially expressed genes was done using the PAM (Partitioning Around Medoids) method using the fpc R library73. Hypergeometric distribution was used to analyze the enrichment of pathways, gene ontology, domain structure, and other ontologies. The topGO R library was used to determine local similarities and dependencies between the GO terms in order to perform Elim pruning correction74. Several database sources were referenced for enrichment analysis, including Interpro, NCBI, MSigDB, REACTOME, and WikiPathways80. Enrichment was calculated relative to a set of background genes relevant for the experiment. Enrichment was calculated relative to a set of background genes relevant for the experiment. Gene enrichment was also analyzed using Metascape (http://metascape.org)22.RNA was extracted using the RNeasy kit (Qiagen), and libraries were prepared for sequencing using the NEBNext Ultra II RNA Library Prep Kit for Illumina with polyA selection. Concentrations were measured using Qubit and checked for quality using Tapestation, then sequenced on Illumina NovaSeq 6000 SP100 flowcell. Data were analyzed by ROSALIND® , with a HyperScale architecture developed by OnRamp BioInformatics, Inc. . Reads were trimmed using cutadapt67. Quality scores were assessed using FastQC68. Reads were aligned to the Homo sapiens genome build hg19 using bowtie282. Per-sample quality assessment plots were generated with HOMER and Mosaics84. Peaks were called using MACS26 85. Peak overlaps and differential binding were calculated using the DiffBind R library86. Differential binding was calculated at gene promoter sites. Read distribution percentages, identity heatmaps, and FRiP plots were generated as part of the QC step using ChIPQC R library and HOMER88. Gene enrichment was analyzed using Metascape (http://metascape.org)22.ChIP experiments were performed as previously described, with the antibodies detailed in the antibody section, and pS213 AR antibody generated previouslyTranswell migration and invasion assays were performed using the CytoSelect 24-Well Migration and Invasion Assay (8 um pores) according to manufacturer’s instructions. Invasion assays used the membranes coated with the extracellular matrix. Three hundred microliter of cells at 500,000 cells/mL were plated for 48 h for migration assay and 72 h for invasion assay. Images were quantified at 10x magnification by counting cells per field for three separate fields.For qPCR, RNA was extracted using the RNeasy kit (Qiagen) and reverse transcribed using the Verso cDNA Synthesis Kit (ThermoFisher) according to manufacturer instructions. cDNA was amplified containing Fast SYBR Green qPCR Master Mix (ThermoFisher) and qPCR performed using Applied Biosystems Quantstudio 6 Flex Real-Time PCR System. Data was analyzed with the DDCT method normalized to RPL19 with three technical replicates for each experiment. Primers for qPCR were as follows:RPL19: Fwd CACAAGCTGAAGGCAGACAA, Rev GCGTGCTTCCTTGGTCTTAGDYNC1I1: Fwd AAAGCTGAGCTAGAGCGCAAA, Rev GTCCTGAACGGGTTCTTTCTTCMMP7: Fwd GAGTGAGCTACAGTGGGAACA, Rev CTATGACGCGGGAGTTTAACATMMP10: Fwd TGCTCTGCCTATCCTCTGAGT, Rev TCACATCCTTTTCGAGGTTGTAGCOL4A6: Fwd CAGCAGCGGGAGAGAAGTC, Rev CAGTAGAGCCAGTGAATCCTTLL2: Fwd GCCATGTGGTTGGGTTTTGG, Rev TGTCAAAGTCGTATGTCTCTCCCPIM1: Fwd GGCTCGGTCTACTCAGGCA, Rev GGAAATCCGGTCCTTCTCCAC14-3-3 ζ: Fwd CCTGCATGAAGTCTGTAACTGAG, Rev GACCTACGGGCTCCTACAACAAR: Fwd TACCAGCTCACCAAGCTCCT, Rev GAACTGATGCAGCTCTCTCGhnRNPK: Fwd CAATGGTGAATTTGGTAAACGCC, Rev GTAGTCTGTACGGAGAGCCTTATRIM28: Fwd CGTGTACTGCTGGCCCTATT, Rev AACTCCTGTGGGGAGCTGTAFor ChIP-PCR experiments, ChIP was performed the same way as in the ChIP-sequencing experiments, with corresponding IgGs as negative controls. DNA was amplified containing Fast SYBR Green qPCR Master Mix (ThermoFisher) and primers which were designed based on peaks identified in the ChIP seq data aligned to hg19. Primers are as follows:MMP7: Fwd CATAGTTTGCCAACTGCTGCT, Rev TCAGTGCTCTTTTCTTATGAAGATTTLL2: Fwd TGGGTCATTTCTATGCCAAGCA, Rev TTGCACTGAGCCTTGCAAAC37. After digestion, beads were pelleted and supernatants containing peptides were transferred to new tubes. After mixing with 5 μl 20% heptafluorobutyric acid, peptides were desalted using C18 spin tips according to manufacturer protocol (Thermo Scientific). Desalted and dried peptides were reconstituted in 12 μl 0.1% formic acid. Peptides concentration was measured spectrophotometrically at 205 nm using NanoDrop One (Thermo Scientific). Peptides were analyzed as described above.Immunoprecipitation for RIME was carried out as described for ChIP-Seq. However, based on Mohammed et al, after washing the beads and prior to eluting, beads with antibody-bound proteins were washed twice with 50 mM NH4HCO3 and resuspended in 50 μl 50 mM NH4HCO3 containing 10 ng/μl trypsin/Lys-C (Promega) followed by overnight incubation at 37 °C with vigorous shaking in thermomixer (Eppendorf)Statistical testing was completed using Prism 8 for Mac OS X, Version 8.0e. Samples were randomly allocated into experimental groups. Samples were randomly allocated into experimental groups, and no data were excluded in the analysis. Error bars indicate standard deviation. Experiments were repeated 2–3 times and had high reproducibility.Further information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Reporting Summary


To evaluate the experience of clients and clinicians in working with a toolto help set goals that are personally meaningful to rehabilitationclients.We have applied the tool in the outpatient rehabilitation setting. Clients’and clinicians’ experiences in working with the tool were evaluated inindividual, semi-structured interviews and focus group interviews,respectively. Thematic analysis was used to analyze the data.A university medical center and a rehabilitation center.n = 8) or multiplesclerosis (n = 10), and clinicians(n = 38).Clients with a first-time stroke  to explorethe client's fundamental beliefs, goals and attitudes and (ii) to identify ameaningful overall rehabilitation goal. The results of that session wereused by the multidisciplinary rehabilitation team (iii) to help the clientto set specific rehabilitation goals that served to achieve the meaningfuloverall rehabilitation goal.Both clients and clinicians reported that the tool helped to set a meaningfuloverall rehabilitation goal and specific goals that became meaningful asthey served to achieve the overall goal. This contributed to clients’intrinsic rehabilitation motivation. In some clients, the meaningfulness ofthe rehabilitation goals facilitated the process of behavior change. Bothclients and clinicians made suggestions on how the tool could be furtherimproved.In the opinion of both clients and clinicians, the tool does indeed result ingoal setting that is personally meaningful. Further development,implementation and evaluation of the tool is warranted. Goal-setting is also thought to have an impact on the rehabilitation process:it is intended to ensure that individual team members work towards the same goals,and that appropriate treatment is provided. Despite it being a core practice, both clients and clinicians have reporteddifficulty in setting goals that are personally meaningful to clients.4 Clients are unlikely to bestrongly motivated for rehabilitation if goals are not personally meaningful. Thisis expected to lead to low adherence and little behavior change.5 Therefore, there is a need fora tool to help set goals that rehabilitation clients experience as personallymeaningful.Goal-setting is a core practice within rehabilitation. Goal setting is thought tomotivate the client for rehabilitation and to increase behavior change. The tool's central tenet is that the client's fundamental beliefs, goals andattitudes need to be explored before setting any rehabilitation goal. Theexploration of these fundamental beliefs, goals and attitudes enables the client to set a meaningful overall rehabilitation goal . Once the meaningful overallrehabilitation goal has been identified, the rehabilitation team and the client setmore specific rehabilitation goals that serve to achieve the overall goal . During the rehabilitationtrajectory, the