To seek efficient aldose reductase inhibitors (ARIs) with excellent in vitro and in vivo biological activities against rat galactosemic cataract.  The method was firstly optimized to screen strong ARIs from nonoriented synthetic compounds and natural extracts. Then, diosgenin was assessed on osmotic expansion of primarily cultured lens epithelial cells (LECs) induced by galactose (50 mM). Diosgenin was administered to galactosemic rats by oral (100 and 200 mg/kg) or direct drinking (0.1%) to evaluate its anticataract effects. 50 of 4.59 × 10−6 mol/L. Diosgenin (10 μM) evidently inhibited the formation of tiny vacuoles and upregulation of AR mRNA in LECs. In vivo, diosgenin delayed lens opacification, inhibited the increase of ratio of lens weight to body weight, and decreased AR activity, galactitol level, and AR mRNA expression, especially in the diosgenin drinking (0.1%) group.  Diosgenin was found as the strongest ARI with IC Diosgenin was an efficient ARI, which not only significantly decreased the LECs' osmotic expansion in vitro but also markedly delayed progression of rat galactosemic cataract in vivo. Thus, diosgenin rich food can be recommended to diabetic subjects as dietary management to postpone the occurrence of sugar cataract, and diosgenin deserves further investigation for chronic diabetic complications. Diabetes, a global epidemic noncommunicable disease, is predicted to rise to 552 million by 2030 . AlthougThe lens, an almost perfect organ, lives in hypoxic microenvironment. The front is aqueous humor and the rear is vitreous liquid, which has neither vascular nutrition nor nerve innervations. In physiological condition, over 80% glucose enters the glycolysis pathway and almost no glucose enters the polyol pathway, the key cascade of sugar cataract , 5. Howe50 at 10−6 mol/L level [In past decades, the once most promising ARIs such as sorbinil, tolrestat, zopolrestat, and ponalrestat were withdrawn from clinical trials because of unsatisfactory efficacy and severe side effects , 7. Now,/L level . Here, wg at 4°C for 30 min. The supernatant was collected and used as an AR source. Lens supernatant should be prepared freshly due to the quick inactivation of the AR enzyme.The whole lenses were instantly removed from normal Sprague Dawley (SD) rats by posterior capsule method after being sacrificed. Each lens was homogenized in 5 × volumes of 50 mM ice-cold phosphate buffer saline , then centrifuged at 15,000340nm can represent the consumption of NADPH. Therefore, we detect the change of OD340nm before and after reaction to screen the effective ARIs or evaluate AR activity. The incubation system contains lens supernatant as an AR enzyme, 0.16 mmo1/L DL-glyceraldehyde as substrate, 1.0 mmo1/L NADPH·Na4 as coenzyme, and 0.1 mol/L PBS (pH = 6.2). The incubation mixture was minimized to a total volume of 200 μL and ongoing in a 96-well ultraviolet plate. In order to ensure accuracy of the screening method, we made all the combinations of the above components and scanned them from 200 nm to 999 nm to validate the OD340nm absorption peak of NADPH.In the first step of polyol pathway, glucose reduction is accompanied by the conversation of NADPH to NADP. While, only NADPH has an obvious spectral absorption around 340 nm and NADP has none; thus, the decrease of OD−3 mol/L and then were diluted to a final concentration of 10−6 mol/L for ARIs screening.As to the ending method, AR, a 36 kD protein enzyme, is easily inactivated by the change of pH value and temperature. Acidation, alkalization, and heating can easily lead to the denaturation of AR, so we think that perhaps cooling is a desirable ending method. After strict comparisons among different cooling methods, we found that the best convenient way was incubation at −20°C for 5 minutes. The appropriate reactive degree is about 80%; the above freezing ending method reserves a chance for the reaction to restart once it is less than 60%. Epalrestat was chosen as the positive ARI; we collected 224 candidates including 180 nonoriented synthetic compounds and 44 natural extracts, all these samples were primarily dissolved in DMSO at 102, followed by positioning it horizontally. After 24 hours, we observed whether new LECs were growing below or around the capsule fragments and added 5 ml of Dulbecco's Modified Eagle's Medium  supplemented with 15% FBS. Each step should be done gently and we tried to avoid shaking during primary cultivation. When the new growing multilayer LECs accumulated around the tissue fragment, they should be digested by 0.1% trypsin for passage. The LECs from the 3rd to 8th passage were used for further studies.Each whole eyeball was immediately removed and soaked into 75% ethanol after sacrificing the beagle dog; then we carefully isolated the intact lens and dissected the capsule into two halves from the equatorial region. All the anterior parts were collected and washed twice by using the artificial aqueous humor. Then, we added 1 ml fetal bovine serum (FBS) to infiltrate them, cut them into tiny pieces and moved them into a flask, and gently shook them to scattered evenly on the flask bottom. In order to enhance the adhesion, the flask was incubated in an erect position for 4 hours at 37°C in presence of 5% COμM), and two DG groups (5 and 10 μM). LECs (the 3rd–8th passage) were firstly harvested and seeded on 6-well culture plates (2 × 104 per well). 24 hours later, galactose was added to control, Epal and DG groups, epalrestat and diosgenin were, respectively, added to Epal and DG groups. After 48 hour incubation, we observed the LECs morphology under an inverted microscope and harvested them, promptly extracted the total RNA by TRIzol reagent and measured the AR mRNA level by real time RT-PCR.The groups were designed as follows: normal group, control group , Epal group : normal group, control group, and three DG groups . All the rats ate normal chew; rats of the normal group drank the purified water, but rats of other groups had ad libitum access to 12.5% galactose solution in the first 7 days, then to 10% galactose solution up to the end of 15 days. Rats of the DG  groups were orally treated with two dosages of 100 and 200 mg/kg, respectively; rats of the DG (0.1%) group directly drank 0.1% diosgenin solution mixed with galactose. All animal procedures were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and approved by the Institutional Animal Ethics Committee.In brief, 21 day normal male SD rats were randomly divided into 5 following groups  at the day of 3, 6, 9, 12, and 15, respectively. Each time, the pupils were fully dilated with a topical ophthalmic solution containing tropicamide 5% and phenylephrine hydrochloride 5%. The anterior segment including the lens was observed and photographed in both eyes of all the rats. Lens opacification was scored as follows : grade 0μmol NADPH per hour per 100 mg protein [Each lens was homogenized according to the above AR preparation method; the supernatant was taken to detect the AR activity. One unit of AR enzyme activity is defined as the amount of enzyme catalyzing the oxidation of 1  protein . The totμL of lens homogenate was fully mixed with 20 μL of 20% TCA, centrifuged at 5,000g for 10 minutes at 4°C, and then the supernatant was collected to determine the galactitol level. The reaction system consisted of the lens supernatant, 12.5 mM periodic acid, 12.5 mM sodium arsenite, and 0.2% chromotropic acid. The absorption peak was recorded at 570 nm to calculate the level of galactitolLenticular galactitol level was detected as follows : 80 μL oP value less than 0.05 was considered to be statistically significant.All data were expressed as mean ± SD, cataract grade was analyzed by Mann–Whitney test, and other data were evaluated with one-way ANOVA (two-tailed test). The 340nm absorption of NADPH. Only after addition of AR for several minutes did a remarkable decrease of OD340nm absorption appear. These results clearly demonstrated that NADPH had an absorption peak at OD340nm, and stimulated AR of normal rat lens could consume NADPH and leads to the decrease of OD340nm absorption.As shown in −11–10−6 mol/L) had significant inhibition on AR activity of normal lens  group.As shown in Figures As shown in −4) than the normal level (3.37 × 10−4); diosgenin treating  significantly inhibit the increase of the ratio at 3.28 × 10−4, 3.64 × 10−4, and 3.40 × 10−4, respectively.In this model, galactosemia resulted in a slow increasing of body weight, but lens weight consistently increased due to severe osmotic expansion especially in the early 7 days, so the two parameters were absolutely negative related. The ratio of lens weight to body weight was the key parameter of lens osmotic expansion in this galactosemic cataract model. At the end , the ratAs shown in Figures Hyperglycemia and the duration of diabetes are the major risk factors of diabetic cataract. To date, no efficient agents, irrespective of glycemic control, are utilized to treat diabetic cataract. Hyperglycemia stimulates AR enzyme, leading to the accumulation of hypertonic sorbitol and subsequent lens osmotic expansion , so any μL ongoing on the 96-well ultraviolet plate, and we made all the combinations of the components. The screening results (200 nm to 999 nm) validated the observation peak of NADPH at 340 nm. The best ending method of “−20°C × 5mins” not only avoids the denaturation of AR by alkalization, acidification, and heating but also keeps a chance to remedy the inadequate reaction if needed. The only commercial ARI, epalrestat, was selected as the positive control to evaluate the accuracy of the new optimized ARIs screening method, the IC50 of epalrestat on normal rat lens AR was 1.32 × 10−8 mol/L, which was on the same level to other researches [50 was 4.59 × 10−6 mol/L, that was on the similar level as other ongoing plant-derived AR inhibitors [At first, we optimized the screening method of ARIs. DL-glyceraldehyde can be reduced to glycerol by the AR enzyme with the conversation of coenzyme NADPH to NADP. The reaction mixture was minimized to 200 searches , 16. Subhibitors .Mechanism of cataracts caused by hyperglycemia or hypergalactosemia are similar, and they are together called “sugar cataract” , 17. ButμM) markedly weakened the formation