The expression of three epidermal growth factor (EGF)-related peptides, transforming growth factor alpha , amphiregulin (AR) and cripto-1 (CR-1), was examined by immunocytochemistry (ICC) in 68 primary infiltrating ductal (IDCs) and infiltrating lobular breast carcinomas (ILCs), and in 23 adjacent non-involved human mammary tissue samples. Within the 68 IDC and ILC specimens, 54 (79%) expressed immunoreactive TGF-alpha, 52 (77%) expressed AR and 56 (82%) expressed CR-1. Cytoplasmic staining was observed with all of the antibodies, and this staining could be eliminated by preabsorption of the antibodies with the appropriate peptide immunogen. Cytoplasmic staining with all of the antibodies was confined to the carcinoma cells, since no specific immunoreactivity could be detected in the surrounding stromal or endothelial cells. In addition to cytoplasmic reactivity, the AR antibody also exhibited nuclear staining in a number of the carcinoma specimens. No significant correlations were found between the percentage of carcinoma cells that were positive for TGF-alpha, AR or CR-1 and oestrogen receptor status, axillary lymph node involvement, histological grade, tumour size, proliferative index, loss of heterozygosity on chromosome 17p or overall patient survival. However, a highly significant inverse correlation was observed between the average percentage of carcinoma cells that expressed AR in individual tumours and the presence of a point-mutated p53 gene. Likewise, a significantly higher percentage of tumour cells in the ILC group expressed AR as compared with the average percentage of tumour cells that expressed AR in the IDC group. Of the 23 adjacent, non-involved breast tissue samples, CR-1 could be detected by ICC in only three (13%), while TGF-alpha was found in six (26%) and AR in ten (43%) of the non-involved breast tissues. These data demonstrate that breast carcinomas express multiple EGF-related peptides and show that the differential expression of CR-1 in malignant breast epithelial cells may serve as a potential tumour marker for breast cancer.


Although 11-ketotestosterone is a potent androgen and induces male secondary sex characteristics in many teleosts, androgen receptors with high binding affinity for 11-ketotestosterone or preferential activation by 11-ketotestosterone have not been identified. So, the mechanism by which 11-ketotestosterone exhibits such high potency remains unclear. Recently we cloned the cDNA of an 11-ketotestosterone regulated protein, spiggin, from three-spined stickleback renal tissue. As spiggin is the only identified gene product regulated by 11-ketotestosterone, the stickleback kidney is ideal for determination of the mechanism of 11-ketotestosterone gene regulation. A single androgen receptor gene with two splicing variants, belonging to the androgen receptor-β subfamily was cloned from stickleback kidney. A high affinity, saturable, single class of androgen specific binding sites, with the characteristics of an androgen receptor, was identified in renal cytosolic and nuclear fractions. Measurement of ligand binding moieties in the cytosolic and nuclear fractions as well as to the recombinant receptor revealed lower affinity for 11-ketotestosterone than for dihydrotestosterone. Treatment with different androgens did not up-regulate androgen receptor mRNA level or increase receptor abundance, suggesting that auto-regulation is not involved in differential ligand activation. However, comparison of the trans-activation potential of the stickleback androgen receptor with the human androgen receptor, in both human HepG2 cells and zebrafish ZFL cells, revealed preferential activation by 11-ketotestosterone of the stickleback receptor, but not of the human receptor. These findings demonstrate the presence of a receptor preferentially activated by 11-ketotestosterone in the three-spined stickleback, so far the only one known in any animal. Androgens have critical physiological roles in male sexual differentiation and in the development of male secondary sex characteristics. Androgens primarily mediate their actions through interactions with receptors belonging to the steroid hormone receptor super-family. Androgen receptors (AR) have been identified in many vertebrates and demonstrate similar binding characteristics in the various species investigated. Most vertebrates have one AR with high specificity for 5α-dihydrotestosterone (DHT). However, some teleosts have two AR with high binding affinities for either testosterone (T) or DHT ,2. 11-keAnguilla japonica), rainbow trout (Oncorhynchus mykiss), tilapia (Oreochromis niloticus) and red seabream (Pagrus major) -KT was equally displaced from stickleback kidney tissue fragments by unlabelled KT or DHT provided no evidence for the presence of a specific KT AR in this species -DHT (92.84 Ci/mmol) and [3H]-KT (98.05 Ci/mmol) were purchased from New England Nuclear  and stored at -20°C. The unlabeled steroids were purchased from either Steraloids, Inc.  or from Sigma Chemical Company . All radiolabelled steroids were stored in 95% ethanol at -20°C. Chemicals and salts used for making the buffers were purchased from Sigma and from Fisher Scientific . The scintillation cocktail was a mixture of 4 L toluene, 16 g PPO , and 0.4 g POPOP .-dCTP radiolabelled AR cDNA fragment (918 base pairs) that was isolated by reverse transcriptase-polymerase chain reaction and sequenced as above, or a randomly primed [α-32P]-dCTP radiolabelled spiggin-α cDNA. Hybridizations were performed at 65°C overnight  SDS, 100 μg ml-1 tRNA, and 5 × Denhardt's solution). The membranes were washed for 2 × 30 minutes at 42°C and 65°C in 0.1 × SCC, 0.1% (w/v) SDS whereafter Hyperfilm™-MP film was exposed at -70°C. The films were visualized using Curix 60 Film Developer. Following AR and spiggin mRNA determination the membranes were stripped and analyzed for 18S rRNA using a cDNA fragment as internal control. Each AR or spiggin transcript was then semi-quantified using Quantity ONE 4.2.3  in relation to its internal 18S transcript.Total RNA was extracted using Tri Reagent™ (Sigma). Aliquots of 10 μg of total RNA were mixed with denaturing solution  formaldehyde) and transferred onto a nylon membrane (Amersham Pharmacia Biotech) using a Minifold II Slot Blot Apparatus . Membranes were probed using either a randomly primed -dCTP radiolabelled partial cDNA encoding stickleback AR, which was isolated and sequenced as described above. Hybridizations were performed at 60°C O/N in 5 × SSC, 0.02% SDS (w/v), 0,1% N-laurylsarcosine (w/v) and 1% blocking solution (w/v) (Roche). Membranes were washed for 2 × 30 min periods at 42°C and 60°C in 0.1 × SSC, 0.1% (w/v) SDS. Membranes were exposed to Hyperfilm™-MP and were visualized as described above.Southern analyses were performed using 20 μg aliquots of male or female stickleback genomic DNA digested with 10 units of either k et al. . MembranHomo sapiens AR (M34233); Mouse, Mus musculus AR (X53779); Rat, Rattus norvegicus AR (M20133); Zebra finch, Taeniopygia guttata AR (AF532914); African clawed toad, Xenopus leavis AR (U67129); Tilapia ARα (AB045211); Tilapia ARβ (AB045212); Burton's mouthbreeder AR, Haplochromis burtoni AR (AF121257); Burton's mouthbreeder ARβ (AY082342); Japanese eel ARα (AB023960); Japanese eel ARβ (AB025361); Red seabream AR (AB017158); Three-spined stickleback ARβ (AY247207); Goldfish AR (AY090897); rainbow trout ARα (AB012095); rainbow trout ARβ (AB012096); Japanese medaka, Oryzias latipes ARα (AB076399). The sequence alignments were performed using Omiga 2.0 (Oxford Molecular Ltd). A phylogenetic tree was constructed using Tree view (Version 1.6.2) -DHT was dried down under nitrogen, re-dissolved in 100 μl ice-cold homogenization buffer and added to assay tubes with or without a 1,000-fold excess of unlabeled DHT. The expressed proteins were diluted 1:100 and aliquots (100 μl) were added to each reaction tube. The tubes were vortexed and incubated over night at 4°C. To stop the binding reaction, 200 μl DCC was added to each tube. The tubes were vortexed and the suspension were incubated for 5 min at 4°C, before spinning at 3,000 × g at 4°C for 5 min. 250 μl of the supernatant was transferred into 5 ml scintillation vials and 4 ml of standard scintillation cocktail was added before the radioactivity in each sample was measured in a liquid scintillation counter .[3H]-DHT (0.42 – 18.2 nM) or [3H]-KT (0.42 – 15.4 nM) was added to each reaction tube with or without a 100-fold excess of unlabeled steroid. For the expressed AR, [3H]-DHT (2.5 – 42 nM) was added to each reaction tube with or without a 1000-fold excess of unlabeled steroid. Nuclear or cytosolic suspensions were incubated with steroids over night at 4°C. The reactions were terminated and the radioactivity measured as described above. Non-linear curve fitting procedures  were used to calculate the dissociation constant (Kd) and to estimate the concentration of binding sites (Bmax) in the different suspensions.[3H]-DHT with or without 400 nM unlabeled steroid. The reaction was terminated at different time-points ranging between 1 min and 24 h. The specific binding at each time-point was determined as described above. In order to investigate the receptor dissociation time, the preparation were incubated over night with 4 nM [3H]-DHT with or without 100-fold excess of unlabelled DHT for determining non-specific binding, before adding 100-fold excess of unlabelled DHT, and binding was subsequently measured between 5 min and 28 h.To investigate the time needed to reach binding equilibrium, cytosolic preparations were incubated in 4 nM [3H]-DHT with or without unlabelled steroid at end concentrations ranging between 1 pM and 100 μM. Steroids used were: KT; DHT; T; E2; 17,20β-dihydroxy-4-pregnen-3-one ; 17,20β,21-trihydroxy-4-pregnen-3-one, (20β-S) and cortisol (F). Following incubation, the reaction was terminated by addition of DCC and centrifugation and the specific binding in each tube was determined as described above.The steroid specificity of receptor binding was examined using a competitive binding assay. Diluted protein suspension (1:100) were incubated over night at 4°C in tubes containing 1 nM [2. The transfection experiments were initiated by replacing the tissue culture medium with fresh phenol-free medium supplemented with charcoal stripped FCS and the cells were seeded on 24-well plates. Transfections were performed at 90–95% confluence using 1,5 μl Lipofectamine 2000  and 0,6 μg DNA per well. Transfected DNA contained 60 ng pRL (Promega), 270 ng androgen response element (ARE) luciferase reporter vector, 270 ng stickleback ARβ2 expression vector or 270 ng human AR expression vector (pCMVhAR) while 270 ng empty TNT cloning vector was used to control background luciferase levels following transfections. Two different ARE containing promoter constructs were tested, one containing 3 separate ARE  and a second one construct (slp-HRE2) containing 4 copies of the ARE with high affinity for R1881, a synthetic androgen, and low affinity for dexamethasone, a synthetic glucocorticoid [Two liver epithelial cell lines were used to test receptor activity. HepG2 cells  were maintained in DMEM supplemented with 10% FCS and non-essential amino acids. ZFL cells (ATCC) were maintained in 50% L-15 Leibovitz, 15% H-12 Ham and 35% DMEM supplemented with 10% FCS  and 50 ng ml-1 EGF (Sigma). Cells were cultured at 37°C (HepG2) or 28°C (ZFL) in 5% COorticoid .Renilla luciferase activity. Each experiment was repeated a minimum of 3 times. The fold-induction is presented as luciferase values normalized against the control. The control levels were arbitrarily set to 1.0 for each cell line.After 16 h the transfection medium was replaced with fresh phenol-free medium, supplemented with charcoal stripped FCS, containing different steroid hormones. The cells were exposed to the hormones for 40 h and luciferase levels were detected using the Dual Luciferase Assay kit (Promega) in a TD 20/20 Luminometer . For each cell line, transfection assay was performed for each concentration with n = 4 and the luciferase value of each assay were normalized to its corresponding t test.All data from semi-quantitative analyses of mRNA are shown as AR in relation to 18S expression and presented as mean ± SD. Significance were determined using ANOVA and Student Implantation of females with 25 μg KA caused a 30-fold increase in spiggin mRNA levels. At this dose, KT was 10 times more effective than DHT and 50 times more effective than T at increasing kidney spiggin mRNA levels , and one shorter sequence (2515 bp) coded for a full length AR, ARβ1 (Gene Bank Accession no AY247207). The ARβ1 sequence contained a 322 bp 5'-UTR (untranslated region) including an in frame stop codon, a 2046 bp coding sequence and a 147 bp 3'-UTR, and the gene coded for a 682 amino acid AR (ARβ1) protein. The ARβ2 sequence differed from ARβ1 in that it contained additional amino acid sequence in the N-terminal region, coding for 735 amino acids and also contained a longer (961 bp) 3'-UTR. The 3'-UTR of both transcripts was identical up to the poly-A tail of the shorter UTR (147 bp), indicating the presence of two poly-adenylation sites in the stickleback AR gene. The sequence of both transcripts exhibited a conserved identity (100%) in overlapping regions at both the nucleotide and protein levels, thus indicating that both were derived from a single locus by alternative splicing. Southern analysis of genomic material from both male and female stickleback demonstrated a hybridization pattern that was compatible with the existence of a single ARβ gene  coded for a partial AR, ARβ2 , or amino acids with possible close contact to DHT , were conserved in the stickleback . An argiin vitro for binding assays and for expression in HepG2 and ZFL cells. S35-Met labeled AR was separated on a SDS-polyacrylamide gel to determine the size of the expressed proteins. From the nucleotide sequence, ARβ1 was estimated to code for a 76.6 kD protein while ARβ2 was estimated to code for a 82.2 kD protein. Both AR migrated as double bands following electrophoresis and the transcript sizes were 52 and 61 kD for ARβ1 and 75 and 87 kD for ARβ2  while both KT and T had lower affinity (42.75 ± 3.42 nM and 68.81 ± 14.45 nM respectively). Binding was also observed for E2 (85.10 ± 17.87 nM), but not for 17,20β-dihydroxy-4-pregnen-3-one , 17,20β,21-trihydroxy-4-pregnen-3-one (20β-S) or F , followed by KT and T (2.18 ± 0.71 nM and 3.03 ± 0.43 nM), while E2 showed lower binding affinity (64.61 ± 0.28 nM) and F showed poor binding .-8 M (P < 0.001 for both cell lines) and 10-6 M (p < 0.05 for both cell lines) steroid. Furthermore, comparison of the stickleback AR with the human AR, in the ZFL cell line, showed that the stickleback AR was preferentially activated by KT while the human AR did not discriminate between androgens . However, no data is available on the function of that receptor. Amino acids L704, M745 and F764 that are considered to have close contact with the ligand in human AR [The stickleback AR shows the greatest similarity to AR from seabream and tilapia, fishes that like the stickleback, but unlike the rainbow trout and eel, belong to Series Percomorpha among the Acanthypterygii . The rechuman AR , are als50: 6 × 10-10 M), for DHT followed by T, while the poorest binding (IC50: 8 × 10-9 M) was observed for KT. In contrast to this, determination of the trans-activation potential using the carp epithelioma papillosum cyprini cell line indicated that the efficiency of activation of the rainbow trout ARα was equal for all three natural androgens [in vitro translated recombinant stickleback ARβ2 (Fig. slp-ARU or the slp-ARE, KT was in both cases approximately 2 to 3-fold more potent than DHT in activating the luciferase reporter gene (Fig. Several previous studies have shown the presence of DHT and T receptors in teleosts, but so far no specific KT receptor has been identified ,2,5-7. Cng IC50:  × 10-9 Mng IC50:  × 10-9 MRβ2 Fig. . Howeverng IC50:  × 10-9 Min vivo [The 3-fold difference in trans-activation potential probably does not entirely explain the observed potency differences between KT and DHT in inducing spiggin in stickleback in vivo . This rein vivo -36, bothin vivo ,35. The in vivo ,13,37.in vivo. As these proteins are not present in the in vitro systems the presently observed differences in ligand dependent transcriptional activation cannot be due to transport proteins.In contrast to estrogen receptors that are auto-regulated by estrogens, AR is not generally up-regulated by androgens and no ARE have been found in the promoter or 5'-flanking region of cloned AR. While AR is generally transcriptionally down regulated by androgens, the protein half-life appears to be increased by androgens ,39. AutoThe present study indicates the presence of a single gene coding for a nuclear AR in the three-spined stickleback. Furthermore, while the results show that the receptor has the highest binding affinity for DHT, it is preferentially activated by KT. While the present study represents the first identification of an AR preferentially regulated by KT, the elucidation of the KT signaling mechanism in teleosts clearly requires further research.


Lolium perenne). We report on the exploitation of a comprehensive EST collection in L. perenne for SSR identification. The objectives of this study were 1) to analyse the frequency, type, and distribution of SSR motifs in ESTs derived from three genotypes of L. perenne, 2) to perform a comparative analysis of SSR motif polymorphisms between allelic sequences, 3) to conduct a comparative analysis of SSR motif polymorphisms between orthologous sequences of L. perenne, Festuca arundinacea, Brachypodium distachyon, and O. sativa, 4) to identify functionally associated EST-SSR markers for application in comparative genomics and breeding.Simple sequence repeat (SSR) markers are highly informative and widely used for genetic and breeding studies in several plant species. They are used for cultivar identification, variety protection, as anchor markers in genetic mapping, and in marker-assisted breeding. Currently, a limited number of SSR markers are publicly available for perennial ryegrass  contained one or more SSRs. Of these SSRs, 955 (3.7%) were non-redundant. Tri-nucleotide repeats were the most abundant type of repeats followed by di- and tetra-nucleotide repeats. The EST-SSRs from the three genotypes were analysed for allelic- and/or genotypic SSR motif polymorphisms. Most of the SSR motifs (97.7%) showed no polymorphisms, whereas 22 EST-SSRs showed allelic- and/or genotypic polymorphisms. All polymorphisms identified were changes in the number of repeat units. Comparative analysis of the L. perenne EST-SSRs with sequences of Festuca arundinacea, Brachypodium distachyon, and Oryza sativa identified 19 clusters of orthologous sequences between these four species. Analysis of the clusters showed that the SSR motif generally is conserved in the closely related species F. arundinacea, but often differs in length of the SSR motif. In contrast, SSR motifs are often lost in the more distant related species B. distachyon and O. sativa.From 25,744 ESTs, representing 8.53 megabases of nucleotide information from three genotypes of L. perenne EST-SSR markers are a valuable resource for genetic mapping, as well as evaluation of co-location between QTLs and functionally associated markers.The results indicate that the  Lolium perenne is one of the major grass species used for turf and forage in the temperate regions of the world. It belongs to the grass family Poaceae. L. perenne (2n = 2x = 14) is taxonomically related to many important plant species in the Poaceae family, including rice (Oryza sativa), wheat (Triticum aestivum L.), barley (Hordeum vulgare L.), maize (Zea mays L.), and sorghum (Sorgum bicolor L.) [L. perenne, including restriction fragment length polymorphism and random amplified polymorphic DNA [L. perenne SSR marker development, most of these markers are currently not publicly available [Poaceae species is based on a limited number of anchor markers [L. perenne.Several anonymous molecular markers have been developed for phic DNA ,3, ampliphic DNA , as wellphic DNA ,6. More phic DNA  have beephic DNA -10. Althvailable ,9. Furth markers , reinforSimple sequence repeats (SSRs) have become one of the most widely used molecular marker systems in plant genetics and breeding. They are widely used for genetic diversity assessment, variety protection, molecular mapping, and marker assisted selection, providing an efficient tool to link phenotypic and genotypic variation -14.SSRs are tandem repeated sequences comprised of mono-, di-, tri-, tetra-, penta-, or hexa-nucleotide units ,16. SSRsThe number of SSR motifs at a locus is variable, because SSRs experience a high rate of reversible length-altering mutations by unequal crossing over and replication slippage, where the transient dissociation of the replicating DNA strand is followed by misaligned re-association ,18. SSRsWhereas earlier studies on SSR marker development primarily utilized anonymous DNA fragments containing SSRs isolated from genomic libraries, more recent studies have used computational methods to detect SSRs in sequence data generated from large-scale EST sequencing projects. About 1 to 5% of ESTs from different plant species have been found to contain SSRs suitable for marker development . EST-SSRThe conservation of grass genomes has been comprehensively documented, and comparative genomics has become an important strategy to extend genetic information from model species to species with a more complex genome, as well as between related species with complex genomes ,32. As EL. perenne for SSR identification. The objectives of this study were 1) to analyse the frequency, type, and distribution of SSR motifs in ESTs derived from three genotypes of L. perenne, 2) to perform a comparative analysis of SSR motif polymorphisms between allelic sequences, 3) to conduct a comparative analysis of SSR motif polymorphisms between orthologous sequences of L. perenne, Festuca arundinacea, Brachypodium distachyon, and O. sativa 4) to identify functionally associated EST-SSR markers for application in comparative genomics and breeding.We report on the exploitation of a comprehensive EST collection in L. perenne cDNA clones from 13 cDNA libraries resulted in 25,744 high-quality ESTs  more tri-repeat than di- and tetra- repeat SSRs, while in the dataset from the genotype NV#20F1-39, there were significantly  more di- and tri- than tetra- repeat SSRs  was observed between genotypes with respect to tri- and tetra- repeat SSRs, while the EST-SSRs derived from the genotype NV#20F1-39 contained significantly  more di-repeat SSRs compared to the EST-SSRs derived from the other two genotypes. The frequency of the SSR motifs (any two complementary sequences considered one motif) are listed in Table In the datasets from the genotypes NV#20F1-30 and F6, there were significantly  between the three genotypes  against 41,834 F. arundinacea EST sequences, 3,818 B. distachyon contigs, and 32,132 full-length O. sativa cDNA sequences, to identify the orthologous sequences of these species. The blast searches resulted in 833, 540, and 26 orthologous sequences of F. arundinacea, B. distachyon, and O. sativa, respectively. A dataset of 19 clusters of sequences containing orthologous sequences from all four species was identified and aligned using ClustalW [An L. perenne and F. arundinacea. The most frequent SSR motif polymorphisms between these two species were changes in the number of repeat units corresponding to 21% of the clusters. However, nucleotide substitutions, additions, and complete loss of SSR motifs were also observed (Table L. perenne was completely conserved in B. distachyon. In six clusters (31%), the SSR motif was completely lost in B. distachyon, and in four clusters (21%) the B. distachyon SSR motif had fewer repeat units. In these four clusters, the B. distachyon SSR motif contained two to three fewer SSR motif units, compared to the corresponding L. perenne SSR motif. Nucleotide