The results demonstrate a significant difference in ACC expression between human and rodents, which may introduce difficulties for the use of rodent models for development of ACC inhibitors.Acetyl-CoA carboxylases ACC1 and ACC2 catalyze the carboxylation of acetyl-CoA to malonyl-CoA, regulating fatty-acid synthesis and oxidation, and are potential targets for treatment of metabolic syndrome. Expression of ACC1 in rodent lipogenic tissues and ACC2 in rodent oxidative tissues, coupled with the predicted localization of ACC2 to the mitochondrial membrane, have suggested separate functional roles for ACC1 in lipogenesis and ACC2 in fatty acid oxidation. We find, however, that human adipose tissue, unlike rodent adipose, expresses more ACC2 mRNA relative to the oxidative tissues muscle and heart. Human adipose, along with human liver, expresses more ACC2 than ACC1. Using RT-PCR, real-time PCR, and immunoprecipitation we report a novel isoform of ACC2 (ACC2.v2) that is expressed at significant levels in human adipose. The protein generated by this isoform has enzymatic activity, is endogenously expressed in adipose, and lacks the N-terminal sequence. Both ACC2 isoforms are capable of  Acetyl-CoA carboxylase alpha (ACC1) and beta (ACC2) catalyze the carboxylation of acetyl-CoA to malonyl-CoA. Malonyl-CoA is a substrate for fatty acid synthase and also inhibits carnitine palmitoyltransferase-1, such that malonyl-CoA is a key molecule regulating both the biosynthesis and oxidation of fatty acids. Thus ACC1 and ACC2 are key regulators of the fatty-acid synthesis and oxidation pathways −/− mutants are embryonic lethal −/− mice have a normal lifespan but higher fat oxidation rate in muscle and adipose tissues and are resistant to diet-induced obesity de novo fatty-acid synthesis. This is supported by observations of high ACC1 expression in rat and mouse lipogenic tissues In mammals, both of ACC1 and ACC2 are multifunctional enzymes, containing biotin carboxyl carrier, biotin carboxylase, and carboxyl transferase domains all within a single polypeptide chain . Calibrated Taqman involves construction of a standard curve derived from dilution series measurements, resulting in highly quantitative measurements permitting cross-gene and cross-tissue comparisons. We find the relative expression of ACC1 and ACC2 transcripts measured by microarray and quantitative PCR  are in gWhen mapped to the human genome, three human and mouse ESTs, human CA392208 and mouse BB854145 and BB866065, suggest the existence of an additional transcribed region between the first and second exons of the RefSeq ACC2 isoform (ACC2.v1) NM_001093 . Other gTo validate transcription of this computational prediction, we designed RT-PCR primers from the novel exon 1 to the known exon 5. The result shows clear expression of the novel band in several tissues . CloningThe relative abundances of ACC1, ACC2.v1 and ACC2.v2 transcripts were measured in five human tissues using calibrated real-time RT-PCR. As shown in To determine if these transcript expression patterns reflected enzyme levels, we assayed ACC1 and ACC2 protein levels by Western blot using streptavidin and antibodies specific to the middle of ACC1 and to the N terminus of ACC2.v1 . A singlin vitro14CO2 fixation assay using lysate from FM3A cells. Overexpression of either ACC2.v2 or ACC2.v1 increases ACC enzymatic activity >20-fold relative to vector alone  activity in FM3A cells overexpressing recombinant ACC clones using [14C]acetate incorporation as a marker for DNL , suggesting that dissection and RNA isolation differences tend to produce relatively small effects on mRNA profiles.We demonstrate the existence of a novel ACC2 isoform (ACC2.v2) that is expressed in human white adipose tissue and has enzymatic and DNL activity. The protein is smaller (∼255 kDa) than that of ACC1 (∼265 kDa) and the known ACC2 protein  , S3. Thim for ACC1 than for ACC2 for acetyl-CoA and citrate et al.−/− mice showed not only increased fatty-acid oxidation but also decreased fat levels in adipose. Finally, our results also suggest ACC2 specific inhibition could result in different physiological effects in humans than in rodents.In human adipose, over 20% of the highly expressed ACC2 mRNA is in the form of ACC2.v2, which is expressed at levels comparable to ACC1. ACC2.v2 and ACC1 transcripts encode proteins that are 76% identical (87% similar), and the biotin carboxylation and carboxyl transferase regions are 84% and 81% identical (93% and 91% similar), respectively. The lack of the first coding exon of ACC2.v1 makes the ACC2.v2 protein sequence more similar to ACC1. Previous enzyme kinetics show only lower K+ mRNA was amplified with a full-length amplification method using random-priming sequences to reproduce the entire transcript Custom oligonucleotide microarrays were purchased from Agilent Technologies . We designed these arrays to monitor the expression of 18,000 genes and associated alternate splicing events Human tissues and cell line samples were purchased as mRNA or total RNA . Each tissue sample was pooled from multiple donors, typically 12. Human adipose tissue consisted of subcutaneous epithelial white adipose tissue. Mouse and rat tissues were purchased from Biochain  and Clontech. Human protein samples were purchased from Biochain and Biopredic International  as tissue lysate or frozen block, Male Sprague-Dawley rats from Japan SLC,  and C57Black/6J mice from CLEA Japan .Alignments to the human genome  were done with sim4 Supplemental TaqMan primer and probe sequences used to monitor ACC1, ACC2.v1, and ACC2.v2 levels quantitatively in human and mouse tissues are shown in Supplemental Total protein samples were separated using 3–8% Tris-Acetate polyacrylamide gel electrophoresis (Invitrogen), and then transferred to PVDF membrane . Membranes were blocked with Block Ace reagent , and then incubated with HRP-conjugated streptavidin  or corresponding polyclonal antibodies followed by HRP-conjugated 2nd antibody according to the manufacturers . Antibody dilutions are shown in Supplemental 5′-GCCTAGGTAATGGTCTTGCTTCTTTGTCTATCTTGTCTG-3′; reverse, 5′-ACCGGTGGTGGAGGCCGGGCTGTCCATG-3′. The amplified product was cloned into a pcDNA5/FRT/V5-His-TOPO vector (Invitrogen) with an added V5 epitope and His tags at the C-terminus, and was confirmed by sequencing. The plasmid was then digested with AvrII and PmeI, and the cDNA fragment was subcloned into the SpeI/EcoRV site of pEFcDNA3 (Invitrogen) vector. The ACC2.v2 cDNA was prepared by replacement of the ACC2.v1 specific N-terminus with that of ACC2.v2 by PCR using the ACC2.v1 cDNA as a template and the following primer pair: forward, 5′-GCCTAGGTAATGAGTCCTGCCAAGTGCAAGATCTGTTTCCCTGATCGCGAAGTAAAGCCGAGCATGTCGGGACTCCA-3′, reverse, 5′-GGCCGGTGCTTCCTCAAC 3′. The PCR product was then digested with AvrII/HindIII and exchanged with that of ACC2.v1 cDNA to construct a full-length ACC2.v2 cDNA clone. The ACC2.v1 and ACC2.v2 expression plasmids and pEFcDNA3 vector were introduced into FM3A (mouse mammary carcinoma) cells  using Lipofectaimine 2000 (Invitrogen) or Effectene Transfection Reagent , according to manufacturers' instructions. The FM3A cells were cultured in RPMI 1640 medium (Invitrogen) containing 10% fetal bovine serum  and 1 mg/ml G418 (Invitrogen) at 37°C in 5% CO2, 95% air to obtain stable transfectants. Cell lysate was prepared and subjected to ACC enzyme assay and Western blot analysis.The full-length human ACC2.v1 cDNA was amplified by PCR from human skeletal muscle Marathon-Ready cDNA (Clontech) was used as the template using the following primer pair: forward, The lysate of human adipose tissue was incubated with affinity-purified anti-ACC2.v2 antibody for 3 hours at 4°C. After adding Protein-A-agarose , the incubation was continued for an additional hour. ACC protein in supernatant was precipitated by 35% saturated ammonium sulfate. The immunoprecipitate and precipitated supernatant were subject to Western blot analysis. Recombinant ACC2.v2 protein was added to the immunoprecipitation reaction as a marker of migration distance. The recombinant ACC2.v2 protein was expressed in FM3A cells and purified with HiTrap chelating column .14CO2 fixation assay which measures the incorporation of [14C]bicarbonate into [14C]malonyl-CoA with a 96-well plate format. Recombinant protein was incubated at 37°C for 40 minutes in a reaction mixture consisting of 50 mM HEPES (pH 7.5), 2 mM DTT, 20 mM MgCl2, 20 mM citrate, 4 mM acetyl-CoA, 5 mM ATP, 4 mM NaHCO3, and 0.08 mM [14C]NaHCO3  at 80 µl/well, and the reaction was terminated by the addition of 20 µl 1 N HC1. Following evaporation of unreacted [14C]NaHCO3, radioactivity in the residue was measured with TopCount Microplate scintillation counter . For kinetic studies, the reaction was run at acetyl-CoA, ATP and NaHCO3 ranging from 3.9 to 2,000 µM, 8.7 to 5,000 µM and 80 to 8,080 µM, respectively. Km values were determined by non-linear regression analysis of initial velocities, according to the standard Michaelis-Menten equation.ACC activity was measured with a standard 2. The Sf9 cells were grown in Grace's Insect Cell Culture Medium  containing 10% fetal bovine serum, 0.1 mg/ml kanamycin, and 0.1% PLURONIC® F-68  at 30° C and 5% CO2. Sf9 cells were collected by centrifugation and resuspended in the culture media at a cell density of 1×107 cells/ml. The Sf9 cells were then incubated with the ACC1 expression virus stock solution (moi = approximately 5) for 1 hour at room temperature. The infected Sf9 cells were diluted with the culture media to cell density of 1×106 cells, and then cultured for 3 days. The recombinant ACC proteins were purified with HiTrap chelating column .The FM3A cells expressing ACC2.v1 and ACC2.v2 were cultured in RPMI 1640, 10% fetal bovine serum, penicillin/streptomycin, and 0.5 mg/ml G418 at 37° C. and 5% CO14C]acetate into the lipids. FM3A cells were suspended in culture medium  in at the density of 0.1×106 cell/50 µl and incubated at 37°C for 1 hour. After the incubation, 50 µl of culture medium were added to the cells and incubated another 1 hour in the presence or absence of 10 µM of a specific dual ACC1 and ACC2 inhibitor, labeled “compound A”. 18.5 kBq of [2-14C]acetate  in 10 µl of culture medium was then added to the cells and the cells were incubated for additional 1 hour. At the end of the incubation, cells were harvested onto 96 well Unifilter GF/C  using Unifilter 96 Harvester and washed with 4 times with ice-cold PBS2−. After dry up of the plate, 30 µl/well of MicroScint 0 scintillant  were added to the plate and then radioactivity in the insoluble lipid fraction were counted by TopCount Microplate scintillation counter .DNL activity in FM3A cells was determined by whole-cell incorporation of [Figure S1Domains in the ACC protein structure (0.03 MB PPT)Click here for additional data file.Figure S2Alignment of the rat and human genomes at the novel exon and the start of the second exon . There are no inserts or deletions after the ‘ATG’ start codon (purple) but many before this start site, suggesting that the protein (underlined) does not extend in the upstream direction of this ATG.(0.02 MB PPT)Click here for additional data file.Figure S3Detection of endogenous ACC2.v2 protein in human adipose samples. Supernatant and immunoprecipitate from a human adipose lysate was probed with streptavidin (anti-biotin) and with an anti-ACC2.v2 antibody Click here for additional data file.Table S1TaqMan probe sequences(0.04 MB DOC)Click here for additional data file.Table S2Antibodies used(0.03 MB DOC)Click here for additional data file.