specific goals can be adjusted or revised, depending on the courseof the rehabilitation process. Our approach has been to involve a chaplain tosupport the client in exploring their fundamental beliefs, goals and attitudes andsetting the overall meaningful rehabilitation goal, although is a possibility totrain other professionals to do this. The tool to help set meaningful goals inrehabilitation is illustrated in We have previously reported on the development of such a tool.After its development, evaluating the tool is the next logical step. The aim of thepresent study was a formative evaluation of the experience of clients and cliniciansin working with the tool. The evaluation focused on meaningfulness of rehabilitationgoals, clients’ motivation and related behavior change, as well as therehabilitation work process. We aimed to conduct the study among clients with afirst-time stroke (a sudden onset condition), multiple sclerosis (a progressivecondition) and cerebral palsy (a lifelong condition), to get an impression about theuse of the tool in conditions with different time course. The study was conducted inthe outpatient setting.In a qualitative study, we evaluated clients’ and clinicians’ experiences in usingthe tool in the outpatient rehabilitation clinics of the Amsterdam UniversityMedical Centers and Reade, Center for Rehabilitation and Rheumatology in Amsterdam.The Medical Ethics Committee of Amsterdam University Medical Centers declared thatthe Research Involving Human Subjects Act did not apply and that official approvalof the study by the committee was not required (reference number 2018.013). Clientswere recruited between October 2018 and August 2019.n = 10), multiple sclerosis (n = 5), orcerebral palsy (n = 5). We expected to get enough information withthese number of clients, without exceeding our capacity to apply and evaluate thetool. Clients with severe communication problems or psychiatric problems wereexcluded. Physicians applied purposive sampling to include both men and women, andboth younger and older clients.At both clinics, rehabilitation physicians were invited to participate in the study,based on oral information and a brief presentation. Rehabilitation physiciansinvited clients to participate in the study. Three chaplains were involved whohelped to explore global meaning and to set the overall meaningful rehabilitationgoal (see next paragraph). We aimed to include adult clients with a first-timestroke , and (ii) together they identified the meaningful overall rehabilitationgoal. Additional support by the chaplain was offered in case the session on globalmeaning raised questions or feelings the client wanted to elaborate on. The resultsof that session were briefly summarized; after a check with the patient, the summarywas included in the client's file and communicated to the other members of therehabilitation team. The other members of the rehabilitation team and the client(iii) set specific rehabilitation goals that served to achieve the meaningfuloverall rehabilitation goal. The specific goals were recorded in the client's file.During the rehabilitation trajectory, the specific goals could be adjusted orrevised, depending on the course of the rehabilitation process. For further detailson the process of setting meaningful goals, we refer to our previous publication.If the client agreed to participate, the researchers sent an information letter and aconsent form. In the letter, the study goal and procedures were explained. After theclient had signed the consent form, the session on global meaning was planned, atthe start of outpatient rehabilitation after the medical intake see . In thatSupplemental file 1). Audio-recording and field notes were made. Twochaplain-researchers (EL and SD) conducted the interviews and focus groups. They hada background in qualitative research, one of them had a PhD, and both were of thefemale gender. As they were also involved in exploring clients’ global meaning andsetting the overall rehabilitation goal, each chaplain-researcher interviewed theother chaplain-researcher's clients, in order to encourage clients to speak freelyabout their experiences with someone who was not involved in the session on globalmeaning. The focus groups were guided by the two researchers together. Occasionally,the researchers attended the team meeting regarding the included clients and madefield notes about goal setting and treatment planning.The experiences of clients were evaluated in individual, semi-structured interviews,in the final phase of their rehabilitation process (3–6 months after the start ofoutpatient rehabilitation). Clinicians’ experiences were assessed inmultidisciplinary focus groups, in the final phase of data collection. Theindividual interviews and focus group interviews were loosely structured using atopic list. The topic list concerned: (i) the conversation about global meaning;(ii) the incorporation of global meaning into the process of goal setting; (iii)re-setting of specific goals during the process of rehabilitation, taking intoaccount the overall goal; and (iv) the impact on the process of rehabilitation, inparticular the impact on the client's motivation for rehabilitation , using the qualitative data analysissoftware Atlas.ti. They collated codes into potential themes, gathering all codesrelevant to each potential theme. They reviewed, defined and named the themes. Thefield notes about goal setting and treatment planning obtained during the teammeetings were added to the data of clinicians. The interviews with clients and focusgroups with clinicians were analyzed separately, in order to ensure an independentanalysis.Transcripts from the interviews and focus groups were analyzed by the twochaplain-researchers, using thematic analysis. First, peer review was applied to enhance credibility. The two researcherscompared and discussed the individual steps in data collection and analysis.Throughout the process, they discussed their opinions and findings, until mutualagreement was reached. Besides that, they regularly discussed their findings withinthe whole research group. Second, regarding reflexivity: the researchers documenteddecisions regarding the analysis and the development of themes. The decisions werebased on a critical discussion with other members of the team. Third, a member checkwas applied. A short summary of the interview and the initial analysis was sent toclients and clinicians, who provided feedback regarding the summary and initialanalysis. Fourth, in the initial phase of the analysis an independent researcher(MO) analyzed two interviews with clients and one focus group. The result of theseanalyses was discussed and compared to the analyses of the two main researchers.To ensure the trustworthiness of the analysis, several strategies were applied.n = 8) or multiple sclerosis(n = 10). Clients with cerebral palsy were not recruited. Threeclients initially agreed to participate, but withdrew from the study he thought study participation ‘would betoo much’, and one because (s)he was referred to another hospital).In total 38 clinicians participated in the study: eight rehabilitation physicians, three chaplains (1 and 2), and 27other members of the rehabilitation team (20 and 7). The category of physiciansalso comprised residents and a physician assistant. The category of othermembers of the team comprised physical therapists , occupational therapists (5 and 2), speech therapists (2 and 0),sports therapists (1 and 0), psychologists (3 and 3) and social workers (2 and1). In total 18 clients participated in the study . Clinicians were asked to report the experiences of other clinicianswith the same disciplinary background as well (only sports therapists were notrepresented). The interviews and focus groups were conducted at the clinic(except for two interviews that were held at home). The interviews ran for about45 min, the focus groups for 1 h.Supplemental file 2 provides an overview of the development ofthe themes.The following themes emerged in the interviews and focus groups: overallevaluation, impact on motivation, impact on behavior change, stability of globalmeaning and