substitutions and additions were observed in five (26%) of the nineteen compared orthologous sequences (Table L. perenne was completely conserved in O. sativa. In eight clusters (42%), the SSR motif was completely lost in O. sativa, and in six clusters the O. sativa SSR motif had fewer repeat units compared to the corresponding L. perenne SSR motif. However, in one cluster the O. sativa SSR motif had more repeat units compared to the corresponding L. perenne SSR motif (Table In six of the 19 clusters (31%), there were no polymorphisms at the SSR motif between the sequences of the two closely related species ed Table . None ofin five 2% of the in five 2% of the L. perenne genome by identification of SSRs in a dataset consisting of 25,744 ESTs from three different genotypes. Random sequencing of cDNA libraries leads to a high proportion of redundant ESTs. In this study, both the redundant and non-redundant dataset of EST-SSRs were included in the analysis. The redundant EST-SSRs were used to characterize the frequency of SSR motifs and to compare SSR motif polymorphisms between three genotypes of L. perenne, while the non-redundant dataset was used to characterize the type and distribution of EST-SSRs in the transcribed region of the L. perenne genome, and for a cross-species comparison of SSR polymorphisms within four species of the Poaceae family.The present study was designed to create an SSR database of the transcribed region of the L. perenne ESTs are a valuable resource for SSR marker development. The transcribed region of the genome of the genotype F6 contains a significantly higher frequency of SSRs. Approximately 10% of the ESTs from the genotype F6 contain an SSR, compared to approximately 3.6% in the other two genotypes, indicating a large genotypic variation in the frequency of SSR motifs. To our knowledge, this is the first report where the frequency of SSRs in ESTs from different genotypes within one plant species has been compared. The results suggest that it would be reasonable to generate a small number of ESTs from different genotypes, to decide which one is the best for EST-SSR development.A total number of 1,458 redundant and 955 non-redundant SSRs were identified, corresponding to 5.66 and 3.71% of redundant and non-redundant ESTs, respectively. Preliminary results exemplified in Figure However, the differences observed in the frequencies of SSR motifs might not only be genotypic differences, but also be due to different cDNA libraries established for the three genotypes, because the composition of expressed genes is likely differing between the thirteen cDNA libraries selected for EST development. NV#20F1-30 and NV#20F1-39 are full-sibs , and mosL. perenne genome is within the same range as previously reported for other plant species [The average frequency of 3.71% non-redundant SSRs in the transcribed region of the  species ,23,41-43L. perenne genome are size-restricted. In addition, 6 bp di-repeats comprise 40 to 64% of the di-repeats in the three genotypes, indicating that di-repeats, which do not perturb the open reading frame are preferred over others. The expansion of SSR repeats in transcribed regions of the genome is limited by functional and evolutionary constraints [Approximately 96% of all SSRs analysed were shorter than 21 bp, indicating that the length of SSR motifs in the transcribed region of the straints ,45, becastraints ,46. Shorstraints ,48. The straints .L. perenne, the most common SSR repeat units were also found to be tri-nucleotide repeats, constituting between 59 and 85% of the repeats in the three genotypes included in this study, while di- and tetra-nucleotide units constitute the majority of the remaining motifs. Only a few penta- and hexa-nucleotide repeat units were identified. A wide variety of tri-nucleotide repeat units were represented at high percentages, however, the abundance of the different types of repeat units differed, especially between the genotype F6 and the two other genotypes. The repeat motif (CCG/CGG)n was highly represented in 42% of EST-SSRs from the genotype F6, while it was represented at a low frequency of approximately 1% in the other two genotypes.Previous studies have shown that tri-nucleotide repeats predominate in coding regions of plant genomes ,50, as wL. perenne, while the other amino acid residues were not represented. The presence of SSRs in transcripts of genes suggests that they may have a role in gene expression or function. In O. sativa, the length of a poly(CT) SSR in the 5'-untranslated region of the waxy gene is associated with amylose content [Z. mays a SSR the 5'-untranslated region of some ribosomal genes, have been suggested to be involved in the regulation of fertilization [In the two genotypes NV#20F1-30 and NV#20F1-39 the most abundant repeat encodes for the amino acid threonine, while the most abundant repeat in the genotype F6 encodes for the amino acid proline. Analysis of all protein sequences from the SWISS-PROT database for single amino acid repeats, tandem oligo-peptide repeats, and periodically conserved amino acids showed that repeats of glutamine, serine, glutamic acid, glycine and alanine seems to be fairly well tolerated in many proteins . Of thes content , and in lization .L. perenne genome. In all contigs containing an SSR motif polymorphism, the polymorphisms identified were changes in the number of repeat units, while no contigs were identified with changes in the repeat type or complete loss of the SSR motif. The majority of the SSR polymorphisms were allelic polymorphisms, and most of the SSR motif polymorphisms were one to two repeat unit changes. All polymorphisms identified, except for polymorphisms in compound SSRs, were changes in the number of repeat units, while no single nucleotide additions or deletions were identified, that otherwise would perturb the open reading frame.A total of 22 contigs containing EST sequences with either allelic- and/or genotypic SSR polymorphisms were identified, corresponding to 2.3% of the non-redundant EST-SSR contigs. The remaining 499 contigs (97.7%) contained no SSR motif polymorphism, indicating a selection against length polymorphisms in the transcribed region of the O. sativa genome sequence provides a rich source of molecular information [O. sativa by applying this knowledge to less studied forage and turf species.Several studies have shown that SSRs developed for one species could be used in related plant species, and that the success of cross-species amplification depends on the evolutionary relatedness . The avaormation . On the L. perenne SSR markers between species of the Poaceae family were performed in silico, to evaluate if the SSRs can be used as anchor markers for comparative mapping and evolutionary studies. SSRs designed from EST sequences are especially valuable owing to their genome location, which implies constraints on length, motif, abundance and flanking regions, the latter of particular interest in this context, because common primers can be designed to conserved flanking regions. However, before primers are designed it is necessary to evaluate if the SSR motif is conserved between related species, and therefore useful for SSR marker development. Blast searches using the 955 non-redundant Lolium perenne EST-SSRs as query sequences against 41,834 F. arundinacea EST sequences, 3,818 B. distachyon contigs, and 32,132 full-length O. sativa cDNA sequences resulted in 833, 540, and 26 orthologous sequences, respectively. However, because the amount of sequence information available differs between the species included in this study, the number of hits cannot be directly compared. A total of 19 clusters were identified containing sequences of all four species. Analysis of the clusters indicates that the SSR motif in general is conserved in the closely related species F. arundinacea apart from differences in the length of the SSR motif. In contrast, the SSR motif is often lost in the more distant related species B. distachyon and O. sativa.The transferability of the F. arundinacea across multiple grass species was investigated [F. arundinacea genomic SSRs were used to screen the six species; F. arundinacea,F. arundinacea var. Glaucescens (tetraploid), F. pratensis, L. perenne, O. sativa, and Triticum aestivum, representing three tribes and two subfamilies of the Poaceae family. Most SSRs could be amplified in all forage and turf grasses but not in cereal species included in that study [L. perenne and F. arundinacea, compared to B. distachyon, and O. sativa.In a previous study, the transferability of genomic SSR markers developed for stigated . A totalat study . These rin silico analysis of SSR motif polymorphisms between the species included in this study. However, the in silico analysis of the conservation of SSR motifs across species is a valuable tool, because it gives an indication of how distant related species can be, when experiments for comparative mapping and evolutionary studies are designed. Furthermore, the results are valuable for estimating how large the chance is, to find SSR motifs as prerequisite for a polymorphic marker, in closely- as well as distant related species.Experimental validation of these hypothetical transferable SSRs and their polymorphism is needed, to validate the results of the L. perenne EST-SSRs presented in this paper, a valuable tool has been developed for further genetic-, genomic-, and plant breeding applications on the intra- as well as on the inter-species level.With the Lolium perenne, one of the major grass species used for turf and forage in the temperate regions.In this study, we present a comprehensive set of publicly available EST-derived SSRs from three genotypes of in silico using clustered and assembled EST data. Tri-nucleotide repeats were the most abundant type of repeats followed by di- and tetra-nucleotide repeats. Approximately 96% of all SSRs identified were shorter than 21 bp, indicating that the length of SSR motifs in the transcribed region of the L. perenne genome are size-restricted.A total of 955 non-redundant SSRs were detected A large variation in the number of SSRs in transcribed regions of the three genotypes was observed, ranging from one SSR per 10.9 kb in genotype NV#20F1-30 to one SSR per 2.7 kb in the genotype F6. This result suggests that several genotypes should be screened to find the best genotype for SSR discovery in transcribed sequences.L. perenne were changes in the number of repeat units. When comparing SSR motifs from L. perenne to SSR motifs in orthologous sequences from F. arundinacea, B. distachyon, and O. sativa changes both in the number of repeats, and complete loss of the SSR motifs were observed. Comparing orthologous sequences of L. perenne and F. arundinacea revealed that the most frequent SSR motif polymorphisms between these two species were changes in the number of repeat units corresponding to 21% of the clusters, while there were no SSR polymorphisms in 31% of the analysed clusters. Thus, the EST-SSRs are suitable for synteny studies between these two species.All allelic SSR polymorphisms identified within L. perenne was completely conserved in the more distant related species B. distachyon and O. sativa. In 31% of the clusters the SSR motif was completely lost in B. distachyon, and in 21% the SSR motif had fewer repeat units. This suggests that the EST-SSRs are less suitable for synteny studies outside the Lolium/Festuca complex.In contrast, none of the SSR motifs identified in With the EST-SSR set, a valuable tool has been made publicly available for numerous further genetic and genomic applications on intra- and inter-species level.L. perenne genotypes: NV#20F1-30, NV#20F1-39 [Thirteen directional cDNA libraries were constructed from a range of tissues and developmental stages Table . Tissues#20F1-39 , and F6 ® Reagent , and the cDNA libraries were constructed using the Creator™ SMART™ cDNA Library Construction Kit , according to the manufacturer's instructions. The cDNAs were cloned directionally into the asymmetric SfiI sites of the pDNR-LIB vector, transformed