Arabidopsis, different types of induced resistance have been defined based on the signaling pathways involved, particularly those dependent on salicylic acid (SA) and/or jasmonic acid (JA).Upon appropriate stimulation, plants increase their level of resistance against future pathogen attack. This phenomenon, known as induced resistance, presents an adaptive advantage due to its reduced fitness costs and its systemic and broad-spectrum nature. In ocp3 plants are impaired in a Pseudomonas fluorescens WCS417r-triggered induced systemic resistance (ISR) against both Pseudomonas syrinagae DC3000 and Hyaloperonospora arabidopsidis, and we show that this impairment is not due to a defect in JA-perception. Likewise, exogenous application of JA failed to induce defenses in ocp3 plants. In addition, we provide evidence showing that the over-expression of an engineered cytosolic isoform of the disease resistance regulator NPR1 restores the impaired JA-induced disease resistance in ocp3 plants.Here, we have assessed the implication of the transcriptional regulator OCP3 in SA- and JA-dependent induced defenses. Through a series of double mutant analyses, we conclude that SA-dependent defense signaling does not require OCP3. However, we found that Our findings point to a model in which OCP3 may modulate the nucleocytosolic function of NPR1 in the regulation of JA-dependent induced defense responses. Arabidopsis mutants that fail to produce, accumulate or perceive SA show enhance susceptibility to biotrophs. Likewise, mutations that disrupt JA signaling result in enhanced susceptibility to necrotrophic pathogens . When tiewed by ,10).vice versa. Indeed, many examples of trade-offs between SA-dependent resistance against biotrophic pathogens and JA-dependent defense against necrotrophic pathogens have been reported [PDF1.2 and VSP2 marker genes and several genes of the octadecanoid biosynthesis pathway have been identified as targets of the SA-mediated suppression of JA-responsive gene transcription [In nature, plants often deal with simultaneous invasion by multiple aggressors, which can influence the primary induced defense response of the host plant -13. Therreported . The JA-cription ,19. Thiscription ,21. HoweArabidopsis, two forms of biologically induced disease resistance have been characterized: systemic acquired resistance (SAR), which is triggered upon infection by a necrotizing pathogen; and induced systemic resistance (ISR), which is triggered by colonization of roots by selected strains of non-pathogenic rhizobacteria [PR) genes and is JA/ET-independent [Arabidopsis) against a wide range of pathogens, little is known about its molecular basis [Arabidopsis, analysis of local and systemic levels of JA and ET revealed that ISR is not associated with changes in the production of these two hormones and neither with major changes in transcript or protein profiles [In addition to basal resistance mechanisms that protect plants against virulent pathogens, plants have the ability to develop an enhanced defensive capacity against a broad spectrum of pathogens after stimulation by specific biological or chemical agents. In bacteria -24. SAR ependent ,26. Moreependent  and Attaependent  have preependent . Despitear basis . In Arabprofiles . This suprofiles . It has profiles . The incprofiles -35.Arabidopsis through several genetic screens for SAR-compromised mutants . Subseqiewed by ,36). DurPR genes . Besidesbolished ). Using bolished . In addis mutant . Also SA [npr1-3 , corrobo [npr1-3 ,39-42.PR gene expression . Furthermore, it indicates that these two induced resistance responses are compatible and additive [Most studies have concentrated on unraveling the role of NPR1 in regulating SA-dependent SAR and iewed by ). Howeveadditive . MoreoveArabidopsis ocp3-1 mutant which shows enhanced disease resistance against the necrotrophic fungal pathogens Botrytis cinerea and Plectosphaerella cucumerina, but is not impaired in basal defense against the biotrophs Hyaloperonospora arabidopsidis and Pst DC3000 [PDF1.2 and the redox-sensitive GST1 marker genes. The enhanced disease resistance to necrotrophs of ocp3-1 mutant plants is fully dependent on COI1, a central regulator of JA-signaling [OCP3 gene encodes a homeodomain transcriptional factor which is constitutively expressed in healthy plants but repressed in response to infection by necrotrophic fungi and exogenous applications of MeJA and ABA [Pseudomonas fluorescens WCS417r-triggered ISR and MeJA-induced disease resistance, but not basal defense. In addition we provide evidence pointing at a plausible mechanism by which OCP3 regulation of this process is based on the modulation of a cytosolic NPR1 function.Most of the mutants affected in the response to JA-mediated disease resistance against necrotrophs have opposite effects on SA-mediated disease resistance against biotrophs. This trade-off is generally explained by the antagonistic effect that exists between SA and JA signaling pathways ,21,44. Pt DC3000 . This phignaling -48. The  and ABA ,49. In tOCP3 plays a role in the modulation of SA-dependent defenses, we examined the effect of the ocp3-1 mutation on disease susceptibility in the context of Arabidopsis genotypes that are impaired in their ability to produce, accumulate or perceive SA in response to Pst DC3000 infection. For this purpose we generated a series of double mutants, including ocp3-1 pad4-1, ocp3-1 NahG and ocp3-1 npr1-1 and evaluated the growth of Pst DC3000. PAD4 encodes a lipase-like protein that participates in a positive regulatory loop for increasing SA levels in response to pathogen attack, thereby activating SA-dependent defense responses; the pad4 mutant thus shows enhanced disease susceptibility to biotrophic pathogens [nahG gene (encoding a SA hydroxylase) are unable to accumulate SA and therefore they are also highly susceptible to biotrophic pathogens [NPR1 are compromised in SA perception and similarly show enhanced disease susceptibility to biotrophic pathogens [pad4-1, NahG, and npr1-1 plants show enhanced susceptibility to Pst DC3000 infection, confirming previous findings [ocp3-1 plants did not differ from Col-0 plants in the level of disease susceptibility to Pst DC3000. In the pad4-1, npr1-1, and NahG background, the ocp3-1 mutation did not alter the level of susceptibility to Pst DC3000. These results indicate that, while OCP3 is critical for the regulation of JA-dependent basal defenses against necrotrophs [Pst DC3000.Previously, we demonstrated that OCP3 functions as a negative regulator of JA-dependent disease resistance to necrotrophic fungi . Mutantsathogens . Transgeathogens ,51. Finaathogens . Figure findings ,50-52. Trotrophs , it seemPst DC3000 enters host plants, usually the leaves, through these natural openings and wounds, and then spreads and multiplies to high population densities in intercellular spaces [Pst DC3000 directly in the apoplast, as we did in the experiment shown in Figure ocp3-1 mutation. This thus may mask our interpretation on the results. To study this effect we repeated the experiments and infected Arabidopsis plants by spraying Pst DC3000 onto the leaf surface and subsequently monitored growth of Pst DC3000 , showed no major differences between Col-0 and ocp3-1 plants. However, this is in contrast to what is observed in npr1-1 plants, which are blocked in PR-1 gene expression . Likewit DC3000 ,56,57. Bire NPR1 . To furtin planta was measured after treatment with MeJA, ocp3-1 plants were clearly compromised in their ability to mount an induced resistance against Pst DC3000  or alternatively a fusion of NPR1 with the hormone binding domain of the rat glucocorticoid receptor (named NPR1-HBD) that is impeded in its translocation into the nucleus [NPR1 rendered enhanced basal resistance towards Pst DC3000 (growth in control-treated plants 0.5 log units lower in Col-0 (NPR1-H) in comparison to Col-0). However, this overexpression had no significant effect on MeJA-induced disease resistance , but the MeJA-induced resistance remained blocked in ocp3-1(NPR1-H) plants  to genetically perform crosses with ocp3-1 plants and generated a homozygous ocp3-1 npr1-3(NPR1-HBD) line. As expected, and as shown in Figure npr1-3 plants had no effect on the basal level of resistance to Pst DC3000. This suggests that the NPR1-dependent basal defenses against Pst DC3000 require the nuclear function of NPR1. By contrast, the MeJA-induced resistance against Pst DC3000 was restored in npr1-3(NPR1-HBD) plants  in an ocp3-1 background recovers the protective effect mediated by treatment with MeJA. Consequently, we further investigate this effect by treating transgenic lines with dexamethasone (DEX) which allows NPR1HBD to enter the nuclei. As shown in Figure npr1-3(NPR1HBD) plants respond to MeJA treatment in combination with DEX with a further enhancement of resistance towards Pst DC3000, this effect on further enhancing resistance is not observed in the case of ocp3-1 npr1-3(NPR1HBD) plants. These results point to a model in which the inability of ocp3-1 plants to mount an effective JA-induced defense response, including that controlling ISR, could be explained by a defect in controlling the cytosolic function of NPR1 in regulating induced plant defense responses.Previously, a cytosolic function