Application of a cell-permeable inhibitor of caveolin-3 scaffolding interactions mimicked the effect of MBCD on phosphorylated phospholamban (pPLB) during β2-AR stimulation, consistent with MBCD acting via caveolae. Biosensor experiments revealed β2-AR mobilisation of cAMP in PKA II signalling domains of intact cells only after MBCD treatment, providing a real-time demonstration of cAMP freed from caveolar constraint. Other proteins have roles in compartmentation, so the effects of phosphodiesterase (PDE), protein phosphatase (PP) and phosphoinositide-3-kinase (PI3K) inhibitors on pPLB and contraction were compared in control and MBCD treated cells. PP inhibition alone was conspicuous in showing robust de-compartmentation of β2-AR-derived signalling in control cells and a comparatively diminutive effect after cholesterol depletion. Collating all evidence, we promote the novel concept that caveolae limit β2-AR-cAMP signalling by providing a platform that not only attenuates production of cAMP but also prevents inhibitory modulation of PPs at the sarcoplasmic reticulum. This article is part of a Special Issue entitled “Local Signaling in Myocytes”.Inotropy and lusitropy in the ventricular myocyte can be efficiently induced by activation of β1-, but not β2-, adrenoceptors (ARs). Compartmentation of β2-AR-derived cAMP-dependent signalling underlies this functional discrepancy. Here we investigate the mechanism by which caveolae  contribute to compartmentation in the adult rat ventricular myocyte. Selective activation of β2-ARs (with zinterol/CGP20712A) produced little contractile response in control cells but pronounced inotropic and lusitropic responses in cells treated with the cholesterol-depleting agent methyl-β-cyclodextrin (MBCD). This was not linked to modulation of L-type Ca ► We investigate the mechanism(s) by which caveolae compartmentalise β2-AR signalling. ► Disrupting caveolae reveals β2-AR cAMP signals in the PKA-RII compartment. ► Disrupting caveolae promotes a selective PKA-mediated phosphorylation of PLB. ► Phosphatases (PP) 1/2a (but not PDE 2/3/4) contribute to caveolar β2-AR compartmentation. ► We propose that intact caveolae attenuate cAMP production and limit inhibitory modulation of PP at the SR. This occurs because different receptors produce changes in cAMP in different compartments of the cell. The idea of cAMP compartmentation is well accepted but the mechanisms responsible are not fully understood.2+ channel β1- and β2-adrenoceptors (AR), the predominant β-ARs in the heart, provide an excellent illustration of compartmentalised cAMP signalling in the adult ventricular myocyte. β1-AR stimulation promotes positive inotropic and lusitropic responses, whereas β2-AR stimulation has minimal functional effects We are interested in the mechanisms that restrict cAMP dependent signalling from the β2-AR.Whilst the β1-AR couples to the stimulatory Gs protein, β2-ARs couple sequentially to Gs and the inhibitory Gi protein. The Gi pathway has been proposed as a key factor responsible for the spatial restriction of β2-AR-cAMP signals to the sarcolemmal compartment, perhaps through stimulation of protein phosphatase activity However, an additional, complementary, explanation for the diverse effects of β1- and β2-AR stimulation on cAMP is that the differential membrane distribution of signalling elements, including receptors, produces cAMP signals in different cellular compartments. The lipid bilayer is not a homogenous structure with randomly distributed proteins, but contains liquid-ordered phases enriched in cholesterol and sphingolipids known as lipid rafts. Caveolae are specialised forms of invaginated rafts characterised by the presence of caveolin and cavin proteins  ICa,L, [Ca2+]i and contraction, and how this is affected by inhibition of key components linked with β2-AR compartmentation. Measuring these different indices gives insight into the mechanism of caveolar control (e.g. cAMP production/cAMP degradation/phosphatase activity) and the region of the cell (e.g. sarcolemma/SR/myofilaments) in which this control operates. We show that spatial restriction of the β2-AR cAMP signal by caveolae occurs at the level of cAMP production and phosphatase activity in the SR. This can be explained by the formation of specific signalling complexes in the caveolar microdomain.The aim of the present study was to reconcile, for the first time, work showing the roles of phosphatases and phosphodiesterases in spatial control of β2-AR signalling with mechanism(s) by which caveolae compartmentalise cAMP in the adult ventricular cell. We report how disruption of caveolae impacts on β2-AR signalling at the level of cAMP, protein phosphorylation, 22.1Animals (Scientific Procedures) Act 1986, the Recommendation from the Declaration of Helsinki and the Guiding Principles in the Care and Use of Animals. All experiments were carried out at room temperature unless otherwise stated and, except for those involving fluorescence resonance energy transfer (FRET), used ARVM on the day of isolation in an extracellular buffer containing (mM): NaCl 137, KCl 5.4, MgCl2 0.5, CaCl2 1.0, NaH2PO4 0.33, glucose 5.5, HEPES 5 (pH 7.4).Adult rat ventricular myocytes (ARVM) were enzymatically isolated from the hearts of male Wistar rats using a standard procedure outlined elsewhere 2.2For FRET imaging experiments only, ARVM were plated in minimum essential medium (MEM) containing insulin–transferrin–selenium complex , bovine serum albumin (1 mg/ml), 2,3-butanedione monoxime (10 mM) and penicillin–streptomycin. After incubation for 2 h, the cells were transduced with adenoviral constructs containing protein kinase A (PKA)- or exchange protein activated by cAMP (Epac)-based cAMP biosensors, as described previously 2.3Selective β2-AR stimulation was achieved with the β2 agonist