into electrocompetent DH10B T1-phage-resistant Escherichia coli cells , and robotically arrayed into 384-well plates. A total of 31,379 random clones were subjected to single-pass sequencing reactions from the 5'end using BigDye® Terminator v3.1 sequencing chemistry and analyzed on an ABI Prism 3700 DNA Analyzer . Colony picking and sequencing was performed by MWG Biotech . Base calling, vector trimming, removal of low quality bases, and clustering and assembly of the ESTs were performed using the PHRED and PHRAP/CROSS_MATCH software packages [RNA was isolated using Tripackages -62. SequAn EST database was developed consisting of 25,744 ESTs corresponding to 8.53 Mb of sequence . Protein functions were predicted by BlastX similarity searches against the protein database in the GenBank , and annL. perenne EST sequences. The parameters for the SSR search were defined as follows. The size of motifs was two to six nucleotides, and the minimum repeat unit was defined as six for di-nucleotides and four for tri-, tetra-, penta-, and hexa-nucleotides. Compound SSRs were defined as ≥ 2 SSRs interrupted by ≤ 50 bases.The Perl script MIcroSAtelitte (MISA)  was usedL. perenne is a diploid (2n = 2x = 14) outbreeding species with self-incompatibility being controlled by two genetic loci. A maximum number of two alleles can therefore be expected in each genotype. The 3,195 L. perenne contigs was queried using MISA to identify SSR containing contigs. The individual sequences within each SSR containing contig was subsequently analysed for SSRs using MISA to identify allelic and/or genotypic SSR motif polymorphisms.L. perenne, F. arundinacea, O. sativa, and B. distachyon. A total of 41,834 F. arundinacea ESTs were downloaded from dbEST in the GenBank [O. sativa full-length sequences were downloaded from KOME [B. distachyon contigs were downloaded from the Genomics and Gene Discovery bEST Resource home page [L. perenne ESTs containing SSRs, to identify the orthologous sequences. A relational database was created and used to store all information related to the DNA sequences of the four species, including DNA sequences, similarity search results, query search results, SSR presence, SSR motif type, and SSR locus polymorphisms between the four species included in this study.The cross-species SSR motif polymorphism analysis was performed by comparing orthologous sequences of  GenBank , 32,132 rom KOME , and 3,8ome page . The seqSequences described have been submitted to GenBank. Submitted sequences are in the accession number range of ES699013 to ES700454.TA and TD constructed the cDNA libraries for EST sequencing. TA and UKF conducted the bioinformatic analysis. TA, KKN, and TL designed and coordinated the study. TA interpreted the data, performed the statistical analysis, and drafted the manuscript. TL assisted in drafting the manuscript. All authors read and approved the final manuscript.


Acute exacerbations of chronic obstructive pulmonary disease (COPD) represent a major burden for patients and health care systems. Respiratory rehabilitation may improve prognosis in these patients by addressing relevant risk factors for exacerbations such as low exercise capacity. To study whether respiratory rehabilitation after acute exacerbation improves prognosis and health status compared to usual care, we quantified its effects using meta-analyses.Systematic review of randomized controlled trials identified by searches in six electronic databases, contacts with experts, hand-searches of bibliographies of included studies and conference proceedings. We included randomized trials comparing the effect of respiratory rehabilitation and usual care on hospital admissions, health-related quality of life (HRQL), exercise capacity and mortality in COPD patients after acute exacerbation. Two reviewers independently selected relevant studies, extracted the data and evaluated the study quality. We pooled the results using fixed effects models where statistically significant heterogeneity (p ≤ 0.1) was absent.We identified six trials including 230 patients. Respiratory rehabilitation reduced the risk for hospital admissions (pooled relative risk 0.26 [0.12–0.54]) and mortality (0.45 [0.22–0.91]). Weighted mean differences on the Chronic Respiratory Questionnaire were 1.37 (95% CI 1.13–1.61) for the fatigue domain, 1.36 (0.94–1.77) for emotional function and 1.88 (1.67–2.09) for mastery. Weighted mean differences for the St. Georges Respiratory Questionnaire total score, impacts and activities domains were -11.1 (95% CI -17.1 to -5.2), -17.1 (95% CI -23.6 to -10.7) and -9.9 (95% CI -18.0 to -1.7). In all trials, rehabilitation improved exercise capacity .Evidence from six trials suggests that respiratory rehabilitation is effective in COPD patients after acute exacerbation. Larger trials, however, are needed to further investigate the role of respiratory rehabilitation after acute exacerbation and its potential to reduce costs caused by COPD. Acute exacerbations of chronic obstructive pulmonary disease (COPD) represent a major burden for patients and health care systems. For patients, acute exacerbations are a common reason for hospital admissions and severely affect health-related quality of life (HRQL)  and progFrom the health care provider's perspective, COPD is resource consuming . A smallRecent position papers of the American College of Physicians and American College of Chest Physicians provided recommendations on the management of acute exacerbations ,11. HoweThus respiratory rehabilitation may have the potential to reduce hospital admissions by improving exercise capacity. It is hence surprising, and in contrast to the large body of evidence supporting respiratory rehabilitation in stable patients ,17, thatWe used five strategies to identify studies including electronic databases, consultations with experts from North America and Europe, our own files, bibliographies of articles that met the inclusion criteria and conference proceedings of the International Conference of the American Thoracic Society and the Congress of the European Respiratory Society.An information specialist conducted electronic database searches in MEDLINE , EMBASE , PEDRO  and the Cochrane Central Register of Controlled Trials . We did not restrict the search to COPD patients with exacerbation only because exacerbation is not indexed as a Medical subject heading term and we feared to miss relevant studies with a narrow search. We used a broad search strategy using the terms "lung diseases obstructive", "chronic obstructive lung disease", "chronic obstructive pulmonary disease", "rehabilitation", "exercise", "exercise movement techniques", "physical endurance", "muscle training", "kinesiotherapy", "clinical trial", "controlled study" and "epidemiologic methods". A detailed search strategy is available on request. We also searched the Science Citation Index database  and the "related articles" function of PubMed  by entering all included studies.We included randomized controlled trials comparing respiratory rehabilitation of any duration after acute exacerbation of COPD with conventional care. Respiratory rehabilitation programmes needed to include at least physical exercise. We included studies if more than 90% of study participants had COPD. Main outcome measure was unplanned hospital admissions and secondary outcomes included exacerbations, outpatient visits, dyspnea, HRQL as measured by disease-specific or generic questionnaires, functional and maximum exercise capacity, mortality and adverse events during rehabilitation. We did not apply any language restrictions.The bibliographic details of all retrieved articles were stored in a Reference Manager file . We removed duplicate records resulting from the various database searches. Two members of the review team  independently scrutinized the titles and abstracts of all identified citations . In anticipation of significant heterogeneity we established a priori hypotheses to explain differences in outcomes across studies. First, heterogeneity may arise from the setting patients were recruited (in- or outpatient treatment of exacerbation), second from different lengths of follow-up, third from different length of the intervention and finally from differences in the methodology of the intervention. Pooled risk ratios and 95% confidence intervals (CIs) were computed by calculating weighted mean differences and pooled risk ratios using STATA .We show the study selection process and agreement on study inclusion in Figure Initial agreement of reviewers on quality assessment was 85% for all items (chance corrected kappa = 0.70). All disagreement could be resolved by consensus. The quality of trials was moderate Table . Three tFigure Three trials assessed HRQL using the Chronic Respiratory Questionnaire (CRQ) ,24 and tMan and Murphy also used generic HRQL instruments and found improvements by respiratory rehabilitation of 10.6 -0.3 to 21.6) and 20.1 (3.3 to 36.8) on the physical composite and mental composite score of the Short-Form Survey 36  and of 0.6 and 20In the trial by Behnke , the meaAll trials showed a significant benefit of respiratory rehabilitation on the six-minute walking distance Figure . We did The individual study relative risks for mortality ranged from 0.40 0.18–0.86) to 1.00 , St. GeCompared to respiratory rehabilitation in stable COPD patients , its effA word of caution is needed when interpreting the current analysis. A clear limitation of the trials is their relatively small sample size. All trials, in particular the trials reported by Behnke  and KirsLarger trials seem justified to challenge the data presented in this article. Such trials should assess the effect of respiratory rehabilitation on unplanned out- and inpatient care but also include data on patient-important outcomes such as HRQL. Conducting trials on respiratory rehabilitation after acute exacerbation is, however, challenging. First, recruitment of patients is difficult because not all of them may want to be randomly allocated to respiratory rehabilitation or usual care in a situation of poor health status. Second, one needs to take into consideration that exercise capacity is particularly low after acute exacerbations so that The present data show that respiratory rehabilitation has the potential to reduce the large COPD-related costs due to hospital admissions. It may not only reduce the number of acute exacerbations but also their severity. Patients may learn to notice imminent exacerbations and seek medical attention earlier leading to a shift from inpatient to the less costly outpatient treatment of acute exacerbations. The significant reduction in hospital readmissions is suggestive of a beneficial cost-benefit balance. However, larger trials should provide the final evidence base for formal cost analyses to test the hypothesis that respiratory rehabilitation after acute exacerbation is cost effective.The data presented in this review are the first to show a survival benefit of respiratory rehabilitation in patients at risk. Although the results should be interpreted with caution, as mentioned above, this study provides the most solid evidence currently available that mortality is reduced. In summary, current evidence suggests that respiratory rehabilitation reduces unplanned hospital admissions and mortality and improves HRQL and exercise capacity when initiated immediately after acute exacerbations.COPD: Chronic obstructive pulmonary diseaseHRQL: Health-related quality of lifeCI: confidence intervalCRQ: Chronic Respiratory QuestionnaireSD: Standard deviationProtocol writing: Puhan, Scharplatz, SteurerAcquisition of data: Puhan, ScharplatzAnalysis and interpretation of data: Puhan, Scharplatz, Troosters, SteurerDrafting of manuscript: PuhanCritical revision of manuscript for important intellectual content: Puhan, Scharplatz, Troosters, SteurerThe author(s) declare that they have no competing interests.Helmut Horten Foundation; Zurich Lung League. Thierry Troosters is a postdoctoral fellow of the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen.