of NPR1 was suggested to play a role in the regulation of JA-dependent defense responses ,38-42. Ws Figure , indicatArabidopsis homeodomain transcription factor that negatively regulates a branche of the JA signaling pathway that leads to basal defense against necrotrophic pathogens [OCP3 is an athogens ,49.ocp3-1 plants show enhanced disease resistance to necrotrophic fungi [Pst DC3000  or an e facilitates its oligomerization, which maintains protein homeostasis in the cytoplasm upon SA induction. Conversely, the SA-induced NPR1 oligomer-to-monomer reaction is catalyzed by thioredoxins (TRXs). Thus, the regulation of NPR1 functions through the opposing action of GSNO and TRX [Recently, it has been demonstrated that pathogen-triggered redox changes finely regulates NPR1 functions via protein modifications ,63. NPR1ocp3-1 mutant plants constitutively express GST1, a glutathione S-transferase implicated in the protection of oxidative stress during several biotic and abiotic plant stresses [ocp3-1 plants show reduced NO accumulation in response to Pst DC3000 infection . This could be linked to the observation that GSNO, a NO donor, mediates S-nitrosylation of NPR1 to maintain protein homeostasis in the cytoplasm upon SA induction [Our current understanding does not allow us to determine how OCP3 could be modulating the NPR1-mediated JA-dependent activation of defenses. However, it could be the case that OCP3 is regulating specific aspects of the oxidative plant cell status that in turn modulates the cytosolic function of NPR1 required for the activation of JA-dependent induced defenses. In this regard, it has been suggested that OCP3 may be functioning as a specific regulator of the redox homeostasis in plant-pathogen interactions . In factstresses . In addinduction . All theArabidopsis thaliana were kept at 4°C for 3 days and sown in jiffy7 peat pellets  or on a turf substrate mix. Plants were grown in a growth phytochamber with a light intensity of approximately 150-200 μE m-2s-1 at 23°C under 10 h light/14 h dark cycles and 60% humidity.Seeds of mutants and wild-type ocp3-1, pad4-1, and NahG were performed as described [npr1-1 mutant allele, the primers used were (5'-ATGTCTCGAATGTACATAAGGC-3' and 5'-CTCAGTTTCCTAATAGAGAGG-3'). Genomic DNA was extracted from young leaves of Arabidopsis as described [NlaIII (New England Biolabs) resulting in 263, 204, 98 and 16 bp in wild-type and 302, 263 and 16 bp in npr1-1. For the npr1-3 mutant allele the primers used were (5'-AGGCCGACTATGTGTAGAAATACTAGTA-3' and 5'-GCAAGTGCAACTAAACAGTGG-3'). The 245 bp PCR product was digested with RsaI (New England Biolabs) resulting in 218 and 27 bp in wild-type and 245 bp in npr1-3. For the detection of 35S:NPR1 and 35S:NPR1-HBD, the primers used were (5'-AATATCCCGGAGCAATGCAA-3' and 5'-CGGTTGATTTCGATGTGGAAG-3').PCR-based detection of escribed . For theescribed . The 581In double mutant analysis the same phenotype was observed for, at least, two independent double mutant lines generated.ocp3-1 (NPR1-H) and ocp3-1 npr1-3 (NPR1-HBD) lines were generated by crossing ocp3-1 plants (in a Col-0 background) with Col-0 (NPR1-H) and npr1-3 (NPR1-HBD), respectively.4 and adjusted to the indicated OD600  Silwet L77 was used as a surfactant). Five-week-old plants were challenge inoculated and three days later, the bacterial growth was measured. Bars represent the logarithm of colony forming units per mg of fresh weight. Error bars represent standard deviation (n = 8)For preparation of the inoculum, bacteria were streaked out from a -80°C glycerol stock onto a plate of King's medium B supplemented with 100 μg/mL rifampicine and grown for 2 days at 28°C. Bacteria were harvested in 10 mM MgSOInduction treatments with salicylic acid (SA), methyl jasmonate (MeJA) and 1-aminocyclopropane-1-carboxylate (ACC) were performed 3 days before challenge inoculation by spraying the leaves with a solution containing either SA, MeJA or ACC in 0.02% (v/v) Silwet L77. Control-treated plants were sprayed with a solution containing only 0.02% (v/v) Silwet L77. When indicated, plants were spray-treated with 5 μM dexamethasone (DEX) (Sigma).-2 sec-1) and a 16 h night cycle. After 10 days plates were photographed and the primary root length was measured using the free software ImageJ 1.36b (Broken Symmetry software). In each case, 5 plates were measured (30 seedlings/plate).Seeds of Arabidopsis were surface-sterilized for 2 min in 70% ethanol and 5 min in 5% sodium hypochlorite, washed five times with sterilized water. Subsequently, seeds were distributed evenly on square Petri dishes containing 2.2 g/L MS (Duchefa Biochemie), 5 g/L sucrose, 6 g/L Agargel  (pH 5.7). MeJA  was added to the autoclaved medium from a filltersterilized 1 mM stock solution (containing 0.96% ethanol). Seeds were pre-germinated in the dark for 4 days at 4°C. The effect of MeJA on primary root growth was determined essentially as described by . Plates g, room temperature, 3 min), 0.4 mL of the supernatant was added to 0.6 mL of acidic methanol. Absorption of the extracts at wavelengths of 530 and 657 nm was determined photometrically . Quantitation of anthocyanins was performed using the following equation: Q (anthocyanins) = (A530 - 0.25 A657) × M-1, where Q (anthocyanins) is the concentration of anthocyanins, A530 and A657 are the absorptions at the wavelengths indicated, and M is the fresh weight (in grams) of the plant tissue used for extraction. The numbers of samples used for the measurements are indicated in each figure. Error bars indicate the SD of the average anthocyanin contents.Extraction and quantification of anthocyanins was performed in accordance with the protocols of , with miPR-1 (AT2G14610): 5'-ATGAATTTTACTGGCTATTC-3' and 5'-AACCCACATGTTCACGGCGGA-3', PR-2 (AT3G57260): 5'-GCTTCCTTCTTCAACCCCACA-3' and 5'-CTGAACCTTCCTTGAGACGGA-3', PR-5 (AT1G75040): 5'-CTCTTCCTCGTGTTCATCACA-3' and 5'-CATCTACGAGGCTCACATCGT-3', PDF1.2a (AT5G4442): 5'-ATGGCTAAGTTTGCTTCCAT-3' and 5'-ACATGGGACGTAACAGATAC-3', eIF1α (AT5G60390): 5'-GCACAGTCATTGATGCCCCA-3' 5'-CCTCAAGAAGAGTTGGTCCCT-3'. qRT-PCRs were carried out with gene specific primers designed using the Primer Express 2.0 software (Applied Biosystems): PR-1: 5'-AAGGGTTCACAACCAGGCAC-3' and 5'-CACTGCATGGGACCTACGC-3'; PDF1.2a: 5'-CTTGTTCTCTTTGCTGCTTTC-3' and 5'-CATGTTTGGCTCCTTCAAG-3'. qRT-PCRs were performed using the SybrGreen PCR Master Mix (Applied Biosystems) in a ABI PRISM 7000 sequence detector. Cts were obtained using the 7000 System SDS Software Core Application Version 1.2.3 (Applied Biosystems) and the data was transformed with the formula 2^(40-Ct). qRT-PCR and RT-PCR analyses were performed at least three times using sets of cDNA samples from independent experiments.RNA was isolated with Trizol (Invitrogen). For RT-PCR, RevertAid M-MuLV Reverse Transcriptase (Fermentas) was used according to the manufacturer's instructions. The resulting single stranded cDNA was then used as template in semi-quantitative PCR (RT-PCR). RT-PCRs were carried out with gene specific primers designed using the Primer Express 2.0 software (Applied Biosystems)  and the mutant ocp3-1 were sown in quartz sand. Two-week-old seedlings were transferred to 60-mL pots, containing a sand/potting soil mixture that had been autoclaved twice for 20 min. Plants were further cultivated, as described [Pseudomonas fluorescens WCS417r was grown on King's medium B agar plates [4 to a final density of 109 colony-forming units (cfu) per mL. ISR was induced by transplanting 2-week-old Arabidopsis seedlings to soil supplemented with a suspension of WCS417r bacteria to a final density of 5 × 107 cfu/g as described [In these experiments, escribed . For trer plates  for 24 hescribed .H. arabidopsidis WACO9 sporangia were obtained by washing sporulating Col-0 leaves in 10 mM MgSO4, collected by centrifugation, and resuspended in 10 mM MgSO4 to a final density of 5 × 104 sporangia per mL as described [H. arabidopsidis by spraying with 10 mM MgSO4 containing 5 × 104 conidiospores per mL. Inoculated plants were maintained at 17°C and 100% relative humidity. Disease symptoms were scored for about 200 leaves per treatment at 7 days after challenge. Disease was monitored by assessing the rate of colonization and sporulation as described [escribed . Three-wescribed . For detescribed  and scor-1) and boiled about 20 min. After several exchanges of hydrate chloral solution, samples were equilibrated in 50% (w/v) glycerol and observed using a light microscopy.Leaves were plunged in lactophenol trypan blue  and boiled at 95°C for 2-3 min and then incubated at room temperature for 1 h. Samples were transferred into chloral hydrate solution (2.5 g mLP. syringae infection assays, participated in the design of the study. SVE carried out the P. fluorescens WCS417r-triggered ISR bioassays. AC participated in the genetic studies and in the JA assays. CMJP participated in the design of the study and critically revised the manuscript. PV conceived of the study, participated in its design and coordination of experiments. VR and PV wrote the manuscript. All authors read and approved the final manuscript.VR carried out the molecular and genetic studies, the