goals, existential distress, and process of setting meaningfulgoals. The same themes were identified for clients and clinicians. Clinicians made morecomments on the process of setting meaningful goals than clients. Clients withstroke and with multiple sclerosis made similar comments for the most part;clinicians also made similar comments about these diagnostic groups. The onlyexception was that some clients with multiple sclerosis stated that the globalmeaning session came too early (see below), while clients with stroke did notmention this as an issue.it was okay but had no impact on myrehabilitation’ to ‘every patient should have aconversation like this, this is what rehabilitation is allabout’.Although some clients were more eloquent than others in putting theirthoughts and beliefs into words, in all cases the global meaning sessionresulted in a summary of the client's global meaning and overallrehabilitation goals that clients recognized themselves in. Reflecting onthe global meaning session and the impact on rehabilitation, clients’experiences ranged from ‘Itwas about me, not only about the multiple sclerosis, it was really aboutme’, ‘it is good to give people food for thought, witha focus on meaning, which is different from what you get from thepsychologist’, ‘I felt respected, seen, andunderstood’, and ‘it was no question-and-answer, butmore like a real conversation’. One client expressed heradmiration that the chaplain was able to ‘put into motion such deepthoughts in such a short time’. Two clients stated that theglobal meaning session was confronting for them, but that in hindsight itwas helpful and enriching. One of them even stated that ‘you shoulddo this with every patient, even if they don't want it, or are not ableto formulate it very well. (…) everybody needs this’.All clients appreciated the time and attention given to them as a person,rather than just rehabilitation issues. This was expressed as: ‘The questions were to the point and it mademe aware of what I myself hoped and expected of myrehabilitation’; ‘I use the summary as a thermometer, Ican see how my condition was when I answered these questions and where Iam now’; and ‘it helps to structurereflection’. On the other hand, one client stated that her goalshad no connection to her global meaning, and she did not think that theglobal meaning session or the summary would be useful to her in thefuture.Several clients emphasized the importance of the global meaning session intheir rehabilitation. ‘this is what I wantto accomplish and this is why’, referring to values orrelationships they talked about in the session on global meaning. One clientused her meaningful overall goals to choose what exercises to give priority.When asked in the global meaning session what would keep him motivated,another client said that he needed to be pushed. This was part of his innerposture. Reflecting on his rehabilitation process he claimed that histherapists had taken notice of this need and had helped him to staymotivated.Clients frequently mentioned the connection between motivation and globalmeaning; several clients explicitly referred to the global meaning session,claiming that the session had made a difference for them. Duringrehabilitation, they regularly told themselves ‘I am someone whowants to win’. After the session and her rehabilitation, it wasbased more on the value of enjoying the activity and on relationships, beingpart of a group: ‘if I can participate in small parts, and enjoy it,then it's okay’.Global meaning and the global meaning session played a varying role inchanging clients’ behavior. Some used the global meaning session to reflecton their goals, but did not change their behavior. For others, behaviorchange was driven by their global meaning. These clients explicitlymentioned the global meaning session as important in their process ofbehavior change. One client, for example, continued to be active in hersports, but in an adapted way, and based on another part of her globalmeaning. At first, her activity in sports was based on the value ofself-enhancement and the identity of a winner: ‘Thinkingabout your life goal, knowing that your children are important to you,and your parents … it gives acknowledgement, trust, you know what you gothrough this trouble for’. This helped her to make decisionsrelating to work and her role in the family.Several clients changed the way in which they looked at themselves and whatthey expected of themselves, now that their possibilities had changed. Oneof them stated that the global meaning session had helped her in the processof saying goodbye to her old self. She said it was good to identify what isimportant to her, and that it helped her to regain trust: ‘Those three goals, whether I can really reach them remainsthe question, but those are the things that make life meaningful to me.That remains the same’. In some clients, however, the changedpossibilities as a result of their medical condition led to the questionwhether their identity  had changed aswell.Overall, clients experienced global meaning and meaningful overall goals asstable: ‘Specific rehabilitation goals were regularly adapted during therehabilitation process using the meaningful overall goal as a reference. Forexample, the meaningful overall goal of self-respect did not change, but thefocus changed: not so much focusing on self-respect based on physicalachievements, but on being able to handle the new situation, or beingempathetic to themselves or to others. Another example: the meaningfuloverall goal of being active in sports did not change, but clients choose toengage in other, less physically demanding sports.miss positivo’. Several other clientsalso mentioned that reading the summary was confronting. All this did notlead to severe problems in the rehabilitation process. In one case, it wasan indication to involve a chaplain in the rehabilitation.In a few clients, we found existential distress related to global meaning.One client, for example, discovered that global meaning as she would haveformulated it earlier was no longer applicable. Her global meaning had notchanged (yet), but the confrontation with the consequences of her medicalcondition led to existential questions regarding her identity and worldview.Another client found the summary of her global meaning so confronting, thatshe did not want her partner or anybody else to read it. It revealed adiscrepancy within her global meaning, between her negative worldview andher inner posture as ‘it helped me prioritize and keep agency over myrehabilitation process’. Some clients stated that the globalmeaning session came too early: they were not prepared to reflect on suchpersonal issues while still in shock about their diagnosis, or when stillfully focused on regaining physical strength. This applied in particular toclients with multiple sclerosis.With regard to timing, the global meaning session came at the right time formost clients : ‘With regard to clinicians involved, one client appreciated that she alreadyknew the physician involved in the global meaning session. Others statedthat they did not have a preference for a specific person to have thisconversation with.it was not particularly fun, they were no easy questions, butit was really helpful, because now I knew what I wanted toachieve’. Some clients appreciated when therapists referred totheir global meaning and meaningful overall goals during therapy and insetting specific rehabilitation goals. Clients would have liked to have thesummary of their global meaning with them during their rehabilitationprocess, not only in their file, but in their own personal papers.With regard to specific goals, several clients stated that their specificrehabilitation goals would not have been different, but that the connectionto their global meaning was important, as this helped to stay motivated:‘That is what we do. I cannot imagine notaddressing meaning in my training and support of people’. Clinicians appreciated the information given in thedescription of the client's global meaning. ‘I think it is a form ofinvestment in the collaboration with the patient when we explicitlyaddress