Timely accessibility to organized inpatient stroke rehabilitation services may become compromised since the demand for rehabilitation services following stroke is rapidly growing with no promise of additional resources. This often leads to prolonged lengths of stays in acute care facilities for individuals surviving a stroke. It is believed that this delay spent in acute care facilities may inhibit the crucial motor recovery process taking place shortly after a stroke. It is important to document the ideal timing to initiate intensive inpatient stroke rehabilitation after the neurological event. Therefore, the objective of this study was to examine the specific influence of short, moderate and long onset-admission intervals (OAI) on rehabilitation outcomes across homogeneous subgroups of patients who were admitted to a standardized interdisciplinary inpatient stroke rehabilitation program.A total of 418 patients discharged from the inpatient neurological rehabilitation program at the Montreal Rehabilitation Hospital Network after a first stroke  were included in this retrospective study. After conducting a matching procedure across these patients based on the degree of disability, gender, and age, a total of 40 homogeneous triads (n = 120) were formed according to the three OAI subgroups: short (less than 20 days), moderate (between 20 and 40 days) or long  OAI subgroups. The rehabilitation outcomes , absolute and relative FIM gain scores, rehabilitation length of stay, efficiency scores) were evaluated to test for differences between the three OAI subgroups.Analysis revealed that the three OAI subgroups were comparable for all rehabilitation outcomes studied. No statistical difference was found for admission (P = 0.305–0.972) and discharge (P = 0.083–0.367) FIM scores, absolute (P = 0.533–0.647) and relative (P = 0.496–0.812) FIM gain scores, rehabilitation length of stay (P = 0.096), and efficiency scores (P = 0.103–0.674).OAI does not seem to affect significantly inpatient stroke rehabilitation outcomes of patients referred from acute care facilities where rehabilitation services are rapidly initiated after the onset of the stroke and offered throughout their stay. However, other studies considering factors such as the type and intensity of the rehabilitation are required to support those results. The demand for rehabilitation services following stroke is rapidly growing since the population at risk for vasculocerebral accident is continuously increasing and the progress in acute clinical management have resulted in higher survival rates over the past few years . Due to The association between the timing of rehabilitation efforts and rehabilitation outcomes has been repeatedly documented -12. NumeIt is important to document the ideal timing to initiate intensive inpatient stroke rehabilitation after the neurological event. Therefore, the objective of this study was to examine the specific influence of short, moderate and long onset-admission intervals (OAI) on rehabilitation outcomes across homogeneous subgroups of patients who were admitted to a standardized interdisciplinary inpatient stroke rehabilitation program.The Montreal Rehabilitation Hospital Network (MRHN) is formed by five publicly-funded rehabilitation hospitals offering standardized interdisciplinary inpatient neurological rehabilitation programs. A total of 749 patients were admitted to the multiple rehabilitation hospitals of the MRHN neurology program offering similar rehabilitation treatments during the study (12-month period) from acute care facilities where inpatient rehabilitation services  are rapidly initiated after admission . The majority of these patients presented with a primary diagnosis of stroke . Admission to these programs is reserved to individuals who experience residual impairments or disabilities following a recent stroke, remain hospitalized in an acute care hospital, and demonstrate a capability to actively participate and tolerate daily intense therapeutic interventions  that are usually initiated within 24 hours from admission. Despite the paucity of beds available in relation to the demand for services, tremendous efforts are made to assure rapid admission to a rehabilitation hospital, usually within 72 hours after the request, to minimize nonmedical bed-days among inpatient rehabilitation candidates waiting in acute care facilities. Rehabilitation hospitals can accommodate to specific needs of each medically stable patient. Readiness for inpatient rehabilitation discharge depends on personal needs of each individual in conformity with their plan of treatment and is periodically reviewed by the interdisciplinary team with input from the patient and its family when indicated.Following approval of this study by the Research and Ethics Committees, medical records were reviewed retrospectively for the 534 clients who registered in the MRHN neurology program with a primary diagnosis of stroke based on clinical history, neurological evaluation or neuroradiologic studies. Patients were excluded if they presented associated medical conditions influencing the course of rehabilitation  was defined as being the number of days elapsed between the onset of the stroke and admission to the interdisciplinary inpatient stroke rehabilitation program. All participants were classified into a short (less than 20 days), moderate (between 20 and 40 days) or long  OAI subgroups. A similar classification has been previously used in a stroke study conducted in Italy . AlthougThe Functional Independence Measure™ (FIM) , which iAll subjects selected were classified into one of 14 Inpatient Rehabilitation Facility-Case Mix Groups (IRF-CMG), specifically developed for individuals with a diagnosis of stroke, based on a Classification and Regression Trees statistical methodology (Table A block design with a matching procedure was used to control for the covariant effects that degree of disability (IRF-CMGs), gender and age may have on the OAI, and ultimately on the rehabilitation outcomes. Three matching variables were selected: IRF-CMGs (14 levels), gender  and age (within 3 years). Each subject was identified by a IRF-CMG, gender, and age. Subjects were selected from each OAI subgroup to generate triads. When multiple subjects were identified within the same OAI subgroups, subjects were matched automatically with the first subject who presented similar characteristics  until no more triads could be created. Subjects for whom there was no exact severity of disability (CMG), age, and gender counterparts were excluded. Given the fact that 120 subjects were selected overall, the final data set consisted of 40 homogeneous triads  made between admission and discharge were determined using an absolute method of calculation which highlights the absolute difference in raw scores between admission and discharge scores. Similarly, relative FIM changes were also calculated (Discharge FIM-Admission FIM/Admission FIM).The length of stay (LOS) corresponds to the net number of days elapsed between admission and discharge from inpatient stroke rehabilitation. This measure is often referred to as the duration of the inpatient rehabilitation stay and represent a useful rehabilitation cost indicator .The total, motor, and cognitive-efficiency scores report the absolute change in FIM score divided by the inpatient rehabilitation LOS. This ratio represents the average amount of FIM gain per day during inpatient rehabilitation.th, 50th (median) and 75th percentiles, were calculated for the categorical data  for the total sample and the three OAI. For these rehabilitation outcomes, between-group differences for participants with different OAI intervals were calculated using a Kruskal-Wallis analysis for non-parametric data. A level of significance of 0.05 was selected for all tests. All data were analyzed using the SPSS® 11.5 statistical analysis software.Descriptive statistics, expressed as mean and one standard deviation (SD), were calculated for the continuous data  for the total sample and the three OAI subgroups. Between-group differences for participants with different OAI intervals were calculated using a one-way independent-samples analysis of variance (ANOVA) for the age, OAI, and LOS after verifying the heterogeneity of variances. When the ANOVA was significant, Tukey post-hoc testing was conducted to locate the differences between the short, medium, and long OAI subgroups. Descriptive statistics, expressed as 25P < 0.001) whereas no mean age difference was observed between the OAI subgroups (P = 0.934). The LOS was not significantly affected by the timing at which inpatient rehabilitation was initiated (P = 0.096) although patients with a long OAI had a tendency to have a longer LOS when compared to those with short or moderate OAI.The final sample included 120 patients equally distributed into three OAI subgroups of each 40 patients: short, moderate, and long Table . As expeP = 0.828), motor (P = 0.972), and cognitive-FIM scores (P = 0.305) were observed between the OAI subgroups. Likewise, comparable discharge total (P = 0.083), motor (P = 0.108), and cognitive-FIM (P = 0.367) scores were found between the OAI subgroups. Both the absolute and relative total (P = 0.647 and 0.812), motor (P = 0.533 and 0.725), and cognitive FIM change scores (P = 0.610 and 0.496) were comparable between the OAI subgroups, respectively.Results of admission and discharge functional assessments along with the absolute and relative changes measured during inpatient rehabilitation are summarized in Table P = 0.120), motor (P = 0.103), and cognitive-efficiency scores (P = 0.674) were reached between the OAI subgroups.Finally, total, motor, and cognitive-efficiency scores generated from total, motor and cognitive absolute FIM gain scores and inpatient rehabilitation LOS measured for all patients and for the short, medium and long OAI subgroups are presented in Table P = 0.120; FIM motor P = 0.103). This small effect definitively deserves attention as this can be of clinical importance even if no statistically significant difference was found. This trend corroborates that individuals who are medically stable following a first CVA should rapidly be transferred to a rehabilitation facility offering an intensive stroke rehabilitation program. Under no circumstance should the admission to a rehabilitation facility from an acute care facility be delayed.Inpatient rehabilitation outcomes  were not influenced by the OAI in this study. Surprisingly, the impact of the OAI on stroke rehabilitation outcomes was found to be suppressed when controlling for the degree of severity, gender, and age. These results challenge the consensus that stroke survivors who are rapidly admitted to inpatient rehabilitation potentially reach more favourable outcomes than those admitted later, and the presumption that delayed inpatient stroke rehabilitation admission may prompt adverse effect on rehabilitation outcomes. Overall, these results confirm that if interdisciplinary rehabilitation services are rapidly initiated after the stroke, OAI loses importance independently of the setting  although a small effect of timing (OAI) might still remain  influence this process. In fact, the average OAI of 30.97 (SD 18.12) days calculated in this study was almost three times more elevated than the average of 11 days recently reported in the United-States of America (USA) following a stroke . This reP = 0.096) among individuals with stroke. Yet, the fact that individuals with a long OAI remained almost 10 days longer