In Europe, theoretical approaches to physical therapy and rehabilitation in multiple sclerosis often appear significantly different. While there is general agreement that rehabilitation plays an important role in maintaining and improving function in persons with multiple sclerosis, no consensus exists on what may be the most effective approach to achieve the best possible functionality within an individual's limitations.The objective of this paper is to initiate an analysis of currently applied physical interventions for people with multiple sclerosis throughout Europe during inpatient or outpatient rehabilitation programs. A study of the content of rehabilitation may show presently performed treatment methods revealing the basic considerations that nowadays guide clinicians implicitly or explicitly in the treatment of persons with multiple sclerosis. Following this first step, comparative studies can be set up. Neurological abnormalities due to multiple sclerosis (MS) manifest themselves with a wide range of symptoms like fatigue, numbness, paraesthesias, muscular weakness and spasticity, double vision, optic neuritis, ataxia, bladder control problems, dysphagia, dysarthria and cognitive dysfunction. These impairments can lead to relevant problems in carrying out activities of daily living, and participation, too. Therefore, rehabilitation is focused on functional disabilities evolving from the reported symptoms such as balance disorders, gait abnormalities, etc. Symptoms of MS are of different severity and thus cause different problems in every stage of the disease -3.There is no curative treatment available for MS yet. Despite the fact that drug-induced immunosuppression and immunomodulation have been shown to decelerate the inflammatory-related progression of MS -7, thereThe processes that lead to functional recovery after rehabilitation have been intensively debated within the last years. Rehabilitation in general "has moved from professional artistry to an evidence-based scientific approach over the last 15-20 years". This very clear and refined statement by D. Richardson  is true A growing body of neuroscientific knowledge on fundamental aspects underlying rehabilitation has emerged including:⇨ • growing knowledge about neuroplasticity and the ability of the central nervous system to trigger and/or promote reorganization of damaged structures and function,• better understanding of the biochemical factors that promote learning and neural remodelling  in a close relationship with the activation or reactivation of neural cell precursors, responsible for reparative processes,• advances in the understanding of neuropsychological factors, such as the systems of memory, executive function and attention ; increasing knowledge of motor control and motor learning ,11.Rehabilitation in general as well as specific physical interventions have been shown to be effective. In the last decade the number, as well as the quality of published scientific studies and systematic reviews in MS rehabilitation have clearly increased [⇨ ncreased ,12. Studncreased  also stuncreased -20.Contextual changes in the rehabilitation field revealing⇨ • an understanding of the importance of properly classifying and recognizing the different health problems that a patient is confronted with, using the International Classification of Functioning, Disability and Health (ICF), a globally-agreed-upon framework of the World Health Organization. It was recently recommended that clinical practice in MS, including rehabilitation, should be based on this classification system which relates the typical spectrum of problems in functioning of PwMS with their personal attitudes and the environmental context in which they live . This ap• the awareness of the necessity of implementing evidence-based knowledge and practice  into rehabilitation,• an increasing interest in Health-related Quality of Life as an essential outcome measure for treatment ,25.Increased political attention towards high-quality rehabilitation for MS and equal chances of access. Rehabilitation in chronically disabled people like PwMS has gained growing attention. This is one of the reasons why the European MS Platform (EMSP) developed and published the "Code of Good Practice" claiming that all PwMS throughout Europe should have "equal rights and access to treatment, therapies and services in the management of Multiple Sclerosis" [⇨ lerosis" . This doFirst, MS is an individually variable and unpredictable disease needing evaluation at different assessment levels , including patient-reported outcome measures. Unfortunately, both researchers and therapists currently use too many different measures or do not address all levels of patient/therapist perspectives, thus making it difficult to directly compare the effectiveness of interventions (about 2600 articles have been found in PubMed about outcome measures in MS). Interestingly, regional differences in using specific outcome measures in MS across Europe were reported in a study of Haigh et al. . The AutSecond, even though an increasing number of studies have been published in the field of physical rehabilitation , there is still restricted conclusive scientific evidence for the efficacy of treatment interventions in MS. This is related not only to the use of different outcome measures or limited sample sizes, but also to the variety in patient characteristics , treatment goals, treatment setting , duration and intensity ("dosage") of treatment as well as time points of measurements  ,19,34-38Third, it is widely agreed that physical rehabilitation includes a variety of techniques and conceptual treatment methods that are not yet studied by rigorous scientific methods but nevertheless may be of value. For example the effectiveness of Vojta reflex locomotion, Proprioceptive neuromuscular facilitation or Perfetti concept and other generally known and accredited methods, has not been scientifically confirmed in MS (no publication was found in PubMed about the above mentioned methods in MS). Education, culture, history and the way of philosophical thinking (how the patient is being perceived); focus on symptomatic or facilitation or task-oriented intervention ) have leFourth, different Health Systems and Policies  have an impact on rehabilitation and physiotherapy approaches and methods. Also the educational systems and highest levels in physiotherapy  are still different in European countries including the access to postgraduate education ,41. ThisFifth, the gap between what is known about effective health services and what is done in real-world practice exists. Deficiencies in the adoption of new strategies and findings in clinical practice were found . It is iIn the last 60 years a great variety of techniques and conceptual treatment methods have been proposed and applied in the clinical field. Some methods have already been used since the 1950 s  and are still in use . OriginaAll methods have in common that they apply internal and external stimuli to achieve better movement, with the aim of improving activities of daily living. The facilitation approach puts the accent on manual application of stimuli  with the aim to facilitate and improve a given motor function, movement pattern or to start a locomotion program, while the quality of execution is carefully controlled . The tasThree main (physio-)therapeutic approaches based on models of motor control are being used and discussed nowadays :▪ muscle re-education, e.g. bio-feedback, aerobic training, and muscle strengthening,▪ neurotherapeutic facilitation, e.g. Vojta reflex locomotion, Brunnström, Rood, Bobath, proprioceptive neuromuscular facilitation, and the▪ task-oriented approach, e.g. Petö concept, Constraint-Induced Movement Therapy, Motor Relearning Programme, "contemporary" (modified) Bobath concept, locomotor training and Dual Tasking methods.The theoretical bases of the different models are partially overlapping and cannot in all cases be strictly separated from each other. In the physiotherapeutic practice it is usually very difficult to define the approach used. Physiotherapists are led by their clinical experience and intuition on the one hand and their knowledge of evidence based medicine on the other. They sometimes combine different therapeutic approaches based on different theoretical models, everybody with the same aim to ameliorate functionality, participation and well-being of PwMS. Nevertheless, it is obvious that the interventions differ not only in content and terminology used, but also in their definitions of content of treatment, partial aims and understanding of therapeutic principles.http://www.rims.be aims at starting an inventory of content of physical rehabilitation both on the level of (i) therapeutic content/philosophy/terminology and (ii) documentation of organisation of care .A Special Interest Group on Mobility, part of RIMS The inventory may be not only a stock-check of the actual MS rehabilitation practice across Europe but may also serve as a basis for further (comparative) scientific work to put MS- rehabilitation on a higher and more widely applied level and so fulfil some of the demands of the Code of Good Practice .The authors declare that they have no competing interests.Each author has participated sufficiently in the work to take public responsibility for appropriate portions of the content. All authors have read the final manuscript.