of tiny vacuoles in LECs and significantly inhibited the upregulation of AR mRNA. Diosgenin could produce predominant inhibition on LECs' osmotic stress induced by galactose in vitro.LECs almost bear the whole metabolism of lens, so they are regarded as “life center” of the lens. AR, the key enzyme of polyol cascade, is also mainly distributed in the cytoplasm of LECs. LECs are a layer of cubic cells just located below the anterior capsule of the lens. This characteristic of LECs seems to be a double-edged sword; the sole type of LECs below the anterior capsule avoids the contamination of other cells, but the filmy anterior capsule and limited numbers of LECs bring great challenge for the primary culture. In order to enhance the adhesiveness of anterior capsule fragments to the flask bottom, we initially added only FBS without any DMEM to the flask, standing incubation lasted for 4 hours, then lied down the flask carefully. As to the osmotic stress, galactose (50 mM) could induce obvious osmotic expansion of LECs in 48 hours, plenty of tiny vacuoles gradually came into being in the cytoplasm especially around the nuclear, and AR mRNA was induced to approximate 2.85-fold of normal level. Our results showed that coincubation with DG , an efficient ARIs screened in present study, is the major raw materials of synthetic contraceptive and steroid hormones . It is ucosidase , 23, but as half . Due to 50 = 4.59 × 10−6 mol/L). In vitro, diosgenin could significantly inhibit the osmotic expansion of LECs induced by 50 mM galactose; in vivo, diosgenin could markedly delay the onset of lens opacification and maturation of rat galactosemic cataract. Therefore, we suggest that plants rich in diosgenin can be regarded as part of dietary management for diabetic patients to postpone the occurrence of sugar cataract, and diosgenin should be further investigated as a potential AR inhibitor to treat life-threatening diabetic complications.In conclusion, the present study demonstrates that diosgenin is a novel AR inhibitor (IC


The mapping precisely identified the two ERK-docking sites in ELK1, the D-box and the DEF  motif, as the essential motifs for its cooperation with AR(A/B) or WTAR. In contrast, the transactivation domain in ELK1 was only required for activation by ERK. ELK1-mediated transcriptional activity of AR(A/B) was optimal in the absence of ELK1 binding partners, ERK1/2 and serum-response factor. Purified ELK1 and AR bound with a dissociation constant of 1.9 × 10−8m. A purified mutant ELK1 in which the D-box and DEF motifs were disrupted did not bind AR. An ELK1 mutant with deletion of the D-box region had a dominant-negative effect on androgen-dependent growth of PCa cells that were insensitive to MEK inhibition. This novel mechanism in which a nuclear receptor impinges on a signaling pathway by co-opting protein kinase docking sites to constitutively activate growth genes could enable rational design of a new class of targeted drug interventions.The ETS domain transcription factor ELK1 is in a repressive association with growth genes and is transiently activated through phosphorylation by ERK1/2. In prostate cancer (PCa) cells the androgen receptor (AR) is recruited by ELK1, via its amino-terminal domain (A/B), as a transcriptional co-activator, without ELK1 hyper-phosphorylation. Here we elucidate the structural basis of the interaction of AR with ELK1. The ELK1 polypeptide motifs required for co-activation by AR  The androgen receptor (AR)1–Most early stage and advanced prostate tumors depend on AR for growth –11. The –12–17–22–28–We have previously reported that the ETS family transcription factor ELK1 is essential for growth in PCa cells that are either dependent on androgen or independent of hormone but still dependent on the AR . In contELK1 belongs to the ternary complex factor subfamily of ETS proteins that characteristically bind to purine-rich GGA core sequences . ELK1 is35–40–A). Typically (e.g. in LNCaP cells), the binding of androgen is necessary for AR to localize in the nucleus, to associate with classical androgen-response elements (AREs), and to activate its gene targets. However, AR has splice variants with carboxyl-terminal truncations that remove the ligand binding pocket; such variants are known to activate growth genes and support growth of PCa cells in a hormone-independent manner. Therefore, we tested the ability of the A/B domain of AR to induce ELK1-dependent gene activation. In contrast to full-length AR, the AR A/B domain does not require bound hormone for nuclear localization.AR includes the following: (i) an amino-terminal A/B domain containing ligand-independent transcriptional activation functions (AF1 and AF5); (ii) a carboxyl-terminal region (E and F domains) containing the ligand binding pocket and a ligand-dependent activation function (AF2); and (iii) internal DNA binding and hinge domains (C and D domains) A. TypicaELK1)2-TATA-LUC). This construct was transfected along with an expression plasmid for either the full-length AR or the N-terminal A/B domain of AR into AR-negative HeLa cells. The full-length AR was able to activate the promoter in an androgen-dependent manner (B). In contrast, the A/B domain activated the promoter to a comparable extent both in the presence and in the absence of hormone (B). When the cells were transfected with the minimal promoter construct in which the ELK1-binding elements were substituted with a canonical ARE, promoter activation only occurred through the full-length AR and in the presence of androgen (C). Western blotting analysis confirmed expression of both the full-length AR and AR(A/B) in the transfected cells (D).We first used a minimal TATA-dependent promoter luciferase construct in which two ELK1-binding cis-elements were placed upstream of the TATA box  previously shown to be activated by AR in an ELK1-dependent manner  known to be activated by androgen (R1881) in an ARE-dependent manner. Androgen activated both classes of target genes in LNCaP cells, which express full-length AR (E). However, when AR(A/B) was ectopically expressed in LNCaP cells in the absence of androgen , only the ELK1-dependent target genes were activated (F). This result demonstrates that the A/B domain of AR is able to recapitulate ELK1-dependent gene activation by androgen plus AR.Next, we compared the abilities of AR(A/B) and the full-length AR to activate genes . Growth of the control cells was dependent on androgen (G). The A/B domain of AR was capable of supporting androgen-independent cell growth, albeit less robustly than androgen (G).Next, we tested whether the A/B domain of AR could support hormone-independent growth in LNCaP cells. AR(A/B) was ectopically expressed in LNCaP cells by lentiviral transduction, and control cells were infected with non-expressing lentivirus G, inset.androgen G. The A/androgen G.Collectively, the data in docking site for ERK, FXFP) motif (–GAL4-TATA-LUC) was stably integrated. These cells also stably expressed the AR(A/B)-VP16 fusion protein. The recombinant HeLa cells were transfected with Gal4-ELK1 fusion constructs in which the DNA binding domain (amino acids 1–86) was replaced by that of Gal4. The reporter readout was used to assess the ability of the Gal4-ELK1 fusion proteins to associate with AR(A/B)-VP16. In parallel, HeLa cells stably expressing GAL4-TATA-LUC alone were co-transfected with each one of the Gal4-ELK1 constructs and an expression plasmid for a constitutively active mutant of MEK1 (CA-MEK1). Activation of GAL4-TATA-LUC by CA-MEK1 entails phosphorylation and functional association of ERK1/2 with Gal4-ELK1; therefore, this parallel test probes the ability of each Gal4-ELK1 deletion/mutation construct to associate with and become activated by ERK1/2.ELK1 consists of several functional domains. The amino terminus comprises the A domain encompassing the ETS DNA binding domain . The B dP) motif –49. SegmA). Expression of the transfected constructs was confirmed by Western blotting, using antibody to the Gal4 DNA binding domain . The reporter assay values were similar to the full-length construct for deletions to positions 187, 261, 287, and 297. However, there was virtually a complete loss of reporter activity for deletions to positions 317, 327, 337, 367, and 397. These results map an element required for association of AR(A/B) to the region downstream of amino acid residue 297 of ELK1 (A and schematic in C). The parallel experiment testing activation of the Gal4-ELK1 constructs by CA-MEK1, showed a virtually identical pattern of activity , a result that is consistent with the position of the D-box region beginning at residue 312 .First, Gal4-ELK1 constructs containing progressive deletions beginning from the amino terminus were tested in the two-hybrid assay A. Expresg domain A, inset.activity B, inset,A). Again, expression of the transfected constructs was confirmed by Western blotting . The reporter assay values were similar to the full-length construct for the deletion made at position 397, but it was lost for deletions to positions 387, 377, and 367. These results map an element required for association of AR(A/B) to the region upstream of amino acid residue 397 of ELK1 (A and schematic in C). The parallel experiment testing activation of the Gal4-ELK1 constructs by CA-MEK1 again showed the same pattern . This result is consistent with the position of the DEF motif, which serves as an additional ERK-docking site .Next, Gal4-ELK1 constructs containing progressive deletions beginning from the carboxyl terminus were tested in the two-hybrid assay A. Again,blotting A, inset. pattern B, inset.A) using the two-hybrid assay. The internal deletions covered segments flanking and within the region mapped above by amino- and carboxyl-terminal deletion analyses. Expression of the Gal4-ELK1 constructs was confirmed by Western blotting . As shown in Fig. 4A, the reporter assay values for the constructs clearly fell into the following two groups: those corresponding to the activity of the full-length construct, and those approaching the background value obtained by transfecting the Gal4 DNA binding domain alone. Based on these data, we were able to confirm and further bracket peptide motifs required for association of AR(A/B) to within the