than those with short or moderate OAI in rehabilitation may represent a clinically and administrative relevant difference. Moreover, there is no evidence suggesting that rehabilitation is ineffective if begun at a later time. In fact, recent publication even proposes that rehabilitation in chronic stroke clients should not be neglected and represents a promising option [Strength of rehabilitation interventions offered in acute care facilities also deserves additional attention to better determine its impact on OAI of clients referred to inpatient rehabilitation programs . This stg option .P = 0.083). More precisely, the comparable FIM efficiency scores found between the OAI subgroups suggest that similar rates of functional improvement may be expected despite differences in OAI. Alternative services, such as ambulatory hospital-based rehabilitation services or early supported discharge with home rehabilitation, may represent interesting clinical approaches to limit the OAI , especially for individuals with mild disability [It is clear that inpatient rehabilitation after a stroke is effective for all individuals although variability is observed among OAI groups. All patients equally gained from inpatient stroke rehabilitation indifferently of OAIs although individuals with short OAI had an extra 8 total-FIM units than those with moderate or long OAI . It is believed that spontaneous recovery usually results from three separate, but interactive, processes which include resolution of diaschisis , behavioural compensation and neuroplasticity ,35. MoreSome evidences suggest a possible association between the severity of the vasculocerebral event and the duration of the spontaneous recovery process . CoursesAlthough this study further advocate the efficiency of inpatient stroke rehabilitation programs, one must be prudent in interpreting these results since the retrospective design of this study, combined to the modest sample size, are inherent limitations. Future prospective study would need to document functional status (FIM scores) at consistent times following stroke between the OAI groups to remove this potential confounding effect. Possible ceiling and bottom effects of the FIM measurement instrument selected to assess functional capacity are also well documented . ClinicaAfter controlling for the degree of disability, gender, and age, this matched block design study showed that OAI may not be a relevant prognostic factor of inpatient stroke rehabilitation outcomes for patients referred from acute care facilities where rehabilitation services are rapidly initiated after a stroke. The impacts of early or delayed initiation of inpatient rehabilitation following a stroke may not favourably or adversely affect rehabilitation outcomes, respectively. However, it certainly remains critical to rapidly initiate rehabilitation services after the onset of a stroke at the acute care hospital, according to specific goals to be achieved for each patient, because any delay may adversely influence rehabilitation outcomes. Perhaps most importantly, these results may have important implications in the continuum of care of individuals with stroke between acute care and rehabilitation hospitals. Finally, the result of the present study not only contrasts from previous literature but also supports the need of future prospective studies focusing on the influence of OAI on inpatient stroke rehabilitation outcomes and their retention, even improvement, over time after discharge.The author(s) declare that they have no competing interests.DG made substantial contributions to the conception and design of this study, to the data acquisition, statistical analysis, interpretation of the results and to the writing of this manuscript. SN participated to the statistical analysis and the interpretation of the results of this study and helped to draft the manuscript. VT was involved in the conception of the study and in drafting and reviewing the manuscript. Following a critical review, all authors read and approved the submitted manuscript.The pre-publication history for this paper can be accessed here:


IPK, a member of the Hsp40 family that inhibits eukaryotic initiation factor 2α (eIF2α), inhibits endoplasmic reticulum (ER) stress and leads to downregulated expression of vascular endothelial growth factor (VEGF) and decreased apoptosis in human retinal capillary endothelial cells (HRCECs).The goal of this research was to determine if P58IPK) and P58 RNA in the plasmid pGIPZ (pGIPZ-P58IPK). The four experimental groups were: (1) non-transfected/non ER stressed control; (2) non-transfected/ER stressed; (3) rAAV2-P58IPK-transfected/ER stressed; and (4) pGIPZ- P58IPK RNAi transfected/ER stressed. ER stress was induced by treating cells with tunicamycin. Expression of P58IPK was determined in transfected cells. Expressions of the following factors were assessed: vascular endothelial growth factor (VEGF), C/EBP homologous protein (CHOP), activating transcription factor 4 (ATF4), and glucose-regulated protein 78 (GRP78). Apoptosis levels were also determined.Recombinant vectors were constructed using P58 in adeno-associated virus type 2 (rAAV2-P58 IPK was detected in cells transfected with rAAV2-P58IPK (0.63±0.02) as compared to those transfected with pGIPZ-P58IPK RNAi (0.23±0.01). P58IPK expression was not different between the control transfected cells (rAAV2-GFP and pGIPZ-GFP). Following ER stress, expression levels of ATF-4, GRP78, CHOP, and VEGF in cells overexpressing P58IPK were not different from those in unstressed control cells. This inhibitory effect of P58IPK on the expression of ER stress-related factors was suppressed in cells transfected with pGIPZ-P58IPK RNAi. Apoptosis was significantly increased in cells transfected with pGIPZ-P58IPK RNAi but not with rAAV2-P58IPK.Significantly increased expression of P58IPK inhibits ER stress and plays an important role in maintaining balance and stability of the ER in HRCECs.The study demonstrates that P58 Endoplasmic reticulum (ER) stress is thought to be a contributing factor underlying many human diseases. In pathologic conditions, accumulation of unfolded or misfolded proteins in the ER can lead to ER stress or initiate the unfolded protein response ,2. If prStudies in mice have indicated that ER stress can lead to islet cell apoptosis through induction of C/EBP homologous protein (CHOP) and contribute to the development of diabetes . ER streIPK, a member of the Hsp40 family, was first discovered as an inhibitor of eukaryotic initiation factor 2α (eIF2α) [IPK plays an important role in ER stress protection [IPK activity were associated with increased expression of several ER stress-inducible genes such as CHOP. It has also been demonstrated that mice lacking the IPKP58 gene exhibit gradual onset of glycosuria, hyperglycemia, and hypoinsulinemia [IPK, leading to a decrease in the number of β-cells and loss of functionality. The ER stress-inhibiting effects of P58IPK appear related to its capacity to inhibit phosphorylation of eIF2α, which is involved in eliciting the previously mentioned unfolded protein response [P58 (eIF2α) . Since totection ,11. For ulinemia . The numresponse .IPK and ER stress in cells isolated from human eyes. Characterizing this relationship is important as it may help identify possible targets for pharmaceutical intervention  in the treatment/prevention of DR. In the present study, we sought to examine the effects of P58IPK on ER stress in human retinal capillary endothelial cells (HRCECs) by assessing the expression of ER stress-related factors, glucose-regulated protein 78 (GRP78), ATF4, and CHOP. We also examined the possibility that P58IPK may be able to ameliorate diabetes-associated blood vessel damage by assessing changes in VEGF expression and cellular apoptosis. Our working hypothesis was that P58IPK would inhibit ER stress in HRCECs.To our knowledge, no study has examined the relationship between P58Donor eyes (12) were obtained from the Tongji Hospital Eye Bank. Donors were healthy accident victims with an average age of 33.5 years .2. Cultured cells were then characterized for endothelial homogeneity by testing for activity to factor VIII antigen (von Willebrand factor), measuring acetyl-LDL (Ac-LDL) receptors in endothelial cells, as described previously [The well established method for isolating and culturing HRCECs has been previously described . The doneviously , and by HRCECs were treated with tunicamycin (an ER stressor) according to a modification of the method described by Shimazawa and colleagues . Cells wIPK was obtained from the human cDNA bank/domain and synthesized using Golden Taq . pSNAV- P58IPK plasmids were then formed after DNA digestion and ligation. Transfection of BHK-21 cells with pSNAV-P58IPK was performed in a 6 well plate using a Lipofectamine 2000 kit . These cells are hereafter referred to as BHK/P58IPK. BHK/P58IPK cells were then transferred to and cultured in a flask  before being infected with HSV1-rc/ΔUL2  to induce rAAV2-P58IPK when the cell number reached 8×108. The cells were detached after 48 h of culture and were then divided into Fernbach culture flasks (250 ml) for further purification. Purified rAAV2-P58IPK was identified by reverse-transcription polymerase chain reaction (RT–PCR). The titer of rAAV2-P58IPK (virus genome/ml) was assessed using in situ hybridization with a digoxin labeled cucumber mosaic virus probe to obtain a final titer of 1×1012.The coding sequence of p58IPK RNAi  sequence inserted was as follows: TGC TGT TGA CAG TGA GCG CAG GTG CTG AAT GTG GAG TAA ATA GTG AAG CCA CAG ATG TAT TTA CTC CAC ATT CAG CAC CTT TGC CTA CTG CCT CGG A. pGIPZ-green fluorescence protein (GFP) was used to evaluate transfection efficiency.The P58IPK, PGIPZ-P58IPK RNAi, pGIPZ-GFP, or rAAV2-GFP. Cells were plated in 24 well culture plates, grown overnight to 70%-80% confluence, and then washed twice with serum-free basal growth medium. While only rAAV2 was added to control group wells, 10×109 copies of rAAV2- P58IPK, or PGIPZ- P58IPK RNAi were added to each well in the experimental groups. In addition, plasmids containing rAAV2-GFP and pGIPZ-GFP were separately added to HRCECs to monitor transfection efficiency.Cultured HRCECs were transfected with rAAV2-P58IPK were as follows: sense, 5′-GAG GTT TGT GTT GGG ATG CAG-3′ and antisense, 5′-GCT CTT CAG CTG ACT CAA TCA G-3′. The PCR reaction for P58IPK was performed in a final reaction volume of 20 μl using the following conditions: a preheating cycle at 95 °C for 3 min then 31 cycles at 95 °C for 30 s, 62 °C for 30 s, and 72 °C for 30 s followed by an elongation cycle at 72 °C for 8 min. Melting curve analysis was performed by monitoring FAM/SYBR fluorescence.HRCECs were harvested, and RNA was extracted using the QiagenRNA extraction kit . After quantification, RNA was reverse transcribed to cDNA using the Super Script II RT kit (Invitrogen Inc.). Real time PCR was performed using the Hotstar Taq Polymerase kit (Qiagen Inc.) with SYBR Green technology . GAPDH was used as an internal control. Primers for GAPDH were as follows: sense, 5′-CCT GTA CGC CAA CAC AGT GC-3′ and antisense, 5′-ATA CTC CTG CTT GCT GAT CC-3′. Primers for P582, 5μM dNTPs, and 1 unit of Platinum Taq (Invitrogen Inc.). PCR conditions were as follows: initial denaturation at 95 °C for 4 min, 30 cycles at 95°C for 30 s, 58°C for 45 s, and 72°C for 30 s, the final elongation step was performed for 5 min at 72°C. After 30 cycles, the final elongation step was performed for 5 min. Equal amounts of PCR products were separated