CONSTITUTIVE EXPRESSOR OF PATHOGENESIS-RELATED GENES5 (CPR5) gene has been previously implicated in disease resistance, cell proliferation, cell death, and sugar sensing, and encodes a putative membrane protein of unknown biochemical function. Trichome development is also affected in cpr5 plants, which have leaf trichomes that are reduced in size and branch number.The Arabidopsis thaliana CPR5 in trichome development was examined. Trichomes on cpr5 mutants had reduced birefringence, suggesting a difference in cell wall structure between cpr5 and wild-type trichomes. Consistent with this, leaf cell walls of cpr5 plants contained significantly less paracrystalline cellulose and had an altered wall carbohydrate composition. We also found that the effects of cpr5 on trichome size and endoreplication of trichome nuclear DNA were epistatic to the effects of mutations in triptychon (try) or overexpression of GLABRA3, indicating that these trichome developmental regulators are dependant on CPR5 function for their effects on trichome expansion and endoreplication.In the work presented here, the role of CPR5 is unlikely to be a specific regulator of pathogen response pathways or senescence, but rather functions either in cell wall biogenesis or in multiple cell signaling or transcription response pathways.Our results suggest that  CONSTITUTIVE EXPRESSOR OF PATHOGENESIS-RELATED GENES5 (CPR5) gene of Arabidopsis thaliana are highly pleiotropic, affecting pathogen responses, cell proliferation, cell expansion, and senescence. The gene was initially identified based on the constitutive pathogen response phenotype of the mutants [NPR1-dependent disease resistance [CPR5 activates pathogenesis-related (PR) gene expression in the RPS2-mediated pathway and not the RPM1-mediated pathway. However, CPR5 appears to play a broader role in plant growth and development as well, because cpr5 mutants exhibit defects in cell proliferation and cell expansion, and the gene has been hypothesized to play a role in programmed cell death [cpr5 mutants are hyper-responsive to glucose and sucrose and prematurely accumulate senescence-regulated transcripts [CPR5 gene encodes a putative membrane protein with five putative transmembrane domains at the carboxy-terminus, a putative bipartite nuclear localization signal at the amino-terminus, and no sequence similarity to other known proteins [Mutations in the  mutants ,2, and all death . In addinscripts . The CPRproteins ,4.cpr5 mutations affect trichome morphology. The trichomes on Arabidopsis leaves are specialized single cells that project from the epidermis, and in wild-type they have an unusual branched shape. In addition, wild-type trichomes replicate their DNA without division during development in a process called endoreplication or endoreduplication, reaching nuclear DNA levels of 16C-32C [cpr5 mutants are smaller and less branched than those of wild-type, and have a lower nuclear DNA content [CPR5 may play a more specific role in trichome development.In contrast to other constitutive pathogen response mutants, Arabidopsis trichomes are used as a model of plant cell differentiation and cell biology ,8, and tgl3 loss-of-function mutants, and these mutants have smaller trichomes with reduced branching, while try mutants have increased levels of trichome endoreplication, and increase trichome size and branching [gl3-sst allele also have large, extra-branched trichomes with enlarged nuclei indicative of an increased DNA content [Several mutants affect endoreplication levels in trichomes, and these mutants reveal that nuclear DNA content, trichome size, and trichome branching are highly correlated, with mutants resulting in higher DNA contents generally having trichomes that are larger and more branched ,10,11. Aranching . Trichom content . These ocpr5 mutants have altered cell walls with a reduced cellulose content in leaves as well as in trichomes, a previously unrecognized aspect of the phenotype. We also find that cpr5 mutations are epistatic to the extra expansion of trichome cells conditioned by either GL3 gain-of function and try loss-of-function. The cpr5 mutations also increase the number of adjacent trichomes formed due to failure of lateral inhibition signaling in try mutants. Our work indicates that the pleiotropy of cpr5 mutants is due to a primary role for the gene product in a general cellular process such as cell wall biogenesis or integrity that impinges on many cellular pathways, rather than a specific role in pathogen response signaling or senescence.To gain insight into the function of CPR5, we examined the role of this gene in the context of the well-understood pathway for trichome development. Here, we show that cpr5-1 [cpr5-2 [cpr5 mutant plants was smaller than wild-type, cotyledons of cpr5 plants senesce sooner than those of wild-type, lesions are present on cpr5 rosette leaves, and both leaf epidermal cell size and cell number were greatly reduced in comparison with wild-type [cpr5-1 plants have a more severe mutant phenotype than cpr5-2 mutant plants. The cpr5-1 mutation is a missense mutation in the fourth exon (G420D), and the cpr5-2 mutation creates a premature stop in the fourth exon at codon 477. Of greatest relevance to this work, trichome branching and size were reduced in plants homozygous for either cpr5 allele , reduced birefringence of trichome cell walls was associated with reduced paracrystalline cellulose in leaves [cpr5-1 rosette leaves (p < 0.002), with walls of the mutant containing approximately 38% of the paracrystalline cellulose of wild-type walls , which averaged nearly three trichomes per cluster, compared to two trichomes per cluster in the try-JC single mutant  had large, highly birefringent trichomes with increased branching , accelerated cell death2 (acd2), and accelerated cell death6 (acd6), and these mutants appeared to have normal birefringent trichome cell walls . Similarly, gl3 loss-of-function mutants, which have reduced endoreplication, produce normally birefringent trichome walls [proGL2:ICK1/KRP1, a construct that induces programmed cell death in trichomes [CPR5 gene product is involved in some general process that is indirectly necessary for trichome cell expansion, pathogen response signaling, and suppressing premature senescence and programmed cell death, rather than being a specialized component of any one of these processes.It is difficult to reconcile our results with the specific roles that have been proposed by others as the most fundamental function of the richomes , also apCPR5 gene product suggested by our data is the cell wall itself. The cell wall is directly involved in several processes related to the cpr5 mutant phenotype, including both cell expansion and pathogen responses [cev1 mutant of Arabidopsis, which has a mutation in the CES3A cellulose synthase gene, was also shown to result in constitutive expression of pathogen response genes and to have enhanced pathogen resistance [rsw1, also results in activation of jasmonic acid signaling, and cellulose biosynthesis inhibitors can mimic the cev1 mutant phenotype in wild-type plants, including the activation of pathogen response genes [cpr5 mutants [cpr5 mutants are comparable in severity to those seen in cellulose synthase mutants [An attractive locus of action for the esponses . Recentlesponses . A cev1 sistance . This muse genes . Jasmoni mutants . And, no mutants ,21. Inde mutants cpr5 mutant phenotype, including the constitutive pathogen response signaling for which it is named. One aspect of the phenotype that is less obviously coupled to the cell wall is the reduced endoreplication seen in cpr5 [Taken together, these results demonstrate that defects in the cell wall itself can lead to many of the specific aspects of the  in cpr5 . However in cpr5 , and it cpr5 mutant phenotype that is the CPR5 protein may be required directly for the function of multiple transcription factors involved in a wide range of distinct processes. Our observation that cpr5 is epistatic to the phenotypes conditioned by try loss-of-function or GL3 overexpression indicates that CPR5 function is required for the cell expansion and increased endoreplication conditioned by these two transcription factors. The CPR5 gene product is predicted to be a Type IIIa membrane protein with five transmembrane domains and a cytoplasmic N-terminus. This N-terminal domain contains a bipartite nuclear localization sequence (NLS), and it has been proposed that the protein may be involved in a signaling cascade in which the cytoplasmic domain is proteolytically cleaved and transported into the nucleus [An alternative model to explain the extreme pleiotropy of the  nucleus ,24. An a nucleus . In this5 in regulation of nuclear transcription, can provide an explanation for the reduced lateral inhibition  seen in the cpr5 try double mutants . Nevertheless, the work presented here suggests that the cell wall may be a unifying locus for CPR5 function, and will provide guidance for further studies. The important role of CPR5 in multiple essential aspects of plant growth and development merits further work to unravel the mechanism of CPR5 function.It is obvious that testing these models will require biochemical analysis of localization and function of the CPR5 protein. Perhaps because it is a membrane protein, little progress has been reported on this front, and our own attempts in this regard have not been fruitful. For example, fluorescent protein fusions to the cpr5-1 allele was obtained from Dr. Xinnian Dong [cpr5-2 derives from our previous work [try-JC allele has been described previously [try-5C" allele. The identity of the cpr5 try-JC double mutants was confirmed by the failure of the double mutants to complement either