amino acid sequences 307–350 and 372–397. Activation of the Gal4-ELK1 internal deletion constructs by CA-MEK1 followed a similar pattern except for the deletion 351–371, which reduced activation by CA-MEK1 but not association with AR(A/B)-VP16 . These results are again consistent with the locations of the ERK-docking sites in ELK1 (D box and DEF motif) . As residues 351–371 lie within the transactivation domain  of ELK1  and include two ERK phosphorylation sites . Western blotting confirmed expression of the constructs . Neither deletion reduced the reporter activity in the two-hybrid assay (B), thus further bracketing the upstream element required for association of AR(A/B) to within amino acid residues 307–330. These internal deletions also had no effect on activation of Gal4-ELK1 by CA-MEK1 . Finally, we tested whether the FQFP motif (FXFP motif) and the D-box region (residues 308–321), both of which direct ERK docking to ELK1, were also required for the association of AR(A/B) to ELK1. We tested a Gal4-ELK1 construct in which the FQFP motif was mutated to FQLA, a Gal4-ELK1 construct in which the D-box region (ELK1 residues 308–321) was deleted, and a Gal4-ELK1 construct with both lesions. All three resulted in loss of function of AR(A/B)-VP16 in the two-hybrid assay , mirroring the loss of activation by CA-MEK1 .To further refine the mapping data for the motif required for association of AR(A/B) within the ELK1 polypeptide segment 307–350, we made additional deletions of residues 331–340 and 341–350 A. Westernstructs B, inset.id assay B, thus f CA-MEK1 C, inset.id assay D, inset,i.e. a deletion within the transactivation domain of ELK1, which disrupted activation by ERK but did not affect association with AR(A/B). The two ELK1 motifs required for association with AR(A/B) equate to the two ERK-docking sites in ELK1.Collectively, the data from A). Each of these constructs, as well as control constructs, was co-transfected with full-length AR in recombinant HeLa cells in which the GAL4-TATA-LUC promoter-reporter was stably integrated. The cells were treated with testosterone or the vehicle control beginning at the time of transfection. Androgen activated the promoter only in cells transfected with Gal4-ELK1 constructs that were found in the preceding sections to have the ability to bind AR(A/B) (A). It was confirmed that both AR and the appropriate Gal4-ELK1 construct were expressed in the transfected cells, as observed by Western blotting using antibody to either Gal4 or AR (B).To further validate the mapping of ELK1 motifs required for association with AR, it was necessary to confirm that the mapping data obtained above by the mammalian two-hybrid assay using AR(A/B)-VP16 applies to the full-length AR. For this purpose we tested Gal4-ELK1 fusion constructs with deletions and mutations that were indicative for bracketing the binding sites for AR(A/B) A. Each o AR(A/B) A. It wasl4 or AR B.C). These results demonstrate that functional association of the full-length AR with ELK1  requires the same ELK1 motifs as those mapped for the binding of AR(A/B) to ELK1.To complement the above data, we co-expressed in HeLa cells AR and WTELK1 or AR and a mutant of ELK1 in which either one of the two ERK-docking sites was disrupted. AR and WTELK1 co-immunoprecipitated in these cells; however, when ELK1 was mutated at either one of its two ERK-docking sites, it was unable to co-immunoprecipitate with AR C. These GAL4-TATA-LUC) was stably integrated in the chromatin. The cells also stably expressed full-length AR and the Gal4-ELK1(87–428) fusion protein. Induction of reporter luciferase activity by testosterone was measured to quantify the binding of AR to ELK1. In these cells, lentiviral transduction with shRNA against SRF resulted in knocking down SRF at both the mRNA and protein levels compared with control shRNA . Knockdown of SRF significantly increased testosterone-induced luciferase activity .Functional interactions between AR and ELK1 could be influenced by the association of ELK1 with its DNA-binding partner SRF. To explore this possibility, we first used recombinant HeLa cells in which a minimal promoter-luciferase reporter construct with Gal4 elements upstream of the TATA box -VP16. Cells were transduced with shRNA against SRF or with control shRNA followed by transfection with the Gal4-ELK1 fusion construct or with the Gal4 control vector. SRF shRNA substantially decreased both SRF mRNA and protein  and increased the luciferase reporter activity 3-fold compared with cells transduced with control shRNA . Taken together, these results demonstrate that SRF is not required for activation of ELK1 by AR; rather, SRF may hinder optimal ELK1-dependent transcriptional activation by AR.As a complementary approach, we used recombinant HeLa cells that stably expressed the AR(A/B) domain fused to the VP16 transactivation domain (AR(A/B)-VP16). Additionally, the GAL4-TATA-LUC promoter-reporter was also stably integrated. These cells were co-transfected with siRNAs against ERK1 and ERK2 or transfected with control siRNA. ERK mRNAs and proteins were depleted within 48 h of siRNA transfection compared with the control siRNA . Following knockdown of ERKs, the cells were transfected again, this time with expression plasmid for the Gal4-ELK1 fusion protein or the control Gal4 vector. In parallel, cells were also co-transfected with Gal4-ELK1 and the expression plasmid for CA-MEK1. The reporter luciferase activity was measured 24 h following the second transfection (72 h following the first transfection), while the knockdown of ERKs persisted . The synergistic activation of the promoter-reporter by AR and ELK1 was unaffected by the combined depletion of ERK1 and ERK2 . In contrast, the CA-MEK-induced hyper-activation of the promoter was abrogated by depletion of ERK1 and ERK2, decreasing the promoter activation to the level observed for cells only expressing AR(A/B) and Gal4-ELK1 .The requirement for MAPK-docking motifs in ELK1 suggested the possibility that the functional association of AR with ELK1 could involve ERKs, the classical ELK1-activating protein kinases, as an essential component of the ELK1-AR complex. As a first test, we used recombinant HeLa cells stably expressing AR(A/B)-VP16 in which the GAL4-TATA-LUC promoter-reporter was stably integrated and that also stably expressed the Gal4-ELK1 fusion protein. As expected, in these cells ectopic expression of CA-MEK1 strongly stimulated expression of the luciferase reporter (G). However, co-expression of AR(A/B) decreased the promoter activation (G), indicating that AR interferes with hyper-activation of ELK1 by ERKs.Second, we tested whether AR(A/B) would compete with ERKs as activators of ELK1. We used recombinant HeLa cells in which the reporter G. Howevetivation G, indica2-TATA-luc reporter and an expression plasmid for either AR(A/B) or CA-MEK1. The (ELK1)2-TATA-luc promoter was responsive to the endogenous ELK1 in the cells. Following the transfections, the cells were treated with the MEK inhibitor, trametinib. As expected, MEK-induced activation of the promoter was inhibited by trametinib (H). In contrast, trametinib did not affect activation of the promoter by AR(A/B). The results from the complementary approaches of depletion and inhibition of ERK1/2 and competition between MEK1 and AR(A/B) as activators of ELK1 all demonstrate that the functional interaction of AR and ELK1 is insensitive to the expression or activation status of ERKs. The data indicate that the association of AR with ELK1 occurs independently of ERK1 and ERK2.In a third approach, the effect of ERK activity on the interaction between ELK1 and AR was tested using HeLa cells co-transfected with the (ELK1)ametinib H. In coni.e. SRF and ERK1/2, interfere with rather than facilitate functional association of AR with ELK1. Therefore, to test whether the association of AR with ELK1 could be due to direct binding, we used surface plasmon resonance (SPR). Purified AR  was immobilized as the “ligand.” His-tagged ELK1 and His-tagged mutant ELK1, in which both the D-box and the DEF motif were disrupted , were affinity-purified to >85% (A) and used as the analyte. The binding kinetics for ELK1 was determined at concentrations of 0, 5, 10, 20, 40, 80, and 160 nm (B). Bovine serum albumin was used as the negative control. Kinetic constants were evaluated using the BIAevaluation software. The equilibrium dissociation constant for the AR-ELK1 interaction was determined to be 1.9 × 10−8m. When the mutant ELK1 was used as the analyte at a concentration of 100 nm, the average response units from triplicate measurements was reduced by ∼90% compared with 100 nm ELK1 (C). The relatively high affinity of binding of purified preparations of AR and ELK1 strongly supports the concept that the in situ interactions of the two proteins are due to direct binding. Moreover, loss of this binding due to disruption of the ERK-docking sites in ELK1 strongly indicates that these are also the docking sites for AR.The studies above indicate that the classical binding partners of ELK1,  to >85% A and used 160 nm B. Bovine nm ELK1 C. The rein situ without disrupting the ability of ELK1 to bind to DNA, we used an ELK1 mutant lacking the D-box region (amino acids 308–321) that should compete with endogenous ELK1 for binding to chromatin. We tested whether the mutant ELK1 would produce a dominant-negative effect on androgen-dependent growth. LNCaP cells were transduced with lentiviral expression vectors for either ELK1 or the ELK1Δ308–321 mutant . Ectopic overexpression of ELK1 did not appreciably influence androgen (R1881)-dependent growth compared with the vector-transduced control (D). In contrast, ectopic overexpression of the ELK1Δ308–321 mutant showed a dominant-negative effect by inhibiting hormone-dependent growth. To confirm that the dominant-negative effect of this ELK1Δ308–321 on androgen-dependent growth was not due to dependence on activation of ELK1 by ERK, we demonstrate that LNCaP growth is insensitive to inhibition of MEK by trametinib (E); as a control in Fig. 8E, the AKT inhibitor AKTi-1/2 completely inhibited androgen-dependent growth. These results further validate the model that docking of AR on ELK1 is an essential component of growth signaling