these things [global meaning]. We show them that they matter,that we really want to know who they are and what is important tothem’ . A psychologist became aware of thecentrality of clients’ meaning: ‘One patient from an Asian countrywas ashamed of his disability. We repeatedly said: you don't need to be,but now I realize that it is part of his culture, of his identity. Myopinion doesn't matter, this is who he is, and this is what we have todeal with’.Clinicians were unanimous in their belief that meaning is important, if notkey, in rehabilitation. ‘The meaningful overall goals Iread in the summary were similar to the ones in my ownassessment’. On the other hand, a physical therapist working inCenter B said that due to the summary, he could start his own specifictreatment much faster.However, clinicians varied in their appreciation of the tool. Some regardedglobal meaning as being informative of setting rehabilitation goals, butthey regarded it not necessary to set the meaningful overall goal in theglobal meaning session. They thought that goal setting could and should takeplace in team meetings. Particularly in Center A, team members felt thatthey already had integrated meaning in their work with clients, andquestioned whether the tool improved their existing practice. Oneoccupational therapist commented: ‘Engaging in this conversation created more awareness andprompted me to ask just that one extra question’, and‘I was able to attune more deliberately to my patient's needsand goals’. Physicians recognized that without the tool theinformation on meaning may have been known in the team, but was not sharedwith other team members on a regular basis. With the tool, the informationon global meaning and meaningful overall rehabilitation goals was explicitlyand coherently recorded in the client's file. This gave direction to thetreatment plan and enabled physicians to monitor whether clients’ specificgoals were still in line with their values. A physician in Center Bexpressed the hope that the tool would change the way a rehabilitation teamworks: not so much focusing on the expertise of each professional, but moreon what is truly important to the client. Other clinicians stated that thetool indeed helped to regard meaning as a team-responsibility, and not thedomain of one specific discipline: ‘I noticed that the whole teamstarted thinking on that level <of global goals> again and notjust focus on their own discipline’ (physician assistant).In particular, physicians were enthusiastic about working with the tool:‘on hold’ because she realized that it wasless important to her at that moment. Another client could not motivateherself to do the prescribed exercises. Her physical therapist read in thesummary of global meaning and overall rehabilitation goal that she desiredto be more independent, and to feel less like a patient. Together theydecided that she would go to the gym with her sister, instead of exercisingwith the physical therapist. This motivated her to regularly exerciseagain.Clinicians noticed in some clients an increased motivation to take agencyover their rehabilitation. For example, one client decided to put one of herspecific goals ‘I think thatglobal meaning can help clarify a person's intrinsic reason for change.It is important to find and use that reason’ . Aphysical therapist said: ‘A patient's being is what should be thesource of their motivation. I would never set a goal which does notinvolve a person's global meaning, because it gives you very littlechance of maintaining the changed behavior’.Clinicians stated that global meaning is an important source of intrinsicmotivation for clients to change their behavior. ‘Clinicians saw changes in clients’ goals, but only in specific rehabilitationgoals, not in meaningful overall goals. They mentioned that specific goalsdo change over time, and often become more clear. Global meaning, however,seemed to remain stable. The exception was when a discrepancy between globalmeaning and life experiences resulted in existential distress.who am I, what is my value inlife, now that I cannot contribute the way I did’. Chaplainssaw the global meaning session as a way to detect existential distress,leading to a referral to a psychologist or a chaplain.Physicians mentioned that some clients suffered from existential problems andspiritual distress, which became clear during the global meaning session.For example in one client, her identity and worldview were challenged by herdiminished physical and mental capacities. This led to confusion anddistress, raising questions, such as ‘I had the idea that it was sort of thesame as what I had already recorded. I did not gain new answers orinsights’. In center B, the larger part of the team waspositive about the tool and how it contributed to their way of providingtreatment. Therapists stated that the tool did influence the way in whichthey approached their clients: ‘For example, the patient that toldus that he needed to be pushed: I wouldn't have known. And it worked forhim’ (physician assistant). One physician said that the globalmeaning session added something to their assessment: ‘We already setgoals, and we inquire about relationships, but identity, values, innerposture are extra’. Other physicians also said they added someof the questions from the tool into their own medical assessment, in orderto deepen their understanding of the client as a person. This fostered theway in which they could attune to the client.With regard to the added value of the tool, in center A reactions were mostlyneutral, apart from the physicians who were positive about the tool. Oneoccupational therapist said ‘go that deep’. ‘Some patients are not open toquestions like that in the beginning of their rehabilitation. In thatcase, we should just start with the treatment and offer a global meaningsession in a later phase’ .With regard to the timing of the global meaning session, some cliniciansemphasized that it should be one of the first things to explore with aclient, while others advocated for the right of the client to refuse to‘With regard to clinicians involved, some physicians wanted to be involvedthemselves, because it enhanced their knowledge of and relationship withclients. Other physicians said that in the future it would be enough to readthe global meaning summary in the client's file. It was commonly believedthat expertise with regard to meaning in rehabilitation should be leading indeciding who is involved, rather than being of a certain discipline.As to for whom the global meaning session would be beneficial, there was noconsensus. Some physicians said it was impossible to know in advance whowould benefit or not, and recommended a global meaning session for everyclient. Other physicians thought that they could tell in advance whether thesession would have added value: they preferred to offer a global meaningsession to some clients in a later stage of their rehabilitation.This qualitative study shows that clients as well as clinicians felt that the toolhelps to set meaningful goals in rehabilitation. They indicated that the explorationof global meaning facilitated the setting of a meaningful overall rehabilitationgoal: as the summary of the client's global meaning and the overall rehabilitationgoal were explicitly recorded in the client's file at the start of therehabilitation, this information could be used to set specific goals. Specific goalswere regularly adapted using the overall goal as a point of reference. Clientsindicated that they were intrinsically motivated to work on specific goals, becausethey were aware of the overall goal: specific goals  becamemeaningful because the specific goals served to achieve the overall goal. Someclients felt that the session on global meaning contributed to changing theirbehavior.10 provides theoretical guidanceon how to translate intentions (motivation) into action. In addition, practicalbehavior change techniques are available that can be used to help motivated clientsactually change their behavior.Overall, clients’ evaluation of the tool was positive: some clients were (very)positive, others were neutral; no client had a