parent mutant after crossing. The early senescence of cpr5 cotyledons was maintained in the double mutants and aided in identifying them. The proGL2:GL3 construct has been previously described [Plants were grown under constant illumination as described previously. All alleles originated in the Columbia ecotype, which was used as the wild-type for these studies, and all alleles had been backcrossed to Columbia at least twice prior to use in this work. The eviously ,26; in teviously , it is mescribed .For carbohydrate analysis, uncrowded plants just prior to bolting were placed in the dark for 24–48 hours to reduce the amount of starch in the leaves. The paracrystaline cellulose content of ethanol-washed cell walls (three washes of 70% ethanol at 70°C) of rosette leaves was determined by the method of Updegraff , using cFor analysis of non-cellulosic wall monosaccharides, cell wall material was prepared by grinding leaf tissue in 80% ethanol, washing residue with 80% ethanol, then 100% ethanol, treating residue for 30 minutes with methanol:chloroform (1:1 v/v), washing residue with acetone, and air drying the residue. Further preparation and monosaccharide composition analysis was provided by the Complex Carbohydrate Research Center at the University of Georgia, Athens, GA. This included hydrolysis using freshly prepared 1 M methanolic-HCl for 16 hours at 80°C and derivatization of the released sugars with Tri-Sil. The samples were analyzed by GC using a Supelco column. Myo-inositol was added as an internal standard.4) for 1 hour. After staining with 1% uranyl acetate for 1 hour, the materials were dehydrated in an ethanol series and embedded in LR White resin (medium grade). Thin sections (70–90 nm) were stained with lead citrate, and observed and photographed with a JEOL 100 X transmission electron microscope.Samples fixed in FAA  were prepared for scanning electron microscopy by standard methods, as described previously . For TEMNuclear DNA contents were measured and normalized to a level of 32C for wild-type trichome nuclei essentially as described previously ,29, excecpr5 alleles, generated the cpr5 try double mutants, carried out the cell wall biochemistry, did much of the electron microscopy, prepared several figures, and drafted the manuscript. RK did the scanning electron microscopy of the pGL2:GL3-containing lines and contributed to the DNA content determinations. LAS did the DNA content determinations and statistical analysis, and prepared the final versions of the figures. VK constructed the pGL2:GL3 cpr5-1 line and did the initial analysis on this line, and contributed to drafting the manuscript. MH was involved in the design of initial studies with the pGL2:GL3 cpr5-1 line and helped with the manuscript. JCL participated in the design of the study, did the work on birefringence of cpr5 trichomes, and helped draft the manuscript. All authors have read and approved the final manuscript.GB backcrossed the


Peripheral arterial disease (PAD) often hinders the cardiac rehabilitation program. The aim of this study was evaluating the relative cost-effectiveness of new rehabilitation strategies which include the diagnosis and treatment of PAD in patients with coronary artery disease (CAD) undergoing cardiac rehabilitation.Best-available evidence was retrieved from literature and combined with primary data from 231 patients.We developed a Markov decision model to compare the following treatment strategies: 1. cardiac rehabilitation only; 2. ankle-brachial index (ABI) if cardiac rehabilitation fails followed by diagnostic work-up and revascularization for PAD if needed; 3. ABI prior to cardiac rehabilitation followed by diagnostic work-up and revascularization for PAD if needed. Quality-adjusted-life years (QALYs), life-time costs (US $), incremental cost-effectiveness ratios (ICER), and gain in net health benefits (NHB) in QALY equivalents were calculated. A threshold willingness-to-pay of $75 000 was used.ABI if cardiac rehabilitation fails was the most favorable strategy with an ICER of $44 251 per QALY gained and an incremental NHB compared to cardiac rehabilitation only of 0.03 QALYs  at a threshold willingness-to-pay of $75 000/QALY. After sensitivity analysis, a combined cardiac and vascular rehabilitation program increased the success rate and would dominate the other two strategies with total lifetime costs of $30 246 a quality-adjusted life expectancy of 3.84 years, and an incremental NHB of 0.06 QALYs  compared to current practice. The results were robust for other different input parameters.ABI measurement if cardiac rehabilitation fails followed by a diagnostic work-up and revascularization for PAD if needed are potentially cost-effective compared to cardiac rehabilitation only. Coronary artery disease (CAD) is the leading cause of mortality and morbidity in the United StatesPatients with CAD, however, frequently have peripheral arterial disease (PAD)(range 19%–42%)The aim of the present study was to evaluate the effectiveness, costs, and relative cost-effectiveness from the societal perspective of new rehabilitation strategies which include the diagnosis and treatment of PAD in patients with CAD undergoing cardiac rehabilitation.A Markov decision model was developed to compare current cardiac rehabilitation with new rehabilitation strategies for patients with CADThe strategies in the model were 1. Cardiac rehabilitation only; 2. Cardiac rehabilitation; if rehabilitation fails ABI measured at rest and post exercise and if needed a diagnostic work-up and revascularization for PAD, after which cardiac rehabilitation is continued; 3. ABI measured at rest and post exercise and if needed a diagnostic work-up and revascularization for PAD prior to cardiac rehabilitation, after which the rehabilitation is started. In the second strategy, patients also entered a cardiac rehabilitation program but now if patients failed, an ABI measurement at rest and post exercise was performed followed by diagnostic subtraction angiography or magnetic resonance angiography (proportion 1∶1) if PAD was present. Next, the lesion was treated with percutaneous intervention or bypass surgery depending on disease severity and level of disease. Suprainguinal percutaneous transluminal angioplasty (PTA) with stent placement, aorto-iliac grafting, infrainguinal PTA, and femoro-popliteal bypass were modelled as revascularization procedures. After revascularization, some patients had complications or procedural failures and were unable to continue cardiac rehabilitation, whereas most patients continued with their cardiac rehabilitation program.In the third strategy, all patients underwent an ABI measurement at rest and post exercise prior to cardiac rehabilitation. If patients had symptomatic PAD, a diagnostic work-up and revascularization with PTA or bypass surgery was performed prior to cardiac rehabilitation.For each of the three strategies, the model kept track of time and costs spent in one of the following health states: (a) pre-rehabilitation; (b) successful rehabilitation; (c) failed rehabilitation; (d) post non-fatal cardiac event; and (e) cardiac death or non-cardiac death. A Markov cycle tree was updated every 6 months after which patients' clinical status and costs were estimated to model life-time health benefits and costs.Effectiveness and cost data for the model were retrieved from the literature and from primary data collection. In addition to estimates derived from the literature and the hospital database that were included directly in the model, some estimates were recalculated and several assumptions were needed. Hazard rates for fatal- and non-fatal cardiac events during follow-up for patients with and without cardiac rehabilitation, were calculated from probabilities derived from representative studies  which were included in a systematic review of Taylor and colleagues [16].Long-term life expectancy was calculated on the basis of age-and sex-specific mortality rates from standard U.S. life-tables of the general populationThe proportion of patients who failed cardiac rehabilitation due to PAD was based on the ABI measured in a subset of our patient sample and based on data retrieved from the literature In our patient sample, 7 out of 39 patients (18%) failed cardiac rehabilitation due to PAD, whereas of all patients who failed cardiac rehab (44%), there was 40% probability that PAD is the cause of cardiac rehab failure . In strategies that included revascularization for PAD, we assumed that in the majority of patients (95%) revascularization was possible and that 90% of these patients would benefit from treatment. In our patient sample, 7 out of 39 patients (18%) failed cardiac rehabilitation due to PAD, therefore we modelled that 15%  underwent successful revascularization for PAD and subsequently continued their cardiac rehabilitation program. Thus, including the possibility of revascularization for PAD in the cardiac rehabilitation strategy, in total 71%  of all patients completed the cardiac rehabilitation program successfully.Costs included in the model incorporated medical and non-medical costs and were assessed from the societal perspective . MedicalNon-medical costs included transportation costs and patient time costs. Transportation costs included parking costs and mean estimated gasoline costs. Patient time costs were determined by multiplying the hourly wage rate ($18.55/hour) by the number of hours or days spent in the hospitalCosts of cardiac rehabilitation only included scheduled cardiac rehabilitation visits, a stress test, follow-up visits, transportation costs, and patient time costs.To take into account time preference, future costs were discounted at the currently recommended nominal discount rate