by androgen/AR in prostate cancer cells.Previous studies using ELK1 depletion methods have demonstrated that both prostate and bladder cancer cells require ELK1 for androgen-dependent growth –33. Howe–1 mutant D, inset. control D. In conametinib E; as a c2-TATA-luc promoter-reporter construct with an expression plasmid for AR-V7 and WTELK1 into the AR-negative HeLa cells. Ectopic AR-V7 activated the promoter-driven luciferase activity well above the basal level presumably associated with endogenous ELK1 (A). Similarly, when the GAL4-TATA-LUC promoter was co-transfected with Gal4-ELK1 and AR-V7, there was strong induction of promoter activity (B). Western blotting analysis confirmed expression of the AR-V7 construct in both cases (C).The ability of the amino-terminal A/B domain of AR to synergize with ELK1 suggested that AR-V7, the major splice variant of AR, would most likely also synergize with ELK1. To test this possibility, we co-transfected the (ELK1)ous ELK1 A. Similaactivity B. Westerth cases C.D, top and bottom panels). These results confirm that the AR-V7 splice variant cooperates with ELK1 as a transcriptional co-activator in the same manner as AR(A/B) and that ELK1 is also necessary for AR-V7-dependent growth of prostate cancer cells.We have previously demonstrated that ELK1 is necessary for androgen/AR-dependent growth of prostate cancer cells. To test whether prostate cancer cells that are dependent on AR-V7 also depend on ELK1, we used shRNA to deplete ELK1 in CWR22Rv1 cells, which depend on endogenous AR-V7. Partial depletion of ELK1 prevented the growth of these cells  in ELK1 as the elements essential for co-activation by AR. Nonetheless, the AR synergy with ELK1 is independent of ERKs and involves AR binding directly to ELK1. Interactions with ELK1 are required for hormone-dependent growth of PCa cells and also account for the ability of the AR A/B domain, as well as the major AR splice variant AR-V7, to support their hormone-independent growth. Indeed the results strongly support the view that synergy with ELK1, via AR docking on ELK1, is a critical component of growth signaling by AR and AR-V7.per se of ELK1 is unimportant for the ELK1-AR synergy.The mammalian two-hybrid mapping data with Gal4ELK1 mutants were clear-cut. Parallel studies using a constitutively active form of MEK to activate ELK1 demonstrated that the regions required for co-activation by AR(A/B) precisely coincided with the D-box and DEF motifs in ELK1 –42. Acti–i.e. SRF and ERKs , strongly supporting the premise that the transcriptional synergy between ELK1 and AR is due to direct binding. Loss of this binding upon mutational disruption of the ERK-docking sites in ELK1 equates the ELK1 motifs required for functional association with AR with those required for direct binding of AR to ELK1.Complementary approaches, including one- and two-hybrid assays, promoter assays, gene knockdown, MEK inhibition, and competition assays, demonstrated that the classical binding partners of ELK1 in the ternary complex factor, and ERKs , are notand ERKs . Similarand ERKs  suggestiXXL motifs in the estrogen receptor are required  and expressing a Gal4-ELK1 fusion protein in which the Gal4 DNA binding domain was substituted for the ETS DNA binding domain of ELK1 were kindly provided by Dr. Johann Hofman . These cells were then stably transduced with a vector expressing the full-length AR. Recombinant HeLa cells were also generated by stably transducing GAL4-TATA-LUC and a vector expressing the AR(A/B) domain fused to the VP16 transactivation domain (AR(A/B)-VP16). LNCaP cells were routinely grown at 37 °C in 5% CO2 in RPMI 1640 medium supplemented with 10% FBS (Invitrogen), 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mml-glutamine mixture (Invitrogen), and sodium pyruvate (1 mm) (Invitrogen). CWR22Rv1 cells were grown in phenol red-free RPMI 1640 medium supplemented with 10% FBS (Invitrogen), 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mml-glutamine mixture (Invitrogen). Parental and recombinant HeLa cells were grown in DMEM supplemented with 10% FBS and 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mml-glutamine mixture (Invitrogen). Additionally, the culture media for the recombinant HeLa cells included one or more of the following selection antibiotics: 100 μg/ml hygromycin (Invitrogen) , 100 or 400 μg/ml geneticin (Invitrogen) (for GAL4-TATA-LUC), and 2 μg/ml puromycin (Sigma) (for AR or AR(A/B)-VP16). For hormone depletion, LNCaP cells were grown in phenol red-free RPMI 1640 medium supplemented with 10% heat-inactivated and charcoal-stripped FBS (Sigma) and 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mml-glutamine mixture for 96 h before each experiment. For hormone depletion, parental and recombinant HeLa cells were grown in phenol red-free DMEM supplemented with 5% heat-inactivated and charcoal-stripped FBS (Sigma) and 2 mml-glutamine for 48 h before each experiment. Affinity-purified rabbit anti-human antibody to AR (sc-7305) and mouse antibodies to Gal4 (sc-510) and GAPDH (sc-47724) were purchased from Santa Cruz Biotechnology . Rabbit monoclonal anti-human antibody to ERK1/ERK2 (ab17942), rabbit monoclonal anti-human to androgen receptor-ChIP grade antibody (ab74272), rabbit monoclonal anti-human antibody to ELK1 (ab32106), and mouse monoclonal anti-human antibodies to the androgen receptor (ab77557) and ELK1 (ab7712) were from Abcam . R1881 was kindly provided by Dr. Steve Patrick . Testosterone was from Sigma. LipofectamineTM 2000 was purchased from Thermo Scientific (product number 78410). Trametinib was purchased from Selleckchem. AKTi-1/2 was purchased from EMD Millipore . The endoribonuclease-prepared siRNAs were purchased from Sigma.LNCaP, CWR22Rv1 cells, and HeLa cell lines were from the American Type Culture Collection ; 293FT cells were from Invitrogen. HeLa cells with a stably integrated minimal promoter-luciferase reporter containing five upstream Gal4 elements  was purchased from Abcam (Cambridge MA). Recombinant His-tagged ELK1 as well as His-tagged ELK1 mutated in both the D-box(Δ308–321) and the DEF motif  expressed from baculovirus-infected Sf9 cells were purified using nickel-agarose affinity chromatography. The proteins were eluted with 200 mN-hydroxysuccinimide, followed by covalent bonding of the ligand to the chip surface via amide linkages. Reference surfaces were prepared in the same manner but blocked with ethanolamine and thus contained no ligand. Kinetic binding analysis was carried out by injecting affinity-purified ELK1 polypeptide at different concentrations (0–160 nm) or mutant ELK1 (100 nm) into the flow cells (ligand and reference cell), and the interaction (response units (RU)) between analyte and ligand was recorded as the ligand RU minus the reference RU. Kinetic values were determined using BIAevaluation software (BIAcore), and the data were fitted with the model showing the closest match (dK) was calculated by dK = koff/kon.Amine coupling kit, CM5 sensor chip, and HBS-N buffer  were used for SPR analysis. The rate and equilibrium binding constants of the interaction of AR with ELK1 or mutant ELK1 were determined using BIAcore 3000 . Affinity-purified AR polypeptide (ligand) was immobilized on a CM5 research grade sensor chip by an amine coupling method . The immst match , 62. A 1GAL4-TATA-LUC plasmid (pG5luc-Promega) and expression plasmid for VP16 and Gal4 were purchased from Promega  . The pRL plasmid encoding Renilla luciferase was purchased from Promega. The PatheDetect pFC-MEK1 trans-reporter plasmid with an S218E/S222E point mutation and internal deletion between amino acid residues 32 and 51 rendering it constitutively active was from Stratagene. The pLVX-AR-V7 plasmid and pLVX control plasmid were the kind gifts from Dr. Yan Dong, Tulane University . Details of all other plasmid constructs generated in this work are provided in the supplemental material.The expression plasmid for human full-length ELK1in the pCMV plasmid was purchased from OriGene . The pLenti-GIII-CMV-hELK1 lentiviral vector was from Applied Biological Materials Inc. The ml-glutamine 24 h before transfection. The following day, cells were transfected with esiRNAs against ERK1  and ERK2  or 2 μg of control siRNA using LipofectamineTM 2000.The appropriate recombinant HeLa cells were plated in a 6-well plate  in DMEM supplemented with 10% FBS and 2 mHeLa cells were transfected with the expression plasmid for AR and co-transfected with expression plasmid for WTELK1 or ELK1Δ308–321 or ELK1 FxLa. Cells were harvested in radioimmunoprecipitation assay lysis buffer and 1× protease inhibitor mixture 48 h post-transfection. Whole cell lysates (500 μg) were pre-cleared for 2 h using protein A-agarose beads . Immunoprecipitation was performed by first incubating 100 μl of the protein A-agarose beads with 20 μg of the anti-rabbit androgen receptor antibody (ab74272) or negative control for 4 h. After washing the antibody-bound beads three times, 500 μg of the cell lysate was added and incubated at 4 °C overnight under rotary agitation. At the end of the incubation, the complexes were washed five times with the radioimmunoprecipitation assay buffer. The Western blotting was probed with mouse monoclonal AR antibody (ab77557) and mouse monoclonal ELK1 antibody (ab7712).Transient transfection and luciferase reporter assays, Checkmate mammalian two-hybrid assay, lentivirus-mediated gene knockdown and gene expression, cell proliferation assay, Western blotting analysis, RNA isolation, reverse transcription, and real time PCR have been described in Ref. t test. The p values are indicated in the figure legends.All experiments were performed in triplicate and repeated at least three times. The error bars in all graphs represent the standard deviation. Statistical analysis was performed using one-way analysis of variance with post hoc and least square differences and/or R. R., P. E. S., and M. R., designed the study and wrote the paper. R. R., S. D., and T. M. performed and analyzed the experiments. R. R., M. P., V. C., and J. S. designed and constructed vectors for expression of mutant proteins. B. L. K. contributed database mining and related analysis. All authors reviewed the results and approved the final version of the manuscript.