zinterol  in combination with CGP20712A , a specific β1-AR antagonist. For all protocols, cells were exposed to CGP for at least 5 min prior to addition of solution containing ZNT and CGP. Baseline recordings were made in the presence of CGP.2.42+-containing isolation solution ) supplemented with 1–2 mM methyl-β-cyclodextrin (MBCD); cultured cells were incubated under the same conditions with MBCD dissolved in MEM-based culture medium. MBCD solution was replaced with fresh solution and treated cells used within 3 h. We have previously shown that incubation of myocytes with 1–2 mM MBCD at 37 °C for 1 h removes cholesterol and Cav-3 from buoyant caveolar membrane fractions Freshly isolated ARVM were incubated for 1 h at 37 °C in Ca2.5Peptides corresponding to the 11-residue trans-activating transcriptional activator sequence (TAT) from HIV-1, N-terminally linked via 4 glycine residues to the 20-residue caveolin scaffolding domain of Cav-3 (C3SD) or a scrambled version of this (Scram) were synthesised . Complete peptide sequences were: TAT-C3SD, YGRKKRRQRRRGGGGDGVWRVSYTTFTVSKYWCYR; TAT-Scram, YGRKKRRQRRRGGGGYWTVYTKVDFCGSRYVRTSW. Lyophilised stock aliquots (0.5 mg) were reconstituted in water and diluted in Ca-IS. ARVM were incubated in 0.5 or 1 μM peptide-Ca-IS solution at 37 °C for 30 min. Peptide solutions ≥ 2 μM caused a marked decrease in the viability of myocyte preparations.2.62+]i transients were recorded simultaneously using digital edge-detection software  and an OptoScan monochromator  in field-stimulated ARVM (0.5 Hz) loaded with 1.5 μM fura-2, following previously described protocols . An average of 10–12 traces under basal conditions, and at steady-state following drug application, were analysed using IonWizard 6.0 software (IonOptix).Unloaded shortening and  glycerol, 2% [w/v] SDS, 1x protease  and phosphatase  inhibitor cocktails). Samples were immediately frozen and stored at − 20 °C until required; 5% (v/v) β-mercaptoethanol was added and samples heated to 95 °C for 5 min before electrophoresis. Proteins were separated by SDS-PAGE, transferred to PVDF membranes and antibody steps were performed as described in Calaghan et al. Myocyte populations used to measure phospho-protein responses to β2-AR stimulation were field-stimulated (0.5 Hz) during drug treatment, then immediately homogenised in immunoblot sample buffer adenine) hydrochloride was from Calbiochem . Fura-2AM was from Molecular Probes. β2-AR, Gαi3, AC5/6, and GRK2 antibodies were obtained from Santa Cruz Biotechnology . PLB, PLB phospho-Ser16, RyR phospho-Ser2809 and RyR phospho-Ser2030 were from Badrilla  23/24 antisera, calyculin-A, milrinone and rolipram were purchased from Cell Signaling Technology . The PKA-RII antibody was from Millipore . The PDE3A antibody was purchased from FabGennix . The PDE4D antibody was a kind gift from Prof. Miles Houslay  All materials were purchased from Sigma-Aldrich  unless otherwise stated. Zinterol hydrochloride was from Tocris Bioscience  and EHNA  was determined by the Student's t-test.3In the last decade, studies of the role of caveolae in cAMP signalling in the ventricular myocyte have been conducted primarily in neonatal cells 2+]i transient amplitude (2.2 ± 3.1%) upon application of ZNT in the presence of CGP in control myocytes. By contrast, MBCD-treated ARVM showed a robust inotropic response to ZNT application (70.2 ± 9.7%) associated with a corresponding increase in the amplitude of the [Ca2+]i transient (21.1 ± 4.1%). A small positive lusitropic response  and increase in rate of [Ca2+]i transient decay (5.0 ± 1.9% decrease in t0.5 decay) were seen with β2-AR stimulation in control cells; this was markedly enhanced (P < 0.001) in MBCD-treated ARVMs (to 13.3 ± 1.3% decrease in t0.5 relax and 16.5 ± 1.9% decrease in t0.5 decay)E, F. ThuThe effects of MBCD can be ascribed solely to its ability to scavenge cholesterol (rather than any non-specific actions of such a large oligosaccharide), as the inotropic and lusitropic responses to β2-AR stimulation were attenuated  by conjugating 1 mM MBCD with cholesterol in a molar ratio of 1:8 G, H.2+ channel have been shown to be closely associated in caveolar microdomains in the ARVM ICa,L was a likely candidate for the observed functional consequences of MBCD treatment. However, as ICa,L in either control or MBCD-treated ARVMs, although both groups showed robust increases in current after application of the adenylyl cyclase (AC) activator forskolin (FSK). These data suggest that the β2-AR contractile effects emerging after cholesterol depletion are not due to modulation of ICa,L.Recent studies have suggested that the cAMP signals generated by β2-AR activation are small in magnitude and transient in nature 2-AR activation in cholesterol-depleted ARVM using different FRET-based cAMP biosensors. Experiments using biosensors necessitated the maintenance of cells in culture for 48–72 h. Although we have previously reported a reduction in total cellular cholesterol in ARVM cultured for 72 h, MBCD is still effective in reducing cellular cholesterol in these cells 2+]i transient amplitude responses to ZNT.Next we sought to describe the temporal and spatial characteristics of the cAMP signal underlying the robust inotropic and lusitropic response to βICa,L; we have recently shown that β1-AR stimulation with isoproterenol (1 nM), which gives an Epac response equivalent to that seen here with ZNT, increases ICa,L by 15% In subsequent experiments, we determined the effect of cholesterol depletion on cAMP responses indexed with a different biosensor engineered around the cAMP binding domain from type 2 exchange protein activated by cAMP (Epac2). As this sensor lacks the normal tethering sequence found in the full-length Epac2 protein, it accesses the cytosolic compartment of the cardiac myocyte and reports