of 3% per yearQuality-adjusted-life expectancy, life time costs, and incremental cost-effectiveness ratios  were calculated for all three strategies. Strategies were ordered according to increasing effectiveness (QALYs). A strategy was considered dominated if another strategy was both more effective and less costly. A strategy was considered extended dominated if another strategy achieved more effectiveness at a lower incremental cost-effectiveness ratio. After eliminating dominated and extended dominated strategies the incremental cost-effectiveness ratios (ICERs) were calculated as the difference in mean lifetime costs divided by the difference in mean QALYs for each strategy compared to the next best non-dominated strategyFurthermore, we transformed costs and QALYs into one comprehensive outcome measure: the net health benefit (NHB)To explore the effect of uncertainty in our parameter estimates, we performed extensive one-way, two-way, and multi-way sensitivity analysis. Using probabilistic sensitivity analysis (second order Monte Carlo simulation), the uncertainty around the outcomes of the strategies was assessed Acceptability curves were used to express the uncertainty in the ICER from the probabilistic sensitivity analyses. These curves show for each predefined WTP-threshold the probability of cost-effectiveness for the three different strategies.In value of information analysis we determined whether more information from future research is necessary to decrease the remaining uncertaintyThe model was developed in TreeAge .In a two-way sensitivity analysis, we varied the success rate of the cardiac rehabilitation program by assuming that patients entered a combined cardiac and vascular rehabilitation program. In this analysis, we assumed that the success rate of current practice increased by 25%. This assumption was based on an 80% success rate as a result of the vascular component of the combined rehabilitation program in patients who would otherwise fail due to PAD. Thus, in this strategy an additional 14%  completed the program successfully. Therefore, in total 70%  completed the new program successfully; hence, the increase in success rate of 25% . This program would be comparable to the cardiac rehabilitation program, except for the aim of the program which is here both improving maximum walking distance and reaching THR by performing different appropriate exercise modalities. The same equipment will be used, but physical therapists need to be trained in order to know how to perform an individualized exercise prescription for aerobic and resistance training in patients with both CAD and PAD making the program more expensive . The results show, that this combined cardiac and vascular rehabilitation strategy would dominate the other two strategies with total lifetime costs of $30 246, a quality-adjusted life expectancy of 3.84 years and an incremental NHB of 0.06 compared to current practice.In another two-way sensitivity analysis we changed both the “probability that PAD is the cause of cardiac rehabilitation failure” and the WTP value. If the probability would be lower, we expected that fewer patients would benefit from the strategy ABI if cardiac rehabilitation fails. Cardiac rehabilitation only was the preferred strategy below a threshold probability of 0.05 and a WTP value of $50 000 with a NHB of 3.35 QALYs. Doing a diagnostic work-up for PAD in all patients prior to the cardiac rehabilitation program would not be beneficial, which was mainly due to the higher costs of the diagnostic imaging and due to the peri-procedural complications. Multi-way sensitivity analysis demonstrated that for a WTP of $50 000 with a 50% increase in peri-procedural complications, 50% increase that the patient has PAD, and below a threshold of 0.10 that PAD is the cause of cardiac rehabilitation failure, cardiac rehabilitation only was the preferred strategy and performing a diagnostic work-up for PAD prior to cardiac rehabilitation in all patients was dominated by all other strategies.For other parameters, we found that alternative assumptions either did not substantially affect the outcomes or affected all strategies similarly. If we lowered, for example, the original estimated rate of cardiac events after cardiac rehabilitation, the NHBs decreased for all strategies. Furthermore, varying the costs of fatal- and non-fatal cardiac events between 50% and 150% of the original estimates affected all strategies similarly and did not change the results of the NHBs.Probabilistic sensitivity analysis demonstrated an incremental NHB of ABI if rehabilitation fails compared to cardiac rehabilitation only of 0.03 QALYs , which implies considerable uncertainty around the outcome. Measuring the ABI in all patients prior to the rehabilitation program demonstrated a loss in NHB of −0.22 QALYs  with 100% of the distribution below zero implying that this strategy is unlikely to ever be cost-effective compared to cardiac rehabilitation only.In this study, we evaluated whether patients with CAD who currently enter a cardiac rehabilitation program would benefit more from the program if treatment for PAD is considered. The results suggest that a strategy with an ABI measurement if cardiac rehabilitation fails followed by a diagnostic work-up and revascularization for PAD if needed was the most attractive. A strategy that included an ABI measurement in all patients prior to the cardiac rehabilitation program did not increase QALYs compared to cardiac rehabilitation only. In a sensitivity analysis we assumed a combined cardiac and vascular rehabilitation approach in which we increased the success rate and the costs of current practice. This new program is expected to be more expensive but can also potentially prevent additional events in CAD patients during follow-up due to its higher success rate, which would lead to a gain in QALYs.Current rehabilitation programs in the United States and in Western European countries consist of either cardiac rehabilitation for patients with CAD or vascular rehabilitation for patients with PAD. A combined program does not exist. Vascular programs range from hospital-based walking on a treadmill to home-based walking in the community until a mild or moderate level of pain is reached. These programs do not induce patients' target heart rate. We showed that it is attractive to develop a new rehabilitation program in which cardiac rehabilitation and vascular programs are combined, or revascularisation for PAD is considered, to decrease the failure rate of cardiac rehabilitation.Due to continuously escalating medical costs, third-party payers demand evidence of cost-effectiveness and cost-related benefits of health care services and programs. With ABI measurement if cardiac rehabilitation fails followed by a diagnostic work-up and revascularization for PAD if needed, many secondary events can be avoided in patients with CAD. Nevertheless, we must interpret these results with caution because of the remaining uncertainty in our analysis. Future clinical research could reduce the uncertainty and patients could potentially benefit from more precise estimates of test characteristics, costs, and treatment effects. To assess whether the remaining uncertainty justifies future research, we performed a value of information analysis. The large population EVPI of $2.4 billion suggests that a substantial investment in future research would be justified.One of the limitations of this study was that certain assumptions were needed in evaluating the rehabilitation strategies in a Markov model, which may have affected our results. For the assumptions, we specified a broad distribution for this model parameters and performed second order Monte Carlo simulation to select random values from this distribution in order to include this uncertainty. In addition, the available evidence regarding costs and effects was extrapolated over the entire remaining lifetime of patients. To explore how changes would affect the lifetime cost-effectiveness, extensive sensitivity analysis was performed and changing costs or effectiveness affected all strategies similarly. If we assumed, for example in our sensitivity analysis, an increase of the success rate of the cardiac rehabilitation probability by assuming a combined cardiovascular rehabilitation program, this strategy dominated the other two strategies. For many other assumptions, we demonstrated that varying the parameter values did not change the results substantially or changed the results for all strategies similarly while the conclusions remained the same. Another limitation of our study was the small subset of our patient sample who participated in the follow-up ABI measurement to determine the percentage failures due to PAD in our study group. However, the patient characteristics between the responders and non-responders were not significantly different and varying the percentage of failures due to PAD in a sensitivity analysis, we demonstrated that the results remained the same.Cardiac rehabilitation programs remain underused in many countries. For example, in the U.S. only 10 to 20 percent of 2 million eligible patients per year who experienced a myocardial infarction or underwent cardiac revascularization procedures participated in a cardiac rehabilitation programIn conclusion, the results suggest that a more aggressive approach to the diagnosis and treatment of PAD in CAD patients undergoing cardiac rehabilitation is warranted. ABI measurement in patients who fail cardiac rehabilitation followed by a diagnostic work-up for PAD and revascularization if needed, could potentially decrease secondary cardiac events and are likely to be cost-effective compared to cardiac rehabilitation only.