In biological vision, contextual modulation refers to the influence of a surround pattern on either the perception of, or the neural responses to, a target pattern. One studied form of contextual modulation deals with the effect of a surround texture on the perceived shape of a contour, in the context of the phenomenon known as the shape aftereffect. In the shape aftereffect, prolonged viewing, or adaptation to a particular contour’s shape causes a shift in the perceived shape of a subsequently viewed contour. Shape aftereffects are suppressed when the adaptor contour is surrounded by a texture of similarly-shaped contours, a surprising result given that the surround contours are all potential adaptors. Here we determine the motion and temporal properties of this form of contextual modulation. We varied the relative motion directions, speeds and temporal phases between the central adaptor contour and the surround texture and measured for each manipulation the degree to which the shape aftereffect was suppressed. Results indicate that contextual modulation of shape processing is selective to motion direction, temporal frequency and temporal phase. These selectivities are consistent with one aim of vision being to segregate contours that define objects from those that form textured surfaces. In natural scenes, objects rarely occur in isolation, more often than not surrounded by other objects or set against textured surfaces. The human visual system is tasked with extracting and recognizing the shapes of objects from such feature-reach textured backgrounds23456789et al.texture-surround suppression of contour-shape’, or TSSCS567813How might this come about? One idea is that neurons in the visual cortex that possess what has been termed ‘extra classical receptive-fields’ (ERFs) are responsible. A significant number of these neurons are suppressed by textural information that lie outside of their classical receptive field. Specifically, they exhibit what is termed ‘iso-orientation surround suppression’, or IOSS, meaning that the maximum surround suppression occurs when the surround elements are oriented similarly to that of the preferred orientation of the neuron’s classical receptive field56simultaneous contrast and aftereffects. With simultaneous contrast, the effect of the surround stimulus on the appearance of a center stimulus is measured, as for tilt1756Studies of center-surround interactions in human vision have traditionally employed two protocols: The shape frequency aftereffect, or SFAE, is our shape aftereffect of choice. Adaptation to a sinusoidal-shaped contour with a particular shape-frequency  causes a shift in the perceived shape frequency of a subsequently viewed test contour with a slightly different shape frequency56242527contour, instead favoring the processing of the adaptor contour as part of a texture. Coupled with the idea that contours and textures are processed by different mechanisms, it follows that an adaptor contour that is encoded as part of a texture will have little adaptive effect on a contour that is processed as a contour.How then can the SFAE be used to study TSSCS? When the adaptor contour is surrounded by a texture made of similar contours, as in the example shown in Although several recent studies have examined the various spatial and photometric (luminance and color) properties of TSSCS and the role they serve7813In different experiments, we investigate the dynamics of TSSCS by examining its dependency on motion direction and timing. To do this, we have measured the SFAE with a center contour adaptor and a surround texture that were either the same or different in one of a number of temporal attributes, specifically motion direction, temporal frequency and temporal phase. The premise is that if the SFAE is significantly greater for different-attribute compared to same-attribute center-surround adaptor combinations, then TSSCS mechanisms are tuned to that temporal attribute.global motion. The center contour and surround texture in the adaptor were separated by a small gap (0.6 deg) to prevent them from spatially overlapping when they drifted in different motion directions and/or with different speeds  obtained with and without the surround texture, respectively. With this metric, 1 indicates complete suppression of the aftereffect while 0 indicates no suppression. Another way to express the results is in terms of the magnitude of surround suppression. To do this we calculate a surround suppression index (SSI), as follows: For each central contour adaptor/test temporal frequency, the data for the shape aftereffect  were subFinally, we submitted the data to a repeated-measures one-way ANOVA to examine the effect of central contour adaptor/test temporal frequency for both same . The adaptor was not coherently moving but its shape-phase was randomly changed every cycle of contrast modulation (i.e. 1.32 Hz) in order to prevent the formation of afterimages and to minimize the effects of local orientation adaptation. The shape-phase of the test contour was also randomly assigned in every test period. We varied the temporal-phase difference between the central contour and surround texture adaptor. We used eleven values of temporal phase difference: 0, ±15, ±30, ±45, ±60 and ±90, with the ‘+’ sign indicating that the central contour preceded the surround texture and the ‘−’ sign indicating that surround texture preceded the central contour. A schematic of the phase difference condition is shown in The data  were subTo our knowledge this is the first study to investigate the motion-direction tuning and temporal properties of center-surround suppressive interactions in shape processing. Our results show that for globally drifting contours, the suppression of the shape aftereffect was maximal when the surround and center adaptor contours were moving in the same direction, at the same speed, and at relatively high speeds. On the other hand, for locally moving stimuli, the suppression was unaffected by the relative motion direction of center and surround. When the contrasts of the contour micropatterns were set to counter-phase, the amount of surround suppression was agnostic to counter-phase temporal frequency when center and surround were in-phase, but increased with counter-phase temporal-frequency when in anti-phase. Finally, again using counter-phasing micropatterns, the surround suppression was broadly tuned for center-surround phase differences, and slightly larger when the surround texture preceded rather than followed the central contour. Altogether, these findings have revealed that center-surround interactions in contour shape processing are tuned to global not local motion-direction and temporal frequency, and tuned to the temporal phase-relationships between center and surround.These results are in keeping with an important functional role of surround suppression, namely to facilitate the segregation of contours that are part of objects from those that are part of textures. A contour that is part of a texture is unlikely to exhibit different dynamic properties from its neighboring contours, and as a result surround suppression will tend to be maximal, preventing the contour from being processed as part of an object. On the other hand, a contour that is embedded in a texture but which exhibits different dynamic properties from its surround, i.e. is likely to be part of an object rather than the texture, will be minimally suppressed, with the result that its shape will be processed as part of the object to which it belongs. It should be emphasized that this functional account of surround suppression relates only to the processing of object contours. The shapes of textures are processed in a separate streamMotion direction selectivity has previously been found for the shape-frequency aftereffect on its own, i.e. with no surround353625Neurophysiological studies have shown that extra-classical receptive field, or ERF neurons, i.e. those subject to influences from outside of their classical receptive fields, or CRFs, are found in many visual areas, such as V1 and V2394041424344454648505132394041424354555678514While some neurophysiological studies report that V4 neurons manifest no selectivity for motion-direction5832505162636450With regard to temporal dynamics, most neurophysiological studies have measured the contrast response functions of V1 neurons using luminance gratings drifting at various temporal frequencies, and find strongest suppression of V1 CRF responses from high temporal frequency surrounds66414244686970We found that for globally drifting contours, TSSCS was maximal when the surround and center adaptor contours were moving in the same direction, at the same speed and at relatively high speeds, while for locally moving stimuli, the suppression was unaffected by the relative motion direction between center and surround. In addition, TSSCS was found to be selective for temporal frequency and temporal phase, and broadly tuned to the temporal phase-difference between central contour and surround texture. These motion-direction and temporal tuning characteristics of surround suppression are consistent with the center-surround motion interactions observed in visual areas such as V1325051626364Eight observers participated in this study, the two authors and six observers who were naive with regard to the experimental aims. Three participants took part in Experiment 1, 3, 4 and four in Experiment 2. All observers had normal or corrected-to-normal visual acuity. Observers gave their written informed consent prior to participating in this study and were treated in accordance with the Declaration of Helsinki. The research protocol was approved by the University of Stirling Ethics Committee and McGill University Health Centre Research Ethics Office.2. Viewing distance was 100 cm.The stimuli were generated by a ViSaGe video-graphics card  with 12-bits contrast resolution, presented on a calibrated, gamma-corrected Sony Trinitron monitor, at 120 Hz frame rate and with a spatial resolution of 1024 × 768. The mean luminance of the monitor was 40 cd/mAdapting stimuli were pairs of sinusoidal-shaped textures and contours made of strings of Gabor elements and presented in the center of the monitor on the mean luminance background and located 4 deg above and below the fixation marker. The upper and lower adapting and test stimuli were set to drift in opposite directions, as shown in global motion. In Experiment 2, the Gaussian envelopes of the Gabor patches were stationary while the carriers moved inside stationary envelopes. This type of motion is termed local motion. These local motions simulate that of a rigid-body contour-shape.The texture adaptors consisted of a central contour flanked on each side by a surround made of 5 contours arranged in parallel . The center contour and surround texture were separated by a small gap of 0.6 deg which was introduced in order to avoid the spatial overlap between contours when they were made to drift at different temporal frequencies in the same or in different directions see . This sm567In different experiments, we varied (i) the motion direction and (ii) the temporal frequency of both the central contour and the surround texture adaptor. There were three adaptor conditions: center contour only, i.e. no surround , center Each session started with an initial adaptation period of 90 s, followed by a repeated test of 0.5 s duration interspersed with