cAMP signals that are qualitatively distinct from those indexed by the PKA-based sensor 16, a modification which relieves its inherent inhibition of SR Ca2+-ATPase, SERCA, and thus promotes re-uptake of Ca2+2+ release channel in ARVM, RyR2, is found predominantly within junctional SR and can be positively modulated by PKA phosphorylation at Ser2809 and Ser20302+ de-sensitising effect via dual phosphorylation of TnI at Ser23 and Ser242+]i transients and shortening in these cells. By contrast, β2-AR activation in cholesterol-depleted ARVM produced a marked increase in Ser16 phosphorylated PLB (pPLB), in agreement with the effects of MBCD on [Ca2+]i transient amplitude and t0.5 decay described in 2809) in MBCD-treated cells. In the latter case this could be related to previous reports of a high background phosphorylation at Ser2809 in the absence of any cAMP-raising stimulus 2030, but we were unable to detect a phospho-RyR signal in either group of cells, even following treatment with 100 nM isoproterenol (data not shown). From the results displayed in Given that PKA-RII is found in many different regions of the adult cardiac myocyte, our next aim was to identify which cellular compartments are accessed by the observed ZNT-mediated mobilisation of cAMP in MBCD-treated cells. We addressed this question by measuring PKA-dependent phosphorylation of 3 distinct targets following β2-AR stimulation. The SR protein PLB is found in non-junctional SR and is phosphorylated by PKA at Ser16 pPLB after β2-AR activation which is not significantly different from that seen under basal conditions (P > 0.05). By contrast, myocytes treated with TAT-C3SD peptide show a markedly enhanced β2-AR pPLB signal relative to CGP background level  which competes with endogenous C3SD for the same intracellular binding partners diminish the effect of PDE inhibition on β2-AR pPLB responses, cAMP compartmentation by PDE 3 or 4 at the level of the SR cannot be reliant on intact caveolae/rafts.Our next aim was to reconcile data showing a role for PDEs Next, using the same approach, we tested the effect of inhibitors of PP  and PI3K (LY294002) on the ZNT-induced phosphorylation status of PLB. In control myocytes there was a trend for both calyculin-A and LY294002 to enhance phosphorylation of PLB during β2-AR stimulation, but this only attained significance for calyculin-A (P < 0.05) H, I. In 2+]i transient amplitude responses to β2-AR stimulation . The identical effects of PDE2 or 3 inhibition in control vs MBCD cells suggest that PDE2/3 inhibition and MBCD exert independent influences on β2-AR cell function. The same comparison between control and MBCD-treated cells was not possible with PDE4 inhibition as application of rolipram (200 nM – 1 μM) elicited spontaneous contractile events in MBCD cells , and further decreased t0.5 relaxation and t0.5 transient decay (by 11.3 ± 4.9 and 15.7 ± 2.2% respectively). The effects of calyculin-A on shortening and [Ca2+]i transient amplitude were significantly attenuated (P < 0.05) in MBCD cells compared with controls (to 51.9 ± 15.4% and 10.8 ± 4.4% respectively)(t0.5 relaxation) A, C. Furaxation) B, but thnt decay D. The atcontrols H. This a2+]i transient magnitude, the rate of relaxation and the rate of transient decay in response to ZNT. None of the LY294002-induced changes in cell shortening and [Ca2+]i transient parameters was significantly different between control and MBCD-treated groups , suggesting that PI3K control of the β2 AR signal is not dependent on caveolae.The effect of PI3K inhibition on functional responses to β2-AR stimulation is shown in 2+]i when PDE4 was inhibited in MBCD-treated cells. Comparison of datasets using pPLB and cell function as indices of cAMP signals demonstrates that we were only able to detect a significant contribution from PI3K to restriction of the β2-AR cAMP signal using functional endpoints. The disparity between these results may reflect the fact that PI3K effects are not mediated through changes in a cAMP compartment that contains PLB. However, an alternative explanation is that the statistical power of our functional experiments is higher because we were able to measure the effect of ZNT alone and ZNT plus inhibitor in the same cell.Together these functional data suggest that PDE3, PI3K and phosphatases make a significant contribution to compartmentation of β2 cAMP signalling in the control AVRM, which is in agreement with previous reports According to our premise that if a particular regulatory element were dependent on intact caveolae, the effect of inhibition of this element would be reduced in MBCD-treated cells, these data identify, for the first time, phosphatases as caveolae-dependent factors which compartmentalise β2-AR signalling.how caveolae control cAMP production and phosphatase activity, we performed sucrose gradient fractionation to look at the distribution of relevant signal components between caveolar and non-caveolar domains in control cells. As shown in Although caveolar-dependent phosphatases contribute to compartmentalisation of β2-AR signalling in the ventricular cell, this cannot be the only mechanism by which caveolae act in this regard because, as shown using FRET-based biosensors, disruption of caveolae with MBCD also increases cAMP levels in a PKA RII compartment. The level of cAMP in the cell will represent a balance between cAMP production (by AC) and degradation (by PDEs). Our data suggest that caveolae do not contribute to compartmentalisation of cAMP through effects on PDE activity, therefore we conclude that caveolae must regulate cAMP production by AC. β2-ARs couple sequentially to Gs and Gi proteins 4Compartmentation of cAMP is a fundamental strategy which allows cells to orchestrate complex receptor signalling with a single second messenger. Over the last decade a