negative evaluation. The variation inclients’ evaluation could mean that for certain clients this tool has no addedvalue, or that the tool should be used in a later stage of rehabilitation. Furtherexploration of this issue is required, as the present study did not provide anyclues in this respect. Besides, further development and improvement of the tool isneeded. This relates in particular to behavior change. Many clients indicated thatthe session on global meaning and the overall rehabilitation goal contributed torehabilitation motivation; however, only some clients indicated that the increasedmotivation translated into behavior change. The application of behavior changetheory and techniques could result in a better translation of motivation into actualbehavior change. For example, the Health Action Process Approach13 The advantageof working with our tool is that global meaning is explicitly addressed; that globalmeaning is addressed in a comprehensive way ; and that the summary of the client's global meaning and theoverall rehabilitation goal is explicitly recorded in the client's file at the startof the rehabilitation process. The present study also indicated that the tool helpedto regard meaning as a team-responsibility, and not the domain of one specificdiscipline. In the future implementation of the tool, we recommend involving allteam members at an early stage, to remove implementation barriers and to increasesupport for working with the tool. The application of a knowledge translation framework and practical techniques such as co-design may help to change the behavior of rehabilitation clinicians.Clinicians also varied in their evaluation, ranging from neutral to (very) positive.Physicians were generally positive about the tool, in both centers. Other members ofthe team were more mixed in their evaluation. Some team members felt that theyalready had integrated meaning in their work, and questioned whether the toolimproved their existing practice, particularly in center A. Indeed, occupationaltherapists, social workers and other disciplines have embraced client-centeredpractice, which entails the exploration of clients’ values and priorities.Some physicians expressed a desire to participate in the global meaning sessionthemselves, as this contributed to their knowledge of and relationship with clients.According to other physicians, another member of the team could participate in thesession . Similarly, the need for achaplain to be involved could be reconsidered. Another member of the team could betrained to explore global meaning . For the session to be successful, the session must include knowledgeof the client's medical condition and care pathway; expertise in exploring globalmeaning is also required. This would argue for the coordinating physician orphysician assistant, and the chaplain or someone with similar expertise to bepresent in the session on global meaning.Overall, clients and clinicians mentioned the same themes in their evaluation of themeaningful goal-setting tool. Clinicians made more comments on the process ofsetting meaningful goals than clients. Possibly, clinicians were more concernedabout the process of rehabilitation. There were no notable differences in theevaluation between diagnostic groups (stroke and multiple sclerosis), not amongclients, nor among clinicians. The only exception concerns the timing of the sessionon global meaning and the overall rehabilitation goal. For some clients withmultiple sclerosis, this came too early: some clients with multiple sclerosis werereferred for rehabilitation soon after receiving the diagnosis; they were still inthe process of accepting the implications of their diagnosis.13 acceptance and commitment therapy and spirituality and religion-based interventions.23 We believe that our approachis more broad and encompassing than these approaches. Our tool is based on theexploration of global meaning, that is, the client's fundamental beliefs, goals andattitudes.17 The session on global meaning concerns the client'srelationships, core values, worldview ,identity and inner posture. We believe that this broad approach contributed substantially to theidentification of a meaningful overall rehabilitation goal, and thereby the successof our approach.In rehabilitation, the role of clients’ values and perspectives has been increasingrecognized over the last decades. Examples of approaches taking clients’ values andperspectives into account include patient-centered care,Several methodological issues need to be considered in interpreting the results ofthis study. First, the study was conducted at the outpatient rehabilitation clinicsof a rehabilitation center and a university medical center, both in the Netherlands.There is a need to explore whether the tool is applicable in other health settings,especially other health care systems. Second, we involved adult clients with afirst-time stroke or multiple sclerosis. We intended to involve clients withcerebral palsy, but none of them had a health problem requiring multidisciplinaryrehabilitation. There is a need to study whether the tool is also applicable inclients with other diagnoses, although we expect the tool to be broadly applicable.Third, not all clinicians could participate in the focus groups, due to schedulingissues. To overcome this, we interviewed clinicians individually, and we askedclinicians to also report the experiences of other clinicians with the samedisciplinary background. Fourth, in this formative evaluation, we used a qualitativemethod and we relied fully on self-report. Furthermore, the chaplain-researchersparticipated in the session on global meaning and they performed the evaluationinterviews. It is, of course, preferable that the evaluation is independent of theintervention. A quantitative evaluation is needed of the impact of the tool onmeaningfulness of rehabilitation goals, motivation, behavior change, and healthoutcomes, using self-reported as well as observed measures, in a study with highmethodological quality.The above considerations result in a number of suggestions on the furtherdevelopment, implementation, and evaluation of the tool. It is suggested (i) todetermine for which clients this tool has added value, and at what stage ofrehabilitation; (ii) to improve the translation of improved rehabilitationmotivation into behavior change; (iii) to consider who participates in the sessionon global meaning; (iv) to involve all members of the team in the implementation ofthe tool, from the very beginning; (v) to determine the effect of using the tool onthe meaningfulness of rehabilitation goals, motivation, behavior change, and healthoutcomes, in quantitative studies.In conclusion, clients as well as clinicians experienced the tool as helpful insetting meaningful goals in rehabilitation. The tool helped to set a meaningfuloverall rehabilitation goal and specific goals that became meaningful as they serveto achieve the overall goal. This contributed to clients’ rehabilitation motivation.In some clients, the meaningfulness of the rehabilitation goals facilitated theprocess of behavior change. The formative evaluation yielded valuable suggestionsfor the further development, implementation and evaluation of the tool.Clients and clinicians both reported that our tool helped to seta meaningful overall rehabilitation goal and specific goals thatbecame meaningful as they served to achieve the overallgoal.Because the rehabilitation goals were experienced as meaningful,clients’ intrinsic rehabilitation motivation increased.The meaningfulness of the rehabilitation goals facilitated theprocess of behaviour change to a limited extent.Click here for additional data file.Supplemental material, sj-pdf-1-cre-10.1177_02692155211046463 for Settingmeaningful goals in rehabilitation: A qualitative study on the experiences ofclients and clinicians in working with a practical tool by Elsbeth Littooij,Suzan Doodeman, Jasmijn Holla, Maaike Ouwerkerk, Lenneke Post, Ton Satink, AnneMarie ter Steeg, Judith Vloothuis, Joost Dekker and Vincent de Groot in ClinicalRehabilitation