top-up adaptation periods of 2.5 s to reinforce the initial adaptation. A schematic representation of the adapting and test procedure is shown in with-adaptation condition we made six measurements, three in which the upper adaptor had the higher shape-frequency and three in which the lower adaptor had the higher shape-frequency. In addition, we measured for each condition the shape-frequency ratio at the PSE in the absence of the adapting stimulus - the no-adaptation condition. To obtain an estimate of the size of the shape aftereffect we first calculated the difference between the logarithm of each with-adaptor shape-frequency ratio at the PSE and the mean of the logarithms of the no-adaptor shape-frequency ratios at the PSE. We then calculated the mean and standard error of these differences across the six measurements. These standard errors are the ones shown in the graphs.A staircase method was used to estimate the PSE. The geometric mean shape-frequency of the two test contours was held constant at 0.35 c/deg while the computer varied the relative shape-frequencies of the two tests in accordance with the subject’s response. At the start of the test period the ratio of the two test shape-frequencies was set to a random number between 0.7 and 1.44. On each trial subjects indicated via a button press whether the upper or lower test contour had the higher perceived shape-frequency. The computer then changed the ratio of test shape-frequencies by a factor of 1.06 for the first five trials and 1.015 thereafter, in a direction opposite to that of the response, i.e. towards the point of subjective equality or PSE. The session was terminated after 25 trials. We used a staircase method that was terminated after a fixed number of 25 trials, rather than a fixed number of reversals. This was done in order to keep the total amount of adaptation for each condition the same. The shape-frequency ratio at the PSE was calculated as the geometric mean shape-frequency ratio of the test that followed the lower shape-frequency adaptor to the test that followed the higher shape-frequency adaptor, averaged across the last 20 trials. Because the aftereffect is defined as the ratio of shape-frequencies at the PSE, its units are dimensionless. For each How to cite this article: Gheorghiu, E. and Kingdom, F. A. A. Dynamics of contextual modulation of perceived shape in human vision. Sci. Rep.7, 43274; doi: 10.1038/srep43274 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


This review examines the delivery of rehabilitation care to cancer patients with relation to disease prognosis. This includes the evaluation when patients are referred for rehabilitation services and the effectiveness of rehabilitation interventions across the cancer continuum.Although prognosticating life expectancy is difficult, referrals for rehabilitation interventions appear to be affected by physician attitudes towards patients with advanced disease, in part because of misconceptions about the nature of rehabilitation for oncology patients. Rehabilitation may also be underutilized in long-term survivors with no evidence of disease. Despite this, our review found that rehabilitation in advanced disease, end-of-life, geriatric cancer patients, and in long-term survivors can be beneficial. There is a relative dearth in studies on rehabilitation interventions specifically at the end-of-life.Cancer rehabilitation can be helpful to patients along the spectrum of cancer prognoses. Examining more accurate ways to prognosticate life expectancy, improving communication and education between oncologists and rehabilitation team members, and modifying survivorship plans to include patient education on functional changes over time may improve the delivery of rehabilitation care. The dynamic and complex nature of oncology patients creates challenges in providing rehabilitation care. As patients progress through treatments and/or their disease advances, the rehabilitation target may move—a goal of independent functioning and returning to work may abruptly change to requiring assistance with activities of daily living and moving to an accessible home. These changes are even more pronounced when a patient’s prognosis changes, as someone who is expected to be a long-term survivor of cancer will undoubtedly have different rehabilitation goals and expectations than someone with a life expectancy fewer than 6 months.The evolution of symptom burden and impaired function as the disease trajectory advances is an ongoing area of study, but it is clear that the rehabilitation needs of patients change as their prognosis does and that all patients across the cancer continuum potentially have rehabilitation needs. For example, Beernaert et al. found that patients with advanced cancer report poorer function and increased fatigue and pain compared to other cancer patients after controlling for age, gender, and type of cancer . AdditioWe sought to evaluate prognosis as it intersects with delivering rehabilitation and supportive care services. Using the search terms listed in Table Despite efforts to improve patient access to rehabilitation and supportive services, physician bias regarding rehabilitation across the spectrum of disease may lead to unmet patient needs. Spill et al. found that only 8% of the medical oncologists—compared to 35% of physiatrists—would recommend a patient with functional goals and advanced cancer  be admitted to an inpatient rehabilitation unit, but 70% of the combined cohorts believed that a patient must fully understand their prognosis before entering into a comprehensive rehabilitation program [In fact, cancer rehabilitation, when focusing on addressing specific impairments, may be quite efficient and is effective in reducing symptom burden . This biPhysician attitudes about prognosis also appear to be the most important consideration in initiating palliative systemic treatment in advanced cancer, as Buiting et al. found that nearly half of medical oncologists documented it as part of their decision making . PerformIn an attempt to standardize when patients are referred for rehabilitation based on prognosis, Jarlbaek et al. suggest that patients who live longer than 2 years post-cancer diagnosis may be better candidates for rehabilitation programs than hospice and end-of-life care programs. In their study of over 24,000 patients, 78% who died did so within 2 years of cancer diagnosis, and many of those that lived longer than 2 years often lived for several more years [It is not clear that providers can accurately predict prognosis. This is particularly worrisome if physician bias about perceived prognosis is a major factor in determining who receives rehabilitation care. A recent study found that using an assessment tool—the palliative prognostic score (PaP)—was significantly more accurate in determining both 30- and 100-day survival when compared to using the tool combined with clinician prediction, and to clinician assessment alone . Other sRehabilitation may be time consuming, and oncologists may be biased against referring patients with limited life expectancies to these services. Because of this, it is important to factor in patients’ goals of care; for example, some patients with less than 3 months to live may desire to undergo 2 weeks of inpatient rehabilitation if it reduces caregiver burden. Unfortunately, patients are generally unsure of their prognosis and tend to be more optimistic than oncologists, as demonstrated in a recent study that found 68% of the patients to be discordant with their oncologists when estimating life expectancy . AnotherUnrealistic expectations about prognosis have the potential to create unrealistic goals of care. This underscores the need for effective communication between patients and their oncology teams about long-term planning, including rehabilitation care. Conversely, patients often experience hope and optimism despite understanding their life expectancy. Reasons for this are multifactorial and not always well-understood by health professionals but may explain why patients seek restorative interventions such as cancer rehabilitation despite a poor prognosis .Understanding when to refer patients for rehabilitation services presents an ongoing challenge. Numerous organizations have advocated and developed criteria for evaluation of patients for rehabilitation needs and other supportive care services without a qualifier of patient prognosis –15. The For patients in whom the disease is presumably cured, the Commission on Cancer (COC) has attempted to improve evaluation and referrals for long-term care by mandating survivorship care plans (SCP). These serve as a way to inform patients about potential short- and long-term complications from cancer or its treatment, and explain how to obtain supportive care services . UnfortuFurthermore, SCPs provide an early opportunity to inform patients about potential rehabilitation needs, but they are not designed for patients with advanced cancer or to inform patients about functional decline as disease advances and prognosis changes. Although plans of care in advanced cancer have been proposed, there exists no standard in providing patients information about symptom management and rehabilitation need .For patients with advanced cancer or higher symptom burden, the National Comprehensive Cancer Network (NCCN) has proposed criteria to determine if patients should be referred for palliative care services in order to provide symptom management. Specifically, the NCCN recommends that if patients display any one of the following six attributes, palliative care referral should be initiated: (1) uncontrolled symptoms; (2) moderate-to-severe distress; (3) serious comorbid physical or psychosocial condition; (4) advanced or progressive disease without available curative therapy; (5) patient/family concerns about decision making; (6) or patient/family request for palliative care . Levy etA recent meta-analysis of outpatient palliative care referrals found inconsistent evaluation and referral mechanisms for patients across the cancer continuum. The most important criteria for referring a patient were physical symptoms  and cancer trajectory (62%), followed by prognosis (33%) and performance status (33%) ••. The aAs outlined above, uncertainty about the benefits and necessity of rehabilitation in cancer care largely center on patients with a poor prognosis. This may be due to multiple factors, including considerations surrounding the appropriateness of time- and resource-intensive services for patients with limited life expectancy. While palliative care and hospice at the end-of-life is generally felt to be important and effective , 28, canRehabilitation at the end of life may cover many facets, including outpatient, inpatient, and hospice-based interventions. Outpatient intervention has the potential to provide rapid assessment and relief of symptoms. Referral to a physiatrist to diagnose and treat neuromusculoskeletal, pain, functional, and other symptoms should be considered as a part of impairment-driven rehabilitation . Many paPatients with bony metastases may need expert assessment of lesions to determine stability and proper management. For example, vertebral metastases often do not require surgical or radiation-based intervention, though this must be first evaluated systematically based on the location, size, and nature of each lesion . PhysiatCentral nervous system lesions also often require