body of evidence has accumulated which suggests that Gi coupling, protein phosphatases and PDEs play essential roles in creating the distinct β2-AR cAMP-dependent signature in the ventricular myocyte. At the same time, a number of groups, including our own, have shown that compartmentation of the β2-AR pathway is dependent on the caveolar microdomain.Here, for the first time, we reconcile these data by showing how the spatial characteristics of the cAMP signal and PKA-dependent phosphorylation following β2-AR stimulation are dependent on caveolae, and how PDEs, PP and PI3K contribute to caveolar control.The 2 essential aspects of our experimental design were to use the most physiologically relevant model (adult ventricular myocytes expressing endogenous β-AR) and to achieve selective activation of the β2-AR (through use of the β2-AR agonist ZNT with a selective β1 antagonist CGP). As we predicted our approach has revealed discrepancies with other studies using neonatal myocytes and and/or other drugs to activate the β2-AR.2+]i transient amplitude or ICa,L in response to β2-AR stimulation with 10 μM ZNT in the presence of 300 nM CGP. This is consistent with other work in these cells using this combination of ZNT and CGP 2+ uptake and/or release, with no contribution from ICa,L .In the present study, control ARVM showed no change in shortening, i transient decay in response to β2-AR stimulation. However, at this stage we cannot exclude the possibility that changes in PP activity could also contribute to altered phosphorylation of PLB.A key question for the present study was how the functional effects of β2-AR stimulation with zinterol in MBCD-treated cells relate to changes in the spatial characteristics of the cAMP signal and the resulting PKA-dependent phosphorylation. Both PDE and PP have been linked with control of the β2-AR signal, and compartmentation may occur at the level of cAMP and protein phosphorylation independently. We used FRET-based biosensors that allow measurement of cAMP in real-time in 2 different cellular compartments to show that disruption of caveolae with MBCD reveals a nascent β2-AR stimulated rise in cAMP specifically in PKA-RII regions of the cell. This rise in cAMP in a PKA-RII compartment is associated with a selective increase in phosphorylation of the SR protein PLB, which suggests that the increase in cAMP occurs specifically in the PLB-containing compartment of the SR. Indeed, this accords with the fact that MBCD enhances the rate of i and contractility together as an index of the sum of all phosphorylation events occurring following β2-AR stimulation, we see an additional contribution from PI3K in control cells, but the pattern of response to all inhibitors between control and MBCD-treated cells is maintained. Together these data show quite clearly that the control of the β2-AR signal by PDEs is independent of caveolae, whilst control by PP is dependent on an intact caveolar domain.Having established phosphorylation of PLB as a robust index of the β2-AR signal seen when caveolae are manipulated by MBCD or TAT-C3SD, we were able to test how the various components linked with compartmentalisation of this pathway are involved in caveolar control. These data show that, whilst PDE3, PDE4 and PP contribute to compartmentation of the β2-AR signal in control cells, only the PP effect is dependent on caveolae. When we take changes in i in MBCD-treated cells. Shown are representative myocyte shortening (A) and [Ca2+]i transient (B) traces from a MBCD-treated ARVM perfused with 300 nM CGP alone (CGP), 300 nM CGP plus 10 μM ZNT (CGP + ZNT), and finally 300 nM CGP plus 10 μM ZNT plus 200 nM rolipram (CGP + ZNT + ROL). Unstable and dysfunctional contraction is matched with aberrant [Ca2+]i transients in the CGP + ZNT + ROL traces.PDE4 inhibition causes dysregulation of [Cavia coupling to Gs; the resulting cAMP/PKA signal may reach PLB, but a counterbalancing level of PP1 activity precludes a functional response (A). In addition, a PKA-induced switch in β2-AR coupling from Gs to Gi abrogates cAMP production, cutting the signal off at the source (B). After disruption of caveolae, a stronger and more propagating cAMP signal tips the balance of PLB phosphorylation/dephosphorylation in favour of phosphorylation (C). The more propagating cAMP signal could also give rise to PKA-dependent phosphorylation and activation of I-1 (D). This would result in inhibition of PP1 and a further imbalance of the kinase/phosphatase activity ratio. As a result, PLB loses its tight association with, and ability to inhibit, SERCA2, so Ca2+ re-uptake into the SR is stimulated. EXT., extracellular; INT., intracellular; SR; sarcoplasmic reticulum.Schematic representation of the mechanisms which govern compartmentation of β2-AR signalling by caveolae. We indicate that β2-AR-containing caveolae are in relatively close proximity to regions of the longitudinal SR containing PLB. Recent evidence promotes the inclusion of PLB in a macromolecular complex with PP1, PP inhibitor-1 (I-1), SERCA2, and PKA, orchestrated by long isoforms of AKAP18 (δ and γ) Supplement R1 FINAL.The following are the supplementary materials related to this article.None declared.