Long noncoding RNA growth arrest‐specific 5 (lnc‐GAS5) is involved in the pathophysiology of acute ischemic stroke (AIS) by regulating vascular stenosis, inflammation, and neurocyte apoptosis. This study aimed to explore the clinical value of lnc‐GAS5 in patients with AIS.Plasma samples were collected from 120 patients with AIS at admission and 60 controls after enrollment, and lnc‐GAS5 expression in the plasma of all participants was assessed by reverse transcription quantitative polymerase chain reaction. In patients with AIS, disease severity was evaluated using National Institute of Health Stroke Scale (NIHSS) score, and plasma inflammatory cytokine levels were measured by enzyme‐linked immunosorbent assay. Recurrence‐free survival (RFS) was calculated during a 36‐month follow‐up period.p < 0.001), and it had the potential to discriminate the controls from patients with AIS . In patients with AIS, elevated lnc‐GAS5 levels were positively correlated with NIHSS score , diabetes mellitus (p = 0.046), and higher levels of tumor necrosis factor alpha , interleukin‐6 , and interleukin‐17A . The expression levels of lnc‐GAS5 were also negatively correlated with the levels of interleukin‐10  and RFS (p = 0.036).Lnc‐GAS5 expression levels were higher in patients with AIS than in the controls (Lnc‐GAS5 is correlated with higher susceptibility to AIS, inflammation, and severity, and can predict an increased risk of AIS recurrence, indicating that monitoring of lnc‐GAS5 might improve the management of AIS. Lnc‐GAS5 correlates with higher AIS susceptibility, inflammation and severity, and predicts increased AIS recurrence risk to some extent. Patients who met the following criteria were eligible for enrollment: (a) newly diagnosed with AIS according to the American Stroke Association guidelines2.2After enrollment, the clinical characteristics of the patients with AIS and controls were documented, including age, sex, body mass index (BMI), and risk factors for stroke. The National Institutes of Health Stroke Scale (NIHSS) scores of the patients with AIS were collected within 24 h after admission to assess disease severity. Peripheral blood (PB) samples were collected from patients with AIS on the day of admission and from the controls on the day of enrollment, and plasma was isolated for subsequent analyses.2.3® Green Realtime PCR Master Mix (Toyobo). Relative lnc‐GAS5 expression was calculated using the 2‐ΔΔCt method, and GAPDH was used as an internal reference. The primers used for qPCR were described in a previous study.The expression levels of lnc‐GAS5 in the plasma of all study participants were determined by reverse transcription quantitative polymerase chain reaction (RT‐qPCR). In brief, total RNA was extracted using the QIAamp RNA Blood Mini Kit (Qiagen) and reverse transcribed using the iScript™ cDNA Synthesis Kit (with random primers) (Bio‐Rad). qPCR was performed using SYBR2.4Enzyme‐linked immunosorbent assays (ELISAs) were performed to measure the plasma levels of inflammatory cytokines in patients with AIS, including tumor necrosis factor alpha (TNF‐α), interleukin‐6 (IL‐6), interleukin‐10 (IL‐10), and interleukin‐17A (IL‐17A). All ELISA kits were purchased from Bio‐Techne China  and were performed according to the manufacturer's protocol.2.5All AIS patients were followed up continuously until stroke recurrence, death, or completion of the 36‐month follow‐up. The final follow‐up date was December 29, 2020. RFS was calculated based on the follow‐up data. Patients who were lost to follow‐up for any reason were censored at the last visit.2.6t‐test, Mann‐Whitney U test, and Chi‐square test. Receiver operating characteristic (ROC) curve analysis and the area under the curve (AUC) were used to estimate the ability of lnc‐GAS5 expression to discriminate patients with AIS from the controls. Correlations between two variables were analyzed using the linear‐by‐linear association test and Spearman's rank correlation test. The Kaplan–Meier method was used to determine RFS, and the correlation of lnc‐GAS5 expression with RFS was determined by the log‐rank test. Statistical significance was determined at a P value of less than 0.05.Statistical analysis was performed using SPSS , and figures were generated using GraphPad Prism 7.02 software (GraphPad Software Inc.). Lnc‐GAS5 expression in patients with AIS was classified based on quantiles: quantile 1 (Q1), 0%–25%; quantile 2 (Q2), 26%–50%, quantile 3 (Q3), 51%–75%; and quantile 4 (Q4), 76%–100%. Comparisons between two groups were determined by Student's 33.1p > 0.05 for all). The patients with AIS had an elevated prevalence of hyperuricemia (p = 0.011) and high‐risk factors (p = 0.009) compared to the controls. More detailed information is provided in Table The mean age of the 120 patients with AIS was 67.7 ± 10.0 years; there were 35 (29.2%) women and 85 (70.8%) men. The mean age of the 60 controls was 66 ± 7.4 years; there were 24 (40%) women and 36 (60%) men. The demographic characteristics were not significantly different between the patients with AIS and the controls . In terms of inflammatory cytokines, the median levels of TNF‐α, IL‐6, IL‐10, and IL‐17A were 85.9 (64.1–115.2) pg/ml, 45.7 (35.4–62.4) pg/ml, 33.6 (24.4–52.4) pg/ml, and 86.9 (64.6–128.2) pg/ml, respectively.3.2p < 0.001; Figure Lnc‐GAS5 levels were higher in patients with AIS (median [IQR]: 2.509 [1.707–4.369]) than in the controls , but not with other comorbidities (Table Increased lnc‐GAS5 expression was correlated with an elevated NIHSS score (3.4p = 0.036), and RFS was significantly shorter in patients with lnc‐GAS5 levels in Q4 than in patients with lnc‐GAS5 levels in Q1  lnc‐GAS5 expression was higher in patients with AIS than in the controls and was a good predictor of AIS risk; (2) lnc‐GAS5 was positively correlated with NIHSS score and pro‐inflammatory cytokine expression , and was negatively correlated with anti‐inflammatory cytokine (IL‐10) in patients with AIS; and (3) lnc‐GAS5 was negatively associated with RFS in patients with AIS.Regarding the role of lnc‐GAS5 in the risk of cardio‐cerebrovascular disease, a previous study showed that lnc‐GAS5 was obviously elevated in the plaques of atherosclerosis patients when compared to the levels in healthy populations.via Notch1 signaling pathway, which might lead to cognitive and functional impairment, consequently increasing the severity of AISThe correlation of lnc‐GAS5 with disease severity or inflammation in AIS has been rarely reported; thus, to explore this issue, for patients with AIS, the NIHSS score were collected to evaluate disease severity, and the levels of some inflammatory cytokines were determined. The results showed that lnc‐GAS5 was positively correlated with disease severity and inflammation. The potential reasons for this might be that: (1) lnc‐GAS5 might accelerate inflammatory responses by regulating Smad1 in AIS, which could lead to increased inflammationRecurrence is still a serious challenge in AIS, as it can result in prolonged hospitalization, unfavorable functional and cognitive outcomes, and increased mortality.This study has some limitations: (1) the follow‐up duration was relatively short, and the correlation between lnc‐GAS5 and the long‐term prognosis of AIS patients could be investigated in a future study; (2) the mechanism by which lnc‐GAS5 through its targets is involved in the pathogenesis and progression of AIS was not investigated, and thus could be explored in future studies; (3) because this study was a single‐center study and the enrolled patients might have been from neighboring regions, there might have been patient selection bias; (4) the sample size of this study was relatively small, larger sample size could be analyzed in the further study; (5) the disease controls such as other neurological diseases apart from stroke, including Parkinson and Alzheimer disease, could be considered in the further study.5In conclusion, lnc‐GAS5 is correlated with higher susceptibility to AIS and greater inflammation and severity and could predict the risk of recurrence to some extent, indicating that monitoring of lnc‐GAS5 might improve the management of AIS.The authors declare that no conflict of interest.