The full set of symposium articles, including recommendations for future research, appear in Archives of Physical Medicine and Rehabilitation.The Rehabilitation Research and Training Center on Measuring Rehabilitation Outcomes and Effectiveness along with academic, professional, provider, accreditor and other organizations, sponsored a 2-day State-of-the-Science of Post-Acute Rehabilitation Symposium in February 2007. The aim of this symposium was to serve as a catalyst for expanded research on postacute care (PAC) rehabilitation so that health policy is founded on a solid evidence base. The goals were to: (1) describe the state of our knowledge regarding utilization, organization and outcomes of postacute rehabilitation settings, (2) identify methodologic and measurement challenges to conducting research, (3) foster the exchange of ideas among researchers, policymakers, industry representatives, funding agency staff, consumers and advocacy groups, and (4) identify critical questions related to setting, delivery, payment and effectiveness of rehabilitation services. Plenary presentation and state-of-the-science summaries were organized around four themes: (1) the need for improved  The growing population of older adults who sustain strokes, hip fractures, joint replacements, and other conditions, Centers for Medicare & Medicaid Services' (CMS) inpatient prospective payment system (PPS), and technical advances in medical and surgical care have led to increasing demand for medical rehabilitation services. Medical rehabilitation provides crucial services that help people with chronic illness and disability learn to live as independently as possible. In inpatient rehabilitation facilities, physician coordinated, multidisciplinary teams focus on reducing impairments, enhancing independence in daily activities and quality of life, and minimizing caregiver burden. As documented in a recent Medicare Payment Advisory Commission (MedPAC) report , the heaOver the past 20 years the cost of postacute services, including postacute rehabilitation services, have grown much faster than overall inflation, reflecting an increased demand for services and growth in number of providers. The U.S. Congress passed a series of laws  intended to reduce Medicare's PAC expenditures by establishing and refining PPSs for rehabilitation hospitals, nursing homes, long-term care hospitals (LTCHs), and home health (HHAs).Changes in payment mechanisms alters providers' incentives and indirectly the organization and availability of PAC. The consequences of payment changes on Medicare beneficiaries' access to high-quality rehabilitation services, independence, and quality of life are unknown. Research on access to, organization of, and the effectiveness of rehabilitation services is needed in order to understand the consequences of new payment mechanisms.Rehabilitation-focused health services research has concentrated on patients' natural recovery in single types of rehabilitation settings – rehabilitation hospitals and units, SNFs, LTCHs, and HHAs. It is often too expensive and unfeasible to evaluate costs and benefits of rehabilitation across sites of care, let alone specific paths of care, such as from hospitals to nursing homes to home. We know that most patients' functional independence improves during rehabilitation, but we know little about the "active ingredients" of rehabilitation and which types of patients are best suited for which setting so that optimal outcomes are achieved at a reasonable cost.Comparing outcomes across postacute settings has been hampered by the lack of a common outcome assessment instrument across settings, or a cross-walk between the instruments used by rehabilitation hospitals, SNFs, LTCHs, and home health agencies (HHAs). Imagine if Maryland's weights and measures differed from California's and Illinois's and Texas's – and we had no way to convert their measures. With only a bit of hyperbole, this is the situation Medicare finds itself in when trying to evaluate the relative effectiveness and cost effectiveness of rehabilitation hospitals, nursing homes, LTCHs, and HHAs.In the absence of scientific evidence and a way to compare outcomes across settings, Medicare has promulgated rules that limit access to IRFs. The so-called "75% rule" and Medicare fiscal intermediaries' "local coverage determinations" are based on expert opinion and on a dearth of scientific evidence. In developing these regulations, Medicare was dependent on anecdotal information. While the 75% rule was written to distinguish rehabilitation hospitals and units from acute care hospitals, Medicare revised inpatient rehabilitation facility (IRF) regulations to require explicit documentation of medical necessity and adopted the 75% rule to limit the types of patients admitted. Beneficiaries' access to rehabilitation services could suffer if the truism that "the absence of evidence of effectiveness does not imply evidence of absence of effectiveness" is not recognized.The need for expanded rehabilitation-focused health services research was addressed during a workshop in 2005 that was sponsored by the National Center for Medical Rehabilitation and Research (NCMRR) within the National Institute of Child Health and Human Development (NICHD) and the CMS [The Rehabilitation Research and Training Center on Measuring Rehabilitation Outcomes and Effectiveness, funded by the National Institute on Disability and Rehabilitation Research (NIDRR), was asked to lead the planning for what became the Symposium on Post-Acute Rehabilitation. The symposium was guided by a planning committee (see Acknowledgments) with representatives from the American Academy of Physical Medicine and Rehabilitation, the American Congress of Rehabilitation Medicine, the Association of Academic Physiatrists, the Foundation for Physical Medicine and Rehabilitation, the American Hospital Association, and the Federation for American Hospitals. The same organizations provided financial support. Major financial and staff support was provided by the American Medical Rehabilitation Providers Association (AMRPA). Additional sponsors included the American Physiatric Education Council, CARF International (formerly the Commission on Accreditation of Rehabilitation Facilities), Casa Colina Centers for Rehabilitation, eRehabData, Fowler Healthcare Associates, HealthSouth Corporation, IT Health Track, Johns Hopkins University Department of Physical Medicine and Rehabilitation, Kessler Institute for Rehabilitation, Moss Rehabilitation Hospital, MetroHealth Rehabilitation Institute of Ohio, the Rehabilitation Institute of Chicago, and UDSMR.The goal for the symposium was to serve as a catalyst for expanded research efforts on PAC rehabilitation so that relevant research can be used as the basis for policy and funding decisions. The planning committee sought to develop an agenda for research that supports an evidence base for PAC rehabilitation, including issues related to measurement and research design, access to PAC rehabilitation services, organization of rehabilitation services, and outcomes attained for beneficiaries of Medicare and other insurers. The objectives were to: (1) describe the current state of our knowledge regarding utilization, organization and outcomes of postacute rehabilitation settings; (2) identify methodologic and measurement challenges to conducting research in this area; (3) foster the exchange of ideas among researchers, policy-makers, industry representatives, funding agency staff, consumers, and members of advocacy groups; and (4) identify critical questions related to setting, delivery, payment, and effectiveness of rehabilitation services that are of the highest priority for investigation.The activities of the symposium were designed to help formulate a research and policy agenda and to stimulate policy discussions, to engage stakeholders who are involved in policy decisions, and to provide emphasis for the need for an evidence base for rational policymaking. Symposium organizers sought balance in perspectives of key stakeholders, including Congress, the CMS and private insurers, providers of rehabilitation services, patients and their advocates, and health service researchers.The planning committee invited research and policy leaders to present plenary and track-specific state-of-the-science summary speakers, and rehabilitation researchers to provide reports on contemporary work funded by AMRPA, the Rehabilitation Research and Training Center and other agencies. The planning committee invited 3 keynote speakers, former Senator Robert Dole; Laurence Wilson, director, Chronic Care Policy Group, CMS; and Steven Tingus, director, NIDRR. Four plenary speakers were invited to address each of the track themes. Articles by Pamela Duncan and Craig Velozo  and discussion leaders  summarized the recommendations which appear as the final report in the series. The remainder of this summary encapsulates key points from the plenary and state-of-the-science presentations followed by the track-specific research recommendations.Patient assessment data are collected in 3 of the 4 PAC settings. SNFs use an instrument called the Minimum Data Set 2.0, HHAs use the Outcome and Assessment Information Set, and IRFs use the Inpatient Rehabilitation Facility Patient Assessment Instrument, which includes the FIM instrument. LTCHs do not have a mandate to use an assessment instrument. Although these instruments include similar items, the item definitions and assessment periods are different. Further, for the functional assessment domain, all 3 instruments were designed with a fixed set of items, regardless of relevance. In their plenary session, Duncan and Velozo  called fResearch priorities suggested by the measurement and methodology track participants included: develop validated measures of rehabilitation treatments; develop stronger cognitive and psychosocial outcome measures; develop long-term outcomes measures; develop robust severity and selection adjusters across the PAC rehabilitation patient population; assess the role of environmental factors on patient outcomes; and continue development of evidence-based treatment guidelines.Buntin  identifiResearch priorities related to access include projecting the PAC needs of the population and determining the range and geographic distribution of existing PAC entities. Research should be directed to understand better how access is influenced by attitudes about family dynamics, social support, and cultural differences, as well as assumptions about the value of improvement for a patient who will not achieve complete independence.Kaplan  describeChan  describeParticipants in the processes of care track suggested that future research include randomized trials that test individual components of PAC care to determine optimal intensity, duration, and frequency of interventions. To overcome the current barriers of conducting research across provider types, the experiences of other health care systems such as the Veteran's Administration and Kaiser Permanente should be examined.Kane  discusseParticipants in the effectiveness group suggested that future research should focus on what kind of treatment, or combination of services, is most effective in achieving specific outcomes for whom across the continuum of care. In addition, better measures of PAC rehabilitation treatments are needed so that key contents or treatments are identified and can be studied systematically and compared across the continuum of PAC. Participants expressed a strong need for a strategic research plan that is shared by payers, providers, research funders, and researchers; a common measurement time period; and collaboration between CMS, National Institutes of Health, the NIDRR, and the research community to provide flexibility within rigorously designed research protocols, because the PPS itself is a primary obstacle to treatment innovation.uSPEQ: Giving Quality a Voice, CARF International's customer feedback service. Keith began his affiliation with Casa Colina in the 1950s. He began volunteering as a clinical psychologist and soon developed a special interest in rehabilitation services, which eventually led him to develop Casa Colina's Research Department. He became a pioneer in the study of rehabilitation outcomes, published extensively, and helped contribute to the development of the industry standard method of assessing functional status. Our accomplishments are a reflection of their dedication and inspiration.Two colleagues who made major contributions to research and policy discussions on rehabilitation outcomes were very much with symposium participants in spirit, though their recent passing leaves us with a major loss. Deborah Wilkerson and Robert Allen Keith made enormous contributions to rehabilitation research. Wilkerson was a researcher, administrator, and national leader on outcomes measurement, rehabilitation services quality, postacute payment policy, and independent living issues. She led the outcomes measurement and performance indicator programs at CARF and spearheaded the research and development for Postacute rehabilitation care is a key component of the health care delivery system, yet we know little about the active ingredients of the rehabilitation process that produce the best outcomes. Well-designed research is needed to develop better measures for case-mix adjustment and outcomes of care. To advance rehabilitation effectiveness research and support the development of evidence-based policies, we must invest in developing new and improve existing measures of patient characteristics, treatment contents, and long-term outcomes. Critical research needs include (1) developing validated measures of rehabilitation interventions and case mix; (2) standardizing PAC measures and timing of routine measurement for payment and quality assurance purposes across sites of care; (3) examining differences in content and processes of care both within facilities of the same type and across types of facilities; (4) identifying patient characteristics that vary by region such as rural and urban mix, cultural characteristics, and provider referral patterns; and (4) implementing a "strategic plan for effectiveness research" that is characterized by collaboration between CMS, federal research funders, researchers, and care sites.The organizers and sponsors of this symposium trust that our goal of catalyzing expanded research on PAC rehabilitation is furthered by the publication of this set of articles. Our nation's health policy requires a solid base founded on compelling evidence. We look forward to the benefits of greater research attention to improved measurement and research design, access to PAC rehabilitation services, organization of rehabilitation services, and outcomes attained for patients, taxpayers, and Medicare and other insurers.Archives of Physical Medicine and Rehabilitation .Supported by the National Institute on Disability and Rehabilitation Research through a Rehabilitation Research and Training Center on Measuring Rehabilitation Outcomes and Effectiveness (grant no. H133B040032).No commercial party having a direct financial interest in the results of the research supporting this article has or will confer a benefit upon the author or upon any organization with which the author is associated.