Microsatellites, or simple sequence repeats (SSRs), are tandemly repeated DNA sequences, including tandem copies of specific sequences no longer than six bases, that are distributed in the genome. SSR has been used as a molecular marker because it is easy to detect and is used in a range of applications, including genetic diversity, genome mapping, and marker assisted selection. It is also very mutable because of slipping in the DNA polymerase during DNA replication. This unique mutation increases the insertion/deletion (INDELs) mutation frequency to a high ratio - more than other types of molecular markers such as single nucleotide polymorphism (SNPs).SNPs are more frequent than INDELs. Therefore, all designed algorithms for sequence alignment fit the vast majority of the genomic sequence without considering microsatellite regions, as unique sequences that require special consideration. The old algorithm is limited in its application because there are many overlaps between different repeat units which result in false evolutionary relationships.To overcome the limitation of the aligning algorithm when dealing with SSR loci, a new algorithm was developed using PERL script with a Tk graphical interface. This program is based on aligning sequences after determining the repeated units first, and the last SSR nucleotides positions. This results in a shifting process according to the inserted repeated unit type.When studying the phylogenic relations before and after applying the new algorithm, many differences in the trees were obtained by increasing the SSR length and complexity. However, less distance between different linage had been observed after applying the new algorithm.The new algorithm produces better estimates for aligning SSR loci because it reflects more reliable evolutionary relations between different linages. It reduces overlapping during SSR alignment, which results in a more realistic phylogenic relationship. However the average of mutations in SSR loci is 10-2 in one generation [SSRs are highly mutable loci . In animeat loci  2- Identify the sequences that do not match the first repeated unit from the beginning of the selected SSR region3- Do this for each repeated unita. Put the tandem repeat in a temporary arrayb. Check if the next nucleotides match the next repeated unitc. If not, put the unmatched nucleotides in another temporary arrayd. Fill the gaps to the longest sequence of the repeats in the same arraye. Merge the temporary arrays4- Put your results instead of the SSR region.See the additional file The sequence case A contained a simple SSR with the tandem TA, which represents 15.4% of the whole sequence. After applying the alignment in the MEGA 4 alignment and our modifications, one major difference was shown clearly in the gap sites in some sequences Figure . HoweverThe sequence case B contained a compound SSR with the tandem TA and CA, which represents 25.2% of the whole sequence. The length was increased from 397 bp to 413 bp after applying the new algorithm. However, the phylogenic trees indicated that 50% of the samples showed a similar cluster before and after the new algorithm being applied . The length of this tandem repeats represents 38% of the whole sequence. The whole sequence length was changed after the new algorithm was applied from 479 bp to 539 bp. The cluster analysis resulted in completely different phylogenic trees before and after applying the new algorithm Figure .The overall pairwise value (PV) for cases A, B, C, and D before applying the new algorithm indicated that these values were increased whenever the sequence contained more repeated units Figure . In contCase E showed a compound-imperfect SSR repeat with the tandems GAA, GAT, and GAGGAT respectively. This imperfect SSR represents 9.4% of the sequence tested in case E. The alignment process showed clear differences before and after the SSR region was treated with the new algorithm Figure . DespiteThe main limitation with the new algorithm is in determining the gap position when applied to an imperfect SSR. According to Kruglyak  and BandWe can deduce from the last examples that (1) the new algorithm could be a powerful tool for compound SSRs, but less so for a simple SSR, (2) it increase the similarity between sequences during alignment by minimizing the overlaps between different repeated units, and (3) it might be necessary to apply it on sequences containing long and complicated SSRs.SALT is a new tool for making an alignment for SSR loci using the new algorithm. It was written using the PERL programming language. Figure The input file should be aligned sequences in fasta format or in .txt format:1. The first line contains the number of samples, followed by any kind of separator (space or tab...) and, subsequently, the number of nucleotides.2. Each of the next lines contains the name of the allele, followed by any kind of separator, then the sequence; thereafter press the Enter button to start the next allele.See the additional file SALT is a new tool to overcome limitations when aligning SSR loci based on the new shifting algorithm proposed in this paper. This tool is essential when aligning compound or imperfect SSRs, which contain many overlaps between repeated units, and when aligning them using the usual methods. The newly developed tool gives a better alignment estimate for such regions.Five different sequences Table  of SSR mThe authors declare that they have no competing interests.AJ planned the study, wrote the PERL script, and developed the phylogenic and alignment analysis. AH was involved in the discussion of the analysis. FCO made substantial contributions towards improving the content and gave final approval to the version to be published. All the authors drafted the manuscript.All authors read and approved the final manuscript.An animation describes the algorithmClick here for fileA compressed file contains the program (SALT.pl) and the sample data used in this research (the folder: Sample DATA). (This file could be run with winrar software https://www.win-rar.com)Click here for file