Objective: Danggui Buxue decoction (DBD), consisting of Angelicae Sinensis Radix (ASR) and Astragali Radix (AR), is a famous prescription with the function of antivasoconstriction. This study intends to probe its mechanisms on the relaxation of the middle cerebral artery (MCA).Methods: Vascular tension of rat MCA was measured using a DMT620 M system. First, the identical series of concentrations of DBD, ASR, and AR were added into resting KCl and U46619 preconstricted MCA. According to the compatibility ratio, their dilatation effects were further investigated on KCl and U46619 preconstricted vessels. Third, four K+ channel blockers were employed to probe the vasodilator mechanism on KCl-contracted MCA. We finally examined the effects of DBD, ASR, and AR on the vascular tone of U46619-contracted MCA in the presence or absence of Ca2+.Results: Data suggested that DBD, ASR, and AR can relax on KCl and U46619 precontracted MCA with no effects on resting vessels. The vasodilator effect of ASR was greater than those of DBD and AR on KCl-contracted MCA. For U46619-contracted MCA, ASR showed a stronger vasodilator effect than DBD and AR at low concentrations, but DBD was stronger than ASR at high concentrations. Amazingly, the vasodilator effect of DBD was stronger than that of AR at all concentrations on two vasoconstrictors which evoked MCA. The vasodilator effect of ASR was superior to that of DBD at a compatibility ratio on KCl-contracted MCA at low concentrations, while being inferior to DBD at high concentrations. However, DBD exceeded AR in vasodilating MCA at all concentrations. For U46619-constricted MCA, DBD, ASR, and AR had almost identical vasodilation. The dilation of DBD and AR on KCl-contracted MCA was independent of K+ channel blockers. However, ASR may inhibit the K+ channel opening partially through synergistic interactions with Gli and BaCl2. DBD, ASR, and AR may be responsible for inhibiting [Ca2+]out, while ASR and AR can also inhibit [Ca2+]in.Conclusion: DBD can relax MCA with no effects on resting vessels. The mechanism may be related to ASR’s inhibition of KATP and Kir channels. Meanwhile, the inhibition of [Ca2+]out by DBD, ASR, and AR as well as the inhibition of [Ca2+]in by ASR and AR may contribute to dilate MCA. Vascular diseases with a high fatality rate include cerebrovascular disease, cardiovascular disease, hypertension, and atherosclerosis, causing millions of deaths worldwide every year . AccordiAt present, the main treatment methods for cerebrovascular diseases  were surgical treatment and drug treatment . Of thesDifferentiation on Endogenous , which was written by Gao Li in the Song dynasty  are in the ratio of 1:5. In the view of Chinese medicine, it could achieve the purpose of “generating blood” through the way of “replenishing Qi” to treat medical miscellaneous diseases related to Qi deficiency and blood stasis  to prevent and treat diseases. TCM scholars had found that different herbs may produce better therapeutic effects according to specific combinations. Danggui Buxue decoction (DBD) was originally derived from  dynasty . Also, id stasis . Modern d stasis , heart pd stasis , immunitd stasis , anti-ind stasis , and antd stasis . The ori2+ channel  (Record No. 2015–03).Healthy male Sprague–Dawley (SD) rats weighing 180–220 g were purchased from Da Shuo Biotechnology Co., Ltd. . All rats were housed under identical conditions . Commercial solid food and tap water were available 2 (no. 342920), glyburide , ethylene glycol-bis (2-aminoethylether)-N, N, N′, N′-tetraacetic acid , and 9, 11-dideoxy-9α, 11α-methanoepoxy prostaglandin F2α  were purchased from Sigma. Astragaloside IV (no. wkq16042601) and ferulic acid (no. wkq17022303) were provided by Weikeqi Biological Technology Co., Ltd. Formononetin (no. DST201025-011) and ligustalide (no. DST200610-007) were procured from Lemeitian medicine.AR and ASR were purchased from the Affiliated Hospital of Chengdu University of TCM and were identified as authentic medicinal materials by Professor Sanyin Zhang of Chengdu University of TCM, and the medicinal materials meet the inclusion requirements of the 2020 Chinese Pharmacopoeia . TetraetThe strict quality control in the preparation of lyophilized powder must be carried out to ensure the consistency of drug quality during the research process. First, AR and ASR were pulverized using a pulverizer . Therefore, we accurately weighed ASR and AR according to the ratio of 1: 5. The mixture with 10 times the volume of distilled water was boiled at 100°C for 1 h, and then, the supernatant was acquired by centrifugation at 5,000 rpm for 10 min. Afterward, the medicinal material precipitation was added with 5 times the volume of distilled water to repeat the above process. The supernatant was placed in a −80°C refrigerator to freeze overnight. Lyophilized powder of DBD was acquired using a freeze dryer . Conformably, ASR and AR freeze-dried powders were prepared by the same method described above. These lyophilized powders were stored in the refrigerator at −20°C. Before the experiment, the concentration of lyophilized powder was prepared in 1 g/ml with distilled water. After centrifugation at 5,000 rpm for 10 min, the supernatant was filtered using a 0.22 μm microporous membrane. According to the minimum and maximum concentrations of DBD in dilating the MCA of rats in our pre-experiment (data were not provided), its series concentrations  were set to the following measures of vascular tension.High-performance liquid mass spectrometry  was employed to determine the contents of astragaloside IV, ferulic acid, formononetin, and ligustalide in lyophilized powder of DBD . First, 2, 1.2 KH2PO4, 1.2 MgCl2, 2.5 NaHCO3, 11 glucose, and 5 HEPES) was inflated to the saturation state with 95% O2 + 5% CO2 gas before the experiment. After sacrificing by neck removal, the skull of SD rats was stripped and the brain tissue was removed after bloodletting. The brain tissue was then placed in a Petri dish containing the 4°C cold PSS liquid. After washing the excess blood on the brain tissue with the PSS fluid, the MCAs of rats were then dissected and isolated under a light microscope . The vessels were cut to 2–3 mm and fixed in the DMT 620 M slot with two 20 μm tungsten filaments, and the temperature was maintained at 37°C. After stabilizing for 20 min, the initial tension of MCA with 1.2 mN was acquired via adjusting the tension button gently four times in a row and being stable for 40 min. Then, the vascular activity was examined by constricting twice with the KPSS liquid , 10 min each time. After each contraction, the vessels were cleaned with the PSS fluid at least three times for 10 min each time to restore the tension as basal tension. The MCA with a vasoconstriction amplitude less than 15% and a vasotension greater than 1 mN between two independent KPSS stimuli was employed for further experiments. The values of vascular tension were recorded by using Labchart Pro professional software v8.3 . The effects of all tested drugs at different concentrations on vascular tone were recorded for at least 10 min and repeated five times with different blood vessels.The PSS liquid  was used to stimulate the MCA contraction . We furt+ channel blockers 4-AP (1 × 10–3 mol/L), BaCl2 (1 × 10–4 mol/L), TEA, and Gli were administered after 60 mM KCl-evoked MCA vasoconstriction. Then, changes in vascular tone were recorded by adding DBD, ASR, and AR (64 mg/ml).Four K2+ from organelles such as the endoplasmic reticulum and mitochondria into the cytoplasmic matrix ([Ca2+]in). After incubating the MCA vessels with EGTA containing the Ca2+-free PSS