physiatric evaluation and management, as primary and metastatic brain tumors can create numerous cognitive and neurologic deficits. Despite the ultimately poor prognosis of these cancers, rehabilitation intervention can improve function and quality of life, safety and independence, and reduce resource utilization by improving care coordination and preventing injury. Difficulties in accurately prognosticating survival in the presence of brain metastases has the potential to reduced appropriate rehabilitation utilization. One study of 2700 cases found that 45% of the estimations were off by 6 or more months, and 18% were off by 12 months. Performance status—which rehabilitation may improve—and age may be the biggest factors in determining prognosis in this patient population ••.Rehabilitation in the setting of brain metastases involves managing the effects of both the tumor(s) and the surgery and/or radiation. Whole brain radiation, for multiple or large lesions, can cause significant long-term cognitive impairment but is becoming less common with the advent of more precise radiation-based interventions . Risk faRegardless of the presence of metastases, inpatient rehabilitation improves function and is safe in patients with advanced cancer. Although patients with advanced cancer may utilize inpatient rehabilitation more than those with controlled disease , Sliwa eIn the end-of-life and hospice setting, rehabilitation interventions have been shown to improve independence, reduce anxiety and depression, and improve fatigue, among other benefits –52. One Finally, it is important to discuss the rehabilitation of geriatric patients with cancer. Even when the cancer is early stage, older patients are at a greater risk for morbidity and mortality, in part due to cancer treatment accelerating the aging process, and goal setting must be done in the setting of both the disease and the patient’s age . NeverthAnother important area of rehabilitation concern is fall risk in the geriatric cancer patient. Spoelstra et al. found that older cancer survivors are 16% more likely to experience a fall than their cancer-free peers ••, whichAlthough much of the controversy about the efficacy and appropriateness of rehabilitation understandably centers on the end of life, it is important to note that patients with the opposite prognosis—those whose cancers have been cured and/or who are expected to have a normal lifespan—may have rehabilitation needs overlooked, because lifesaving treatment has completed, and it is presumed that patients will recover from deficits over time. This is not always the case, as one study found that 38.2% of the unmet needs in cancer survivors 2-, 5-, and 10-year post-treatment were physical needs . The lonThe decline in function related to cancer treatment occurs early. For example, breast cancer patients experience increased fatigue and lower physical function and activity during and after chemotherapy, neither of which improve significantly without intervention . AdditioWhile rehabilitation and exercise have consistently been shown to be beneficial across the cancer continuum including in long-term survivors , 72, 73,Physiatrists and other rehabilitation providers are uniquely suited to manage neuromusculoskeletal and cognitive deficits in cancer survivors, and their involvement may reduce unmet rehabilitation needs in this population . ImproviFinally, rehabilitation has been consistently shown to help cancer survivors return to work, which is beneficial both economically and for patient well-being, as cancer survivors who have job-loss related to a health issue have lower health-related quality of life . FortunaA patient’s medical prognosis has the ability to impact rehabilitation and supportive measures in many ways, including physician bias and resultant lack of proper referrals. Despite this, rehabilitation interventions appear to be generally effective in improving function and reducing symptom burden in cancer patients across the spectrum of different prognoses. Unfortunately, studies evaluating interventions at the end of life are limited, and more likely to be short series or case reports, making the literature imbalanced in favor of interventions in the earlier stages of disease . This crAdditionally, most of the available research focuses on palliative care, which can include management of symptoms treated by rehabilitation providers, such as pain and functional impairment, but is a broad topic, and the studies are often nonspecific about what palliative care interventions are offered. Although there is overlap between cancer rehabilitation and palliative care, it is important to note that there are distinctions in the two fields, primarily that palliative care typically addresses end of life considerations in more than just cancer patients—including spiritual and social or financial concerns—and cancer rehabilitation emphasizes restoring function and reducing symptom burden through identification and management of specific impairments in cancer patients .It is important that rehabilitation be considered in all cancer patients who require it, regardless of prognosis. The challenge facing clinicians and researchers is developing actionable rehabilitation referral mechanisms and interventions specifically tailored to patients at different points of the disease continuum.


To develop an evidence and theory-based complex intervention for improving outcomes in elderly patients following hip fracture.Complex-intervention development  framework phase I) using realist literature review, surveys and focus groups of patients and rehabilitation teams.North Wales.Surveys of therapy managers (n=13), community and hospital-based physiotherapists (n=129) and occupational therapists (n=68) throughout the UK. Focus groups with patients (n=13), their carers (n=4) and members of the multidisciplinary rehabilitation teams in North Wales (n=13).The realist review provided understanding of how rehabilitation interventions work in the real-world context and three programme theories were developed: improving patient engagement by tailoring the intervention to individual needs; reducing fear of falling and improving self-efficacy to exercise and perform activities of daily living; and coordination of rehabilitation delivery. The survey provided context about usual rehabilitation practice; focus groups provided data on the experience, acceptability and feasibility of rehabilitation interventions. An intervention to enhance usual rehabilitation was developed to target these theory areas comprising: a physical component consisting of six additional therapy sessions; and a psychological component consisting of a workbook to enhance self-efficacy and a patient-held goal-setting diary for self-monitoring.A realist approach may have advantages in the development of evidence-based interventions and can be used in conjunction with other established methods to contribute to the development of potentially more effective interventions. A rehabilitation intervention was developed which can be tested in a future randomised controlled trial (MRC framework phases II and III).ISRCTN22464643, Pre-results. A complex intervention for hip fracture rehabilitation was developed, which was evidence based and theoretically underpinned  framework phase I).Programme theories were developed from a realist review of the literature. A survey added context, and focus groups provided data on the experience, acceptability and feasibility of rehabilitation interventions.The methods used to develop this rehabilitation programme may be applicable to the development of other complex interventions.The feasibility and acceptability of the developed intervention are reported separately, but evidence of effectiveness and cost-effectiveness requires testing in a randomised controlled trial (MRC framework phases II and III).Proximal femoral fracture, more commonly referred to as hip fracture, is a common, major health problem in the elderly.Rehabilitation programmes for hip fracture are complex interventions due to their multifaceted nature and the involvement of many heterogeneous factors including individual patient circumstances and comorbidities, healthcare professionals, rehabilitation setting and social influences.Realist reviews provide a flexible way of exploring causal relationships, thus aiding our understanding of intervention mechanisms and supporting the development of potentially more effective interventions.We therefore undertook a realist review of the available evidence for hip fracture rehabilitation to develop theory on the context, mechanism and outcomes of existing rehabilitation programmes, with this forming the basis for the development of our own evidence-based intervention for subsequent testing in a feasibility study.A summary of the methods used is presented below. Further detail can be found in the final report to the funder. The development of the community-based rehabilitation package was informed by three complementary work packages. A coherent theoretical basis for the intervention was developed from a realist literature review. Initial findings from the review were used to develop the questions for a survey. A survey of current services described usual practice, and was an additional source of relevant theories that contributed to the realist review. The initial findings from the review were also used to develop topic guides for the focus groups and the initial framework for their analysis. Focus groups provided data on the experience, acceptability and feasibility of rehabilitation interventions.A realist review was used to identify the evidence base and develop a theoretical understanding. A scoping search of systematic reviews,The list of questions was formulated into statements that described how these different domains interact. These statements were subsequently refined during discussions between members of the research team  and with other researchers from the School of Healthcare Sciences, Bangor University, in two realist review workshops.Feedback from experts in health psychology, rehabilitation and implementation research were combined with initial survey and focus group findings into candidate programme theories. These intermediate working theories were used as the basis for bespoke data extraction forms.The literature search strategy used by the NICE guideline review of multidisciplinary rehabilitation programmes for hip fracture10.1136/bmjopen-2016-014362.supp1Participants of interest were elderly adults with proximal hip fracture. The intervention of interest was multidisciplinary rehabilitation following proximal hip fracture. Outcomes of interest were mortality, pain, functional status, quality of life, health utility, health service use, costs and patients’ experiences. A working definition of multidisciplinary rehabilitation was adapted from a review of intermediate care servicesData were extracted by one reviewer (NUD) and checked for accuracy by a second (NHW). We assessed study quality using the mixed methods appraisal tool.Theories were refined through an iterative process comparing individual study programme theories in turn. Data for each individual study were examined in terms of the identified programme theories and the interaction between mechanisms, context and outcomes, starting with data extracted from studies that were conceptually ‘rich’ and continuing with those that were conceptually ‘thick’. The