Spinal and bulbar muscular atrophy , a late-onset neuromuscular disorder, is caused by expansion of the polymorphic polyglutamine tract in the androgen receptor (AR). The AR is a ligand-activated transcription factor, but plays roles in other cellular pathways. In SBMA, selective motor neuron degeneration occurs in the brainstem and spinal cord, thus the causes of neuronal dysfunction have been studied. However, pathogenic pathways in muscles may also be involved. Cultured cells, fly and mouse models are used to study the molecular mechanisms leading to SBMA. Both the structure of the polyglutamine-expanded AR (polyQ AR) and its interactions with other proteins are altered relative to the normal AR. The ligand-dependent translocation of the polyQ AR to the nucleus appears to be critical, as are interdomain interactions. The polyQ AR, or fragments thereof, can form nuclear inclusions, but their pathogenic or protective nature is unclear. Other data suggests soluble polyQ AR oligomers can be harmful. Post-translational modifications such as phosphorylation, acetylation, and ubiquitination influence AR function and modulate the deleterious effects of the polyQ AR. Transcriptional dysregulation is highly likely to be a factor in SBMA; deregulation of non-genomic AR signaling may also be involved. Studies on polyQ AR-protein degradation suggest inhibition of the ubiquitin proteasome system and changes to autophagic pathways may be relevant. Mitochondrial function and axonal transport may also be affected by the polyQ AR. Androgens, acting through the AR, can be neurotrophic and are important in muscle development; hence both loss of normal AR functions and gain of novel harmful functions by the polyQ AR can contribute to neurodegeneration and muscular atrophy. Thus investigations into polyQ AR function have shown that multiple complex mechanisms lead to the initiation and progression of SBMA. AR point mutations, insertions, and deletions lead to varying degrees of AIS, the underlying cause of SBMA is an expansion of the polymorphic AR CAG repeat encoding a glutamine tract  that alters the AR’s normal functions and physiological roles. This article refers to the polyglutamine-expanded AR as the polyQ AR, in order to differentiate it from mutant ARs found in other diseases, and primarily focuses on investigations into the molecular pathogenesis of SBMA published since our last review  gene are associated with spinal and bulbar muscular atrophy , an adult-onset neuromuscular disease, androgen insensitivity syndrome (AIS), and prostate cancer , dentatorubral-pallidoluysian atrophy (DRPLA), and six spinocerebellar ataxias [SCA1, SCA2, SCA3 (Machado-Joseph disease), SCA-6, SCA-7, SCA-17  confirms a diagnosis of SBMA   Table  (Thomas m) Table , as the Tfm mice. These results lead to the conclusion that the abnormalities in testicular morphology in AR113Q were mediated not only by a partial loss of AR function, but also reflect a “toxic” gain-of-function due to AR polyglutamine tract expansion. Unexpectedly, transgenic mice that overexpress the WT rat AR (22 Q) under control of the human skeletal α-actin (HSA) promoter solely in their skeletal muscle fibers reproduced many neuromuscular features of SBMA model mice  had a relatively limited amount of stable secondary structure. However, for the AR-NTD45Q, a small, but measureable increase in α-helix content and small decrease in the β structures was noted  exhibited delayed onset and reduction of motor deficits compared to AR112Q mice. The nuclear accumulation of ARdNLS112Q in the spinal motor neurons of the ARdNLS112Q mice was also deferred relative to the AR112Q mice. Interestingly, in PC12 cells expressing a polyQ AR with a constitutive-acting NLS (NLSX3-AR76Q), although the NLSX3-AR76Q protein localized to the nucleus in the absence of DHT, neither intranuclear inclusions nor cell death were observed. However, upon DHT treatment of the NLSX3-AR76Q cells, inclusions consisting of N-terminal AR fragment formed, and levels of cell death increased.Drosophila model, transgenic flies that expressed the AR73Q protein with mutations in the NLS  were generated  can directly affect aggregation rate, the SDS solubility and the subcellular localization of aggregates  resulted in increased Akt activation, reduced polyQ AR aggregation and pathology in muscles, improved motor function and increased survival  within the AR hinge region is regulated by several acetyltransferases including p300, p300/CBP-associated factor (P/CAF), and TIF60 (Tat-interactive protein). Androgen-induced acetylation of AR can regulate DNA-binding and enhances AR transcriptional activity in a promoter-dependent context  to proteins, is mediated by activating, conjugating and ligating enzymes, and reversed by a family of SUMO-specific proteases (SENPs)   expression can also be altered due to the polyQ AR  gene and increased expression of the CUGBP1 RNA-binding protein has been demonstrated in SBMA AR113Q knock-in mice  minigene construct was transfected into HeLa cells as a reporter to determine the effect of the wt or polyQ AR on RNA splicing. Expression of the AR24Q in the presence of ligand or the AR112Q in the absence or presence of ligand resulted in increased cTNT exon 5 inclusion. Transcription and RNA processing of the mouse mammary tumor virus (MMTV) driven-calcitonin/calcitonin gene-related peptide (CT/CGRP) minigene is affected by steroid hormone receptors  and p54nrb , which processes short lived and misfolded proteins for degradation  decreased when HEK293-GFPu cells expressing the polyQ AR were treated with androgens, suggesting that proteasome activity was inhibited by the polyQ AR , an ER protein quality control pathway, was found to be induced in skeletal muscle from SBMA patients and male AR113Q knock-in mice -null mouse embryonic fibroblasts did not increase Hsp70 and Hsp40 levels, but still prevented AR112Q aggregation, possibly by inhibiting Hsp90-mediated retrograde trafficking  has been broadened to include the possibility that this disorder may also have a myogenic contribution. The AR113Q knock-in mice developed early androgen-dependent neuromuscular weakness, and myopathic