fluid for 10 min to remove intracellular Ca2+, the vessels were then administered with the EGTA- and Ca2+-free PSS fluids containing DBD, ASR, and AR (64 mg/ml) for 10 min. Subsequently, 1 μM U46619 was employed to stimulate vasoconstriction and maintained for 10 min. Second, 2.5 mM Ca2+ was added to observe whether the above three tested drugs would resist the MCA vasoconstriction induced by the influx of extracellular Ca2+ ([Ca2+]out). The laboratory technology roadmap is shown in According to the previous experimental method , we evalp < 0.05 was considered statistically significant.The experimental data were expressed as the mean ± standard error of the mean (S.E.M). Statistical differences among groups were evaluated by one-way ANOVA with the Tukey–Kramer multiple comparison test using Graph Pad Prism 6.0. Compared with the retention time of the corresponding standards , four coAs shown in p < 0.05). Amazingly, 256 mg/ml of them hardly continued to relax the MCA as compared to the concentration of 128 mg/ml. However, the concentrations of 128 and 256 mg/ml of ASR did not continue to dilate the MCA compared with the concentration of 256 mg/ml  and AR  were added to the MCA vessels constricted by KCl in the prescribed compatibility ratio. Consistently, both ASR and AR can dilate MCA in a concentration-dependent manner, in which the maximum vasodilation rate of ASR at the concentration of 48 mg/ml was 71.28 ± 16.18% , while tp > 0.05).Similarly, we further investigated the dilatation of DBD, ASR, and AR on U46619-induced MCA contraction in rats. Compared with the control group , DBD, AS+ concentration via the inhibition of K+ channel opening, we incubated the KCl-constricted MCA with four K+ channel blockers: TEA , Gli , BaCl2 , and 4-AP . Subsequently, 64 mg/ml of DBD, ASR, and AR were added to investigate their effects on various K+ channels. The results showed that the above four kinds of K+ channel blockers had no significant effect on the vascular tone of KCl-constricted MCA in rats . However, addition of ASR may dilate MCA in a way similar to those of GLI and BaCl2 (p < 0.05) but not TEA and 4-AP.To probe whether the vasodilator effect of DBD, ASR, and AR is related to the decrease of intracellular K in rats . In the blockers  , with the maximum contraction from 1.22 ± 0.07 mN to 0.45 ± 0.39 mN and 0.47 ± 0.38 mN, respectively, indicating the potential inhibition of organelle Ca2+ release partly by ASR and AR (p > 0.05). To demonstrate whether the dilating MCA effect of DBD, ASR, and AR was related to the inhibition of [Ca2+]out, 2 stimulated the further contraction of MCA. However, the three drugs inhibited the CaCl2-mediated secondary contraction of MCA (p < 0.05) with the maximum contraction from 1.92 ± 0.13 mN to 1.15 ± 0.22 mN (DBD), 0.52 ± 0.39 mN (ASR), and 0.46 ± 0.4 mN (AR)  . The abo+ channel. However, ASR can dilate MCA by the inhibition of Kir and KATP. In addition, ASR and AR can inhibit both the [Ca2+]in and [Ca2+]out, but DBD can only inhibit the [Ca2+]out.DBD is a TCM prescription used in the treatment of cerebrovascular diseases with a long history. However, there is no evidence to reveal its regulation on the tension of MCA. The results of this experiment revealed the following: 1) vasodilator active ingredients of DBD may be astragaloside IV, ferulic acid, formononetin, and ligustalide. 2) DBD, ASR, and AR have no relaxing effect on the MCA of rats under resting tension. 3) DBD, ASR, and AR can relax the MCA vessels precontracted by KCl and U46619 in a concentration-dependent manner. The vasodilator effect of ASR is characterized by stronger relaxation at low concentrations and rapidly reaches the maximum relaxation rate with the increase of concentration. However, AR has a lower relaxation effect with the relaxation rate slowly increasing. The relaxation effect of DBD is the same as that of AR, but it is stronger than AR at all concentrations. 4) The relaxing effect of DBD on MCA is not the superimposition of ASR and AR. DBD can exert much stronger relaxing effect analogous to ASR at low concentrations as well as the stable relaxing effect analogous to AR on KCl and U46619 precontracted MCA of rats. 5) The vasodilator effect of both DBD and AR may be not related to the KCerebrovascular disease was a global disease that caused millions of deaths worldwide every year . In recein vitro in the range of 3.75–15 mg/ml , ferulic acid (0.1942–0.5825 mg/ml), formononetin (0.0027–0.0268 mg/ml), and ligustilide (0.001–0.4 mg/ml) have been reported to exhibit excellent relaxation on isolated vessels or smooth muscles , which activates the opening of voltage-dependent Ca2+channels  for the experiment. At this concentration, the three drugs all show good vasodilation effects. It may be that the vasodilator effect of AR at this concentration can compensate for the effect of blockers on ASR, which may be the reason why DBD is not affected by K+ channel blockers.Krization . There a+ ions but also the increase of [Ca2+]in, which can stimulate vasoconstriction (2+, preincubation of DBD, ASR, and AR will not affect vascular tension, but both ASR and AR can inhibit U46619-induced vasoconstriction, while DBD has no obvious effect. On the other hand, the addition of exogenous Ca2+ can cause further contraction of vascular smooth muscles. Preincubation of the three drugs can inhibit the secondary contraction induced by exogenous Ca2+. DBD, as a prescription for the compatibility of ASR and AR, did not show a more comprehensive inhibitory effect. The reason for this result may be related to the release of ingredients during prescription preparation and freeze-dried powder preparation. Experiments have shown that DBD decoction can promote the release of astragalus components, and the process of making freeze-dried powder may cause certain changes in the components of DBD (The contraction and relaxation of vascular smooth muscles are affected by not only Ktriction . This exs of DBD . Simultas of DBD .Paeonia lactiflora, Ligusticum chuanxiong, Pheretima aspergillum, Carthamus tinctorius, and Prunus persica had a tendency to decrease the neurological function score, separately, with no significant difference (Ligusticum chuanxiong and Pheretima aspergillum, Radix Sophorae Flavescentis, and Acorus calamus Linn, as well as AR and ASR in improving the memory capacity of vascular dementia rats (in vitro investigation of prescription disassembly, both Xiaobanxia decoction and Zingiber officinale Roscoe can counteract the isolated ileum contraction induced by acetylcholine, 5-hydroxytryptamine, and histamine in guinea pigs in a concentration dependent manner, and there was almost no difference in their relaxing effects (in vitro uterine contractions of mice (60Coγ -ray radiation in mice (Collectively, our data demonstrated that DBD, ASR, and AR can dilate the rat MCA. In MCA with KCl contraction, the vasodilatation was ASR > DBD > AR. For U46619-contracted MCA, the vasodilatation was ASR > DBD > AR at concentrations less than 128 mg/ml, while DBD > ASR > AR at concentrations greater than 128 mg/ml. Nevertheless, the deep reason why TCM prescription and decomposed prescription show inconsistent pharmacological activities is worth further exploration. In the ischemic stroke model established by middle cerebral artery occlusion, a 14-day administration of Buyang Huanwu decoction can significantly ameliorate the neurological function score of rats. Compared to the model group, groups AR, and the combination of ASR, fference . Guiqi Ctia rats . For in  effects . Evidenc of mice . Meaning in mice . In term in mice . The abo in mice . Maybe t in mice .