data were then examined across the different studies to detect patterns and themes. A second set of refined data extraction forms was used to test each theory in turn, and adjudicate between components of the final programme theories.A UK-wide web-based survey was conducted, targeting physiotherapists, occupational therapists and hip fracture centre therapy service managers working in the rehabilitation of patients over 65 years of age who have had surgery for proximal hip fracture. NICE guidance on hip fracture rehabilitationThree versions of the survey were developed for hip fracture centre managers, physiotherapists and occupational therapists. The managers’ survey focused on the organisation of services, while the therapists’ questionnaires focused on aspects of clinical practice such as session content, frequency and location, and how assessments were conducted. The therapists’ versions were further subdivided according to healthcare setting: acute hospital, community hospital or community-based team.The survey was open for 7 weeks from 6 August 2013 to 25 September 2013. We surveyed a sample of senior managers who had a strategic role in rehabilitation services for this group of patients and aimed to achieve a 10% sample of all UK centres performing hip fracture surgery. Centres in Wales, Northern Ireland and England were identified from publicly available information on the National Hip Fracture Database (NHFD). Hospitals in Scotland were contacted separately and directly. We purposively sampled for geographic spread and centre size, contacting centres by telephone and through advertising on the NHFD. Twenty-four centres from across the UK agreed to take part.Descriptive statistics were used to provide frequency  data concerning current services and practice, where the answer format provided predetermined response options (ZH). Where the response format was open ended, responses were coded and categorised into themes (MW). The integrated care pathways and physiotherapy exercise sheets returned to the team were qualitatively reviewed to provide description of commonalities and differences (MW).Focus groups were completed at the three acute hospital sites across North Wales within Betsi Cadwaladr University Health Board. Three focus groups of members of the multidisciplinary rehabilitation teams in the community and the hospital, and four focus groups for patients and their carers were organised. Informed consent procedures were followed for recruitment, as approved by UK NHS North Wales Research Ethics Committee.People who were over 65 years, were receiving rehabilitation following surgical repair of a proximal hip fracture within the last 3–12 months, were living independently prior to fracture and were able to provide informed consent were eligible to take part.Eligible participants were identified from the NHFD, through the medical and nursing staff who were responsible for maintaining the database at each site.Discussions were semistructured and run by a moderator (CAH) and comoderator  using a topic guide containing open-ended questions regarding experiences, perceptions and beliefs about rehabilitation following proximal hip fracture. In the professionals’ focus groups, patient scenarios were also used to stimulate discussion about the sort of rehabilitation patients would be likely to receive. In the later patient focus groups, we explored initial ideas for the intervention to gain feedback. The focus group discussions were digitally recorded and fully transcribed into the speaker’s original language, with any portions in Welsh subsequently translated into English for analysis. The interviews were thematically analysed using the Framework approach.The final programme theories and the results from the survey and the initial focus groups were discussed by all of the research teams in order to identify the important components of the rehabilitation intervention. The intervention components were discussed and refined in the final focus groups.A summary of the results is presented below. Further detail can be found in the final report to the funder.‘In the context of patients with a great range and variety of pre-fracture physical and mental health co-morbidities affecting their ability to meet rehabilitation goals, a tailored intervention incorporating increased quality and amount of practice of exercise and activities of daily living in addition to usual rehabilitation leads to better confidence, mood, self-efficacy, function, mobility and reduced fear of falling.’This was then broken down into three component programme theories described in Elderly patients with proximal hip fracture presenting with a range of prefracture physical and mental functioning and a variety of comorbidities need a rehabilitation programme that is tailored to individual needs in order to achieve appropriate outcomes such as improved physical functioning, greater mobility, reduced disability and independent living.Findings from the realist review indicated that tailoring of patient care requires a detailed assessment of patients' prefracture level of functioning,In the survey, tailored rehabilitation was also identified as an important aspect of service provision. Respondents from all categories reported that the frequency of rehabilitation visits received was influenced by individual patient need . Survey Proximal hip fracture results in poor physical functioning, fear of falling, low mood and lack of self-efficacy requiring improved quality and increased amount of practice of physical exercises, activities of daily living and psychological tasks in order to gain mastery and control to improve confidence, mobility and physical functioning.Enhancing the practice and quality of exercise and activities of daily living has both physical and psychological components.According to the survey, more rehabilitation staff resources were needed to provide this support. Although patients’ cognitive status, mood, self-efficacy and fear of falling were assessed, routine assessments using validated tools were not performed everywhere and the frequency that progress was assessed varied . The impThe diversity of services provided by different disciplines, across sectors from a variety of funders requires a coordinated provision of the multidisciplinary rehabilitation programme in order to deliver appropriate physical, functional and psychological interventions to patients in a timely manner.The coordination of multidisciplinary care from the acute hospital into the community required good communication between rehabilitation professionals and careful discharge planning. Patients valued the help and support they received from healthcare teams during their recovery and regarded this as the single most important factor in their recovery, so the provision of consistent and reliable care was vital.Most respondents in the survey from both acute and community hospital settings reported that routine clinical practice was following the latest NICE (2011)The fourth focus group theme was a need for more information for patients and their carers about what to expect following the hip fracture and how to access all of the available resources. The complexity in programme provision and the often poor communication between different sectors meant that rehabilitation was neither smooth nor seamless, and because of this lack of consistency, patients felt unsupported in their recovery. Patients and their carers required reassurance from qualified professionals about which activities were safe to perform in order to overcome these barriers, highlighting the role of the therapist as a mediator to improve their self-efficacy.identify individual goals with help from a therapist;enhance self-efficacy;increase the opportunity to practise prescribed exercises and activities of daily living;support the self-monitoring of progress towards identified goals;give encouragement and support from professionals;provide information on what to expect during rehabilitation;provide reliable and consistent care;signpost to other available services.In order to address these, we developed a rehabilitation programme comprising both physical and psychological components . The phyConsidering these findings the rehabilitation intervention needed to:The additional sessions were also an opportunity for patients to obtain reassurance and guidance from a qualified healthcare professional. Similarly, the outcome of the psychological components aimed to increase confidence and self-efficacy that would affect patient’s ability and willingness to perform exercises, thus improving their physical outcomes.A detailed logic model of the intervention activities, their proposed long and short-term goals and how these target different components of the International Classification of Functioning framework has previously been published,There were three programme theories from the realist review: improving patient engagement by tailoring the intervention according to individual needs and preferences; reducing fear of falling and improving self-efficacy to exercise and perform activities of daily living; and coordination of services and sectors delivering the rehabilitation. These were reflected in the survey data highlighting that while routine clinical practice was broadly in line with current guidance, there was variability in the provision of services, especially in the community, and that important psychological mediators such as self-efficacy and fear of falling were not routinely assessed using validated tools. They also agreed with the four focus group themes of: variation in rehabilitation care provided; the need for more information; facilitators and barriers to rehabilitation; and the psychosocial impact of hip fracture. These findings informed the development of a community-based rehabilitation intervention consisting of a psychological component delivered using a workbook and a patient-held goal-setting diary and a physical component comprising additional rehabilitation sessions.Other studies have acknowledged the benefits of using realist review in intervention development,The findings from the individual work packages are supported by existing literature. A qualitative study of physiotherapists’ perceptions of rehabilitation also showed that tailoring of care to patient’s individual needs, based on their own goals and level of support available, was an important component of successful rehabilitation.This was the first realist review of rehabilitation following hip fracture and the first UK-wide survey aiming to describe rehabilitation for patients following hip fracture across acute and community settings since the introduction of NICE recommendations for rehabilitation in 2011.Important implications for practice are the routine assessment of psychological variables and the inclusion of psychological components in rehabilitation interventions. This study demonstrated the potential benefits of using a realist approach to complex intervention development and how a realist review could be used in conjunction with other established methods to provide an evidence base for a hip fracture rehabilitation intervention. This approach may be beneficial for developing complex interventions in other clinical areas and can be used to provide theories of how specific intervention components will facilitate their intended outcomes. The next phase in the MRC framework for evaluating complex interventions