on 1.5% agarose gels and visualized by ethidium bromide staining. GAPDH was used as an internal control.For semi quantitative (sq) PCR, 1 μl of single-stranded cDNA was added to a 20 μl PCR reaction mixture containing 10 pmol of a gene specific primer pair, 1X PCR buffer (Invitrogen Inc.) containing 3μM MgClIPK transfection (upregulation)/ER stress group; and (4) a P58IPK RNAi (inhibition)/ER stress group. cellular apoptosis was evaluated using propidium iodide (PI) to stain the DNA. Cells were trypsinized, pelleted, and resuspended in one volume of ice-cold phosphate-buffered saline (PBS). The suspension was gently agitated while three volumes of ice-cold 95% ethanol were slowly added. Cells were then pelleted and resuspended in equal volumes of 30 μg/ml PI and 100 μg/ml RNase A (both in PBS). Stained cells were stored overnight at 4 °C and protected from light until analysis. Flow cytometry was performed to determine the percentage of cells in apoptosis.There were a total of four experimental groups in this study: (1) a control group (no ER stress or transfection); (2) an ER stress group (no transfection); (3) a P58HRCECs from each experimental group were collected for detection of VEGF, GRP78, ATF4 and CHOP expression. Total RNA was isolated for quantification. RNA was then reverse transcribed to cDNA using a Super Script II RT kit (Invitrogen Inc). Real time PCR was performed using the Hotstar Taq Polymerase kit (Qiagen Inc.) with SYBR Green technology (ABI Inc.). Primers for VEGF, GRP78, ATF4, CHOP, and GAPDH are described in Total protein was extracted from the HRCECs and quantified using bicinchoninic acid. The protein was mixed with the loading buffer, denatured for 6 min at 60 °C, cooled, centrifuged for 5 min, and then separated by sodium-dodecyl sulfate PAGE (SDS–PAGE). Monoclonal anti-CHOP , polyclonal anti-ATF4 , monoclonal anti-GRP78 ,and monoclonal anti-VEGF (Santa Cruz Biotechnology Inc.) were used to probe the proteins. A secondary antibody (1:1000 dilution) was then applied, and the signal was revealed by chemiluminescence. The same polyvinylidene fluoride (PVDF) membranes were reused to detect β-actin  by incubating with the mouse anti-human actin antibody . The bands observed on the films were analyzed by automatic image analysis, and the integrated optical density (OPTDI) of each protein band was normalized to the OPTDI value of the corresponding β-actin band from the same sample.Normally distributed data were compared by one-way ANOVA while non-normally distributed data were compared by Kruskal–Wallis test. When significant differences were detected between groups, multiple comparisons of means were performed using the Bonferroni procedure, with type-I error rate at a maximum of 0.008 (0.05/6) adjustment. Statistical analyses were performed using SPSS 15.0 Statistics software , and differences were considered significant when p<0.05. Data are presented as the mean±standard error of the mean (SEM).IPK in HRCECs transfected with rAAV2-GFP, pGIPZ-GFP, rAAV2- P58IPK, or pGIPZ- P58IPK RNAi as determined by semi-quantitative polymerase chain reaction (sq PCR) and real time PCR are presented in IPK/GAPDH among the four transfection groups (p<0.001). sq PCR result show that the highest level was in the group transfected with rAAV2- P58IPK (0.63±0.02) while the lowest was in the group with pGIPZ- P58IPK RNAi (0.23±0.01). No significant differences in P58IPK/GAPDH levels were found between the rAAV2-GFP and pGIPZ-GFP groups. The results of real time PCR show that the ratio of P58IPK/GAPDH was 1.45±0.05 in the rAAV2- P58IPK group and 0.41±0.03 in the pGIPZ- P58IPK RNAi group.The transfection rate of rAAV2-P58IPK was 85% and pGIPZ- P58IPK RNAi was 87% two days after transfection . Gene expression levels of P58IPK). Apoptosis levels were significantly higher in group 4 (co-transfected with pGIPZ- P58IPK RNAi and rAAV2- P58IPK) compared to all other groups .There was a statistically significant overall difference in apoptosis levels two days after transfection . There wIPK and pGIPZ- P58IPK RNAi and treated with tunicamycin). In contrast, expression levels of these genes in Group 3 cells (transfected with rAAV2- P58IPK and treated with tunicamycin) were not significantly different from those in control cells  when compared to the normal control group  were also significantly higher than those in Group 3 (transfected with rAAV2- P58IPK). No significant differences were detected between Group 3 (transfected with rAAV2- P58IPK and treated with tunicamycin) and Group 1  for these variables  and apoptosis. We found that HRCECs overexpressing P58IPK exhibited decreased expression of the ER stress-related factors, GRP78, ATF4, and CHOP, following ER stress. Importantly, when P58IPK expression was silenced by co-transfecting HRCECs with RNAi against P58IPK, the previously noted inhibition of ER stress was abolished i.e., expression of the aforementioned factors were increased. These findings suggest that P58IPK plays an important role in maintaining the balance and stability of the ER in HRCECs.Taking into account previously reported findings; we conducted a study of HRCECs transfected with P58IPK is purported to be an important factor in maintaining balance within the endoplasmic reticulum [IPK expression was increased by transfection, expression of ATF4 and VEGF was decreased in HRCECs following ER stress. Given these findings, we speculate that P58IPK has the capacity to downregulate the activity of the ATF4/VEGF pathway during ER stress. We also noted that increased P58IPK expression was associated with decreased CHOP expression in HRCECs. Given that CHOP is an initiator of apoptosis, it is hardly surprising that apoptosis levels also decreased in cells that overexpressed P58IPK. With regards to the development of DR, the current findings lead us to suggest that enhanced P58IPK expression may help to decelerate or prevent the process of retinal vessel damage caused by upregulated VEGF expression.P58eticulum . In the IPK on ER stress in human retinal capillary endothelial cells in vitro. The results of the present study indicate that the overexpression of P58IPK in HRCECs can effectively ameliorate ER stress by inhibiting expressions of GRP78, ATF4, VEGF, and CHOP and decreasing apoptosis. Further study is warranted to clarify precisely how P58IPK interacts with VEGF-biologic pathways during ER stress. Confirmation of these findings using an in vivo animal model is also needed.In summary, to our knowledge, this is the first paper to describe the effects of P58


Demand for rehabilitation services is expected to increase due to factors such as an aging population, workforce pressures, rise in chronic and complex multi-system disorders, advances in technology, and changes in interprofessional health service delivery models. However, health human resource (HHR) strategies for Canadian rehabilitation professionals are lagging behind other professional groups such as physicians and nurses. The objectives of this study were: 1) to identify recruitment and retention strategies of rehabilitation professionals including occupational therapists, physical therapists and speech language pathologists from the literature; and 2) to investigate both the importance and feasibility of the identified strategies using expert panels amongst HHR and education experts.Recruitment and Retention and the other on Education were convened to determine the importance and feasibility of the identified strategies. A modified-delphi process was used to gain consensus and to rate the identified strategies along these two dimensions.A review of the literature was conducted to identify recruitment and retention strategies for rehabilitation professionals. Two expert panels, one on Recruitment and Retention and Education expert panels as being important and feasible for the development of a HHR plan for recruitment and retention of rehabilitation professionals. Seven were categorized under the Quality of Worklife and Work Environment theme, another seven in Financial Incentives and Marketing, two in Workload and Skill Mix, thirteen in Professional Development and five in Education and Training.A total of 34 strategies were identified by the Quality of Worklife and Work Environment, Financial Incentives and Marketing and Professional Development.Based on the results from the expert panels, the three major areas of focus for HHR planning in the rehabilitation sector should include strategies addressing  Demand for rehabilitation services is expected to increase within the next decade primarily due to factors such as an aging population, workforce pressures, rise in chronic and complex multi-system disorders, advances in technology, and changes in health service delivery models -4. In CaHealth Personnel Trends in Canada from 1993 to 2002 report, numerous factors have been suggested to influence demand for physiotherapy and occupational therapy services. Factors that may influence increase demand for physiotherapy include: shift in health service delivery models from hospital to community care; earlier patient discharge; increased expectations from aging Canadians concerning more active lifestyles; growing private practice sector and continued shortages for PTs in both private and public sectors in rural, remote and urban settings across Canada ; (2) Workload and Skill Mix [n = 6]; and (3) Financial Incentives and Marketing [n = 15]. The two Education strategy themes were: (1) Education and Training [n = 11] and (2) Professional Development (n = 13).There was a paucity of peer-reviewed studies obtained exploring rehabilitation HHR strategies, therefore the majority of strategies were selected from grey literature reports from international, national and provincial health organizations. From the literature review, 107 potential strategies were identified according to their relevance to HHR issues for rehabilitation in Ontario. These strategies were then categorized into two broad groups: A) Labrador  formed tRecruitment and Retention (n = 8) and the other for Education (n = 9)  Table . The sizRecruitment and Retention panel were sent an electronic survey containing the 40 strategies identified from the literature review and the Education panelists were also sent an electronic survey with 24 strategies. For Round 1, each panel was asked to rate the strategies using a nine-point Likert-type rating scale that ranged from "none" (1) to "maximum" (9), on two key dimensions: Feasibility and Importance. Feasibility was defined as the practicality and cost implications of the strategy and was rated from the respondents' perspective. Importance was defined as how valuable, appropriate and useful the strategy could be for rehabilitation HHR planning in Ontario. At the end of the survey, panelists were given the opportunity to suggest additional strategies that they felt were appropriate to consider. Once completed, panelists were asked to return the survey to the study office by email or fax prior to the expert panel meeting in Round 2. Data from each questionnaire were entered into a spreadsheet and tabulated. Descriptive statistics were calculated for each strategy using frequency distributions and proportional percentages of respondents. Importance and feasibility rankings were based on the percentage of expert panelists' low, medium and high ratings.A modified-delphi technique was then used for the expert panel process, ,47 basedAfter the independent completion of the survey, each panel was convened separately for a one-day meeting for final