and neurogenic skeletal muscle pathology was observed before neuronal intranuclear inclusions were seen in the spinal cord  signaling due to transcriptional dysregulation of TGF-β receptor II (TβRII) has been described. The effects of TGF-β are mediated by transmembrane receptor serine/threonine kinase complex consisting of TβRI and TβRII. TGF-β binding to the complex results in phosphorylation of Smad 2 and 3 (pSmad2/3), which then translocates to the nucleus to regulate gene transcription. Nuclear translocation of pSmad2/3 was suppressed in the spinal cord motor neurons of AR97Q transgenic mice and SBMA patients  or transcription factors activating expression of these mitochondrial proteins, or by interacting with proteins that affect mitochondrial function  is known to regulate mitochondrial biogenesis and function. In ligand-treated AR65Q MN-1 cells, PGC-1β mRNA levels were decreased relative to cells expressing the wt AR , and is essential for the growth and survival of neurons. Abnormalities in axonal transport have been implicated in neurodegenerative diseases , the Drosophila homolog of human retinoblastoma protein (Rb), was found to be a neuroprotective factor  could be identified. Although an interaction of the polyQ AR with a number of genes was proposed, there are a number of post-transcriptional and translation regulatory mechanisms that can alter gene expression profiles. Nevertheless, the approaches taken to carry out high-throughput screens in Drosophila are resulting in novel observations of polyQ AR functions that may contribute to SBMA.Specific RNAi lines for candidate genes selected from known AR coregulators described in the AR mutations database [Given that SBMA is a ligand-dependent polyglutamine disease, clinical trials to reduce testosterone or DHT levels in men have been attempted, but showed only limited improvements in certain disease features (Ranganathan and Fischbeck, It is known that androgens can be neurotrophic and are important in muscle development. In retrospect, it is perhaps not surprising that there are both neurodegenerative and neuromuscular components in SBMA, which is caused by polyglutamine tract expansion in the receptor that mediates the actions of androgens. The increase in the length of the polyglutamine tract in the AR is somewhat analogous to the insertion of a new domain through alternative splicing, which generates a novel protein. The polyQ AR retains many of the function of the wt AR, however, it fails to perform as well as the wt AR in certain roles, and gains novel, often deleterious, properties Figure ; Table 2The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ar depleted mice (MARKO) by combining the floxed allele of Ar with the MMTV-cre transgene on an MMTV-NeuNT background and compared them to littermate MMTV-NeuNT, fl/+Ar control females. Heterozygous MARKO females displayed reduced levels of AR in mammary glands with mosaic AR expression in ductal epithelium. The loss of AR dramatically accelerated the onset of MMTV-NeuNT tumors in female MARKO mice. In this report we show that accelerated MMTV-NeuNT-dependent tumorigenesis is due specifically to the loss of AR, as hormonal levels, estrogen and progesterone receptors expression, and MMTV-NeuNT expression were similar between MARKO and control groups. MMTV-NeuNT induced tumors in both cohorts displayed distinct loss of AR in addition to ERα, PR, and the pioneer factor FOXA1. Erbb3 mRNA levels were significantly elevated in tumors in comparison to normal mammary glands. Thus the loss of AR in mouse mammary epithelium accelerates malignant transformation rather than the rate of tumorigenesis.Androgen receptor (AR) is commonly expressed in both the epithelium of normal mammary glands and in breast cancers. AR expression in breast cancers is independent of estrogen receptor alpha (ERα) status and is frequently associated with overexpression of the ERBB2 oncogene. AR signaling effects on breast cancer progression may depend on ERα and ERBB2 status. Up to 30% of human breast cancers are driven by overactive ERBB2 signaling and it is not clear whether AR expression affects any steps of tumor progression in this cohort of patients. To test this, we generated mammary specific  The majority of breast cancers originate in epithelial cells that line the ducts of the mammary glands. A number of alterations lead to the development of distinct tumor types from these cells. Two of the best described and therapeutically exploited drivers of transformation are estrogen receptor alpha (ERα) and v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian), ERBB2, also known as human epidermal growth factor receptor 2 (HER2/Neu). However, additional markers are sought that denote molecular signatures that are better able to predict tumor progression, therapeutic response, and probability of recurrence.Erbb3 in mammary epithelial cells caused a decrease in ERBB2 induced tumor incidence from 93.3% to 6.7% Erbb4 ablation did not affect latency or histological grade of MMTV-Neu mouse mammary tumors According to different reports, 15 to 30% of breast cancers are driven by overexpression of ERBB2 Esr1 or Pr genes knocked out fail to develop functional mammary glands Estrogen, progesterone, and androgen receptors  are highly expressed in mammary epithelium and are essential for mammary gland development, function, and carcinogenesis. ERα inhibitors, such as tamoxifen, have been a mainstay of cancer prevention trials AR is expressed in the epithelium of the normal mammary gland and in approximately 70–90% of invasive breast cancers, a frequency comparable to that of ERα (70–80%) or PR (50–70%) Ar allele specifically in the mammary gland epithelium (MARKO). Remarkably, the first tumors in female MARKO mice were detected almost 100 days earlier than in control mice. Similar to human ERBB2 driven breast cancer, tumors in ERBB2 transgenic animals show simultaneous increase in endogenous ERBB3 but not ERBB4 expression Erα, Pr, Ar and their pioneer factor Foxa1. Our data suggest that ERBB2/ERBB3 driven tumorigenesis is opposed by AR and its loss results in earlier tumor development.Since AR is frequently expressed in ERBB2 amplified tumors, we asked how the loss of AR signaling would affect tumor incidence and progression in this cohort. We described here analysis of breast tumorigenesis in mice with overexpression of an activated form of ERBB2 and conditional deletion of the ad libitum. Transgenic mice (FVB-Tg(MMTV-ErbB2)NK1Mul/J) containing the activated rat ERBB2 oncogene targeted to the mammary epithelium by the MMTV-LTR promoter (hereafter referred to as MMTV-NeuNT) flAr) were described previously ERBB2 and cre transgenes and wild-type, floxed and deleted (ΔAr) Ar alleles as described in the original publications. MMTV-cre, MMTV-NeuNT, fl/+Ar females and MMTV-cre, MMTV-NeuNT, fl/ArY males  and control MMTV-NeuNT, fl/+Ar females and MMTV-NeuNT, fl/ArY males were produced according to the breeding scheme shown in Animal studies were conducted in accordance with the humane standards of animal care, as outlined in the US National Institutes of Health Guide for the Care and Use of Laboratory Animals and all procedures were approved by the Florida International University Institutional Animal Care and Use Committee. All mice were maintained in a temperature controlled room, with 12-h light, 12-h dark photocycle and fed Teklad global 18% protein rodent diet chow  and fresh water MARKO females (n = 19) and males (n = 33) and littermate controls  were used in the survival study. All females were kept as virgins for the entire period of study. Mice were palpated at least twice a week to detect tumors. Tumor bearing mice were kept until they met euthanasia criteria which included tumor burden of 10% of the body weight or more, significant loss of weight, visible signs of distress, huddled posture, immobility, moribund appearance. The age of mice when a tumor was first detected (incidence) and the age of mice when euthanized due to tumor burden  were recorded. At the time of euthanasia serum was collected, tumors and mammary glands were removed for histological analysis, RNA and DNA isolated, and other organs were examined for metastases. All female mice were sacrificed at 400 days of age and at 450 days for males. Kaplan-Meier survival plots were generated and compared by Log-rank test statistical analyses using GraphPad Prism software . P<0.05 was considered statistically significant.2O2 in methanol for ten minutes. Endogenous biotin was blocked using the Avidin/Biotin Blocking Kit , followed by blocking with 10% normal goat serum in TBS for 45 minutes. Immunostaining for AR , ERα , and ERBB2  were performed overnight at 4°C diluted in 3% BSA in TBS. Ki67 sections were incubated in primary antibody  in TBS for one hour at room temperature. AR, ERα, and ERBB2 stained sections were incubated with biotinylated goat anti-rabbit secondary antibody for 30 min at room temperature and then with streptavidin conjugated peroxidase for 30 min at room temperature. Ki67 was labeled using the Vectastain ABC kit . Staining was developed for all proteins using the ImmPACT DAB Peroxidase Substrate Kit (Vector Laboratories) and all slides were counterstained with haematoxylin.Mammary glands and tumors were fixed in 4% paraformaldehyde at 4°C overnight. Deparaffinized 5 µm sections of both tumors and normal mammary glands were immunostained for ERα, AR, ERBB2, and Ki67. All washes were done in TBS with 0.05% Tween-20. Antigen retrieval for ERBB2 was achieved by boiling for one minute, followed by fifteen minutes at 90°C in 1 mM EDTA pH 8.0. ERα and AR antigen retrieval was done by heating to 99°C in pH 6.0 10 mM sodium citrate buffer for fifteen minutes, with the addition of 1 mM EDTA for AR. Endogenous peroxidase activity was blocked using 1% HKi67 staining was quantified by counting the number of Ki67 stained cells in random fields of a total of >300 cells. Final counts were expressed as a percentage of cells positive for Ki67. AR staining was quantified by counting the number of positively stained luminal epithelial cells from randomly selected ducts. AR staining was determined as a percentage of the total number of luminal epithelial cells.Largest cellular compartment of the mouse mammary gland is fat. To remove fat cells, excised mammary glands were incubated in 1% w/v collagenase  dissolved in Earle’s Balanced Salt Solution at 37°C with constant agitation for 1 hour. Dissociated tissue was centrifuged at 1000 rpm for 5 min and upper layer of fat cells and overlaying buffer removed. An aliquot of pelleted cells was taken for DNA extraction using DNeasy Blood & Tissue Kit  and the remaining cells were resuspended in TRIzol reagent . RNA was extracted according to the manufacturer’s instructions. Five micrograms of total RNA was reverse transcribed to cDNA using the GoScript cDNA synthesis kit . Quantitative PCR analysis of gene expression was performed using Roche Universal ProbeLibrary assays on a Roche 480 LightCycler. For a complete list of primers see Mice were anesthetized by isoflurane inhalation , and blood collected by ocular orbital bleeding. Serum was isolated by centrifugation and stored at –20°C until analysis. Testosterone and estradiol measurements were performed by radioimmunoassay (RIA) and ELISA  respectively in the University of Virginia Center for Research in Reproduction Ligand Assay and Analysis Core . Sensitivity of the testosterone RIA assay was 10 ng/dL. Sensitivity of the estradiol ELISA assay was 7 pg/ml.t-test for two groups and one-way ANOVA for multiple group comparisons were used to assess significance