discussions, debates and consensus voting to decide on strategies . A stratOn the day of the meeting, the panelists were given a copy of the aggregated survey results indicating the ratings of all of the strategies. High and low rated strategies were not discussed as there was already consensus, whereas all medium rated strategies were subject to discussion. Using a nominal group process , each stRecruitment and Retention panel reached consensus on 12 strategies. However, 14 had a combination of high/medium importance and feasibility ratings and 14 had medium ratings on both dimensions, therefore it required further discussion. An additional strategy regarding family relocation programs was added by this panel.Following Round 1 rating of the 40 identified strategies, the Education Panel ranked 16 of 24 strategies with high importance and feasibility after Round 1. Since there were only eight strategies with medium ratings, this expert panel decided to review all the strategies at the face-to-face meeting to discuss the rationale that would explain why some of the highly rated strategies were not already implemented and to come to a consensus on the other eight medium rated strategies. They also added an additional strategy for career paths.The Recruitment and Retention and Education expert panels as being important and feasible for the development of a HHR plan for recruitment and retention of rehabilitation professionals. Under the Recruitment and Retention theme, seven were categorized as Quality of Worklife and Work Environment; two were Workload and Skill Mix, and another seven were Financial Incentives and Marketing. As for the Education panel, five were categorized as Education and Training strategies while the other thirteen were related to Professional Development.A total of 34 strategies were identified by both the Recruitment and Retention panel had a total of 16 highly important and feasible strategies, 8 high/medium importance and feasibility, 8 medium and 9 low ratings for both dimensions. The Education panel on the other hand had a total of 18 high, 1 high/medium, 3 medium and 3 low rating strategies.As indicated in Table Quality of Worklife and Work Environment (44%) and Financial Incentives and Marketing (44%), followed by Workload and Skill Mix (12%). It should be noted that some strategies had equal rankings; therefore the total number of rankings did not equal the total number of strategies.Table Recruitment and retention strategies that had a combination of high and medium ratings in either of the two dimensions included: sense of empowerment in promoting healthy work environments; team-building exercises; developing participatory decision-making systems; improving rural working conditions; recognizing work-life balance; creating an environment where staff are valued; optimizing scope of practice and work-management autonomy. Strategies that had medium importance and feasibility ratings included: resolving concerns about liability and accountability in collaborative practice; recruiting international trained therapists; opportunity to work in different settings; interprofessional payment schemes; family leave; staff recognition and creating a position for a provincial health professional recruiter. Low importance and feasibility strategies included: word of mouth references; bursaries and retention bonuses; exchange employment opportunities; health promotion; retention workshop; 80–20 staffing model ; using recruitment agencies and providing recruitment bonuses Table .Professional Development (72%), more so than Education and Training (28%).Education strategies that were rated highly important and feasible are described in Table The medium rated education strategies included: expand interprofessional education; provide incentives for students interested in rural practice; summer mentorship programs for high school students; and aboriginal student support program. The strategies that were considered neither feasible nor important included: using return of service contracts after professional development; create a tiered pathway approach through modular education and laddered credentialing and in accreditation standards allow greater use of rural practice sites Table .Since the purpose of the panel was to identify recruitment and retention and education strategies that could inform the development of a HHR plan for rehabilitation professionals, there was also discussion about contextual factors that would influence a plan. Panelists commented that key factors to consider prior to implementation of these strategies should include workplace setting, geographical location  and gender issues.Quality of Worklife and Work Environment, Financial Incentives and Marketing and Professional Development are the three major areas of focus when developing a competitive HR plan in the rehabilitation sector.The purpose of this study was to identify recruitment and retention strategies to inform the development of HHR planning for rehabilitation professionals in Ontario. This study highlights that Quality of Worklife and Work Environment strategies ranked among the top category for recruitment and retention of rehabilitation therapists. This has also been found among nurses [g nurses  where itg nurses . Specifig nurses .In addition to the above, ensuring open and timely communication between employer and worker was also ranked highly among the recruitment and retention strategies. Examples of strategies include: open door policies, employee advisory committees and regular staff meetings and evaluations. Although these strategies were reported to be used among organizations providing services to persons with developmental disabilities in Alberta, there was no description of the organizations, sample size or methodology . SimilarAnother area that was ranked highly was marketing strategies to increase high school student and public awareness of rehabilitation careers. These specific strategies were also recommended by the Ontario Hospital Association (OHA) in order to establish a competitive position for Ontario hospitals in respect of recruitment and retention of health care professionals . SimilarWorkload and Skill Mix strategies only two were highly rated: implementing a caseload management database and using support personnel. Caseload management has been identified in the literature as an issue affecting all three rehabilitation professions. For example, in physiotherapy, Christie's study [Of the six 's study  found th's study . A liter's study . TherefoWorkload and Skill Mix strategy was the use of support personnel (i.e. physiotherapy assistants or exercise therapists) to increase efficiency of utilization of scarcer and higher order rehabilitation competencies. Considerations for implementing this strategy include addressing key issues such supply, standards of education, standards of practice and accreditation. These are highlighted in an article by Salvatori [The other highly rated alvatori  who repoalvatori . Since talvatori .Education and Training strategies targeted rural and remote practices underscores the importance of specific strategies for rural and remote areas in the development of a HHR plan. The need to build on existing mechanisms to expand the availability of rural and remote clinical placements by providing financial and accommodation support was ranked among the top two most important and feasible education strategies for rehabilitation professionals. Not only has this strategy been used as a recruitment tool for rehabilitation students, it has also been reported by Solomon et al. [Given that 60% of highly rated n et al.  to be efn et al. . Althougn et al. . TherefoOur findings indicate that the theme with the largest number of strategies that were considered important and feasible to implement as part of HHR planning was professional development. Many were specific to rural and remote areas. Although the importance of continuous professional development (CPD) in recruitment and retention is well recognized, a Canadian study reported several barriers to its implementation . In the Limitations of this study should be noted. First, the majority of the strategies were obtained from the grey literature that is not subject to the same scrutiny as the peer-reviewed literature. Second, almost none of the strategies were specifically developed for rehabilitation professionals and in many cases had to be re-worded to fit the rehabilitation context. There is a lack of research on rehabilitation clinicians' perspectives on recruitment and retention strategies; therefore future research should focus on investigating this area. Third, during the face-to-face meeting, bias could have resulted from panelists whose opinion may have influenced others significantly, especially if members came from similar workplace settings. The facilitator of the expert panels however, followed a strict process for managing the discussion and ensured that all panelists were given the opportunity to express their opinions.Finally, although some strategies such as competitive wage packages, training/growth opportunities and professional development are viewed as both a recruitment and retention incentive, other strategies do not overlap and are appropriate for only one of the two tasks. For example, increasing public awareness of rehabilitation careers, providing rural and remote orientation packages and family relocation programs are only appropriate for attracting a worker while ensuring open and timely communication may be seen as a strategy only for retention. Future research should therefore consider studying recruitment and retention strategies separately so that a distinction between the two can be made.Quality of Worklife and Work Environment, Financial Incentives and Marketing and Professional Development, key factors that need to be considered in the context of implementation include: workplace setting, geographical location and gender issues. While this is the first study to our knowledge that provides a comprehensive list of recruitment and retention strategies relevant to rehabilitation professionals, more information is needed for the development of a HHR plan. Information on trends in labour force participation as well as knowledge regarding the use and effectiveness of recruitment and retention strategies for rehabilitation professionals is needed. More importantly, the success of implementing and sustaining such strategies requires future research to validate these strategies from the perspective of rehabilitation clinicians and human resource decisions makers  so that specific barriers and challenges can be identified.This study identified 34 strategies that should be considered as important and feasible for implementation as part of HHR planning for rehabilitation professionals. Although the highest ranked strategies focused on areas of The authors declare that they have no competing interests.DT participated in the design of the study, conducted the literature review, analyzed and interpreted the results and drafted the manuscript.LMH, AD, DB and KB were involved in providing feedback and editing the content of the manuscript.MDL recommended participants for the expert panel and was involved in providing feedback and editing the content of the manuscript.SJ participated in the design of the study, recommended participants for the expert panel, organized and consolidated strategies, interpreted the data and edited the content of the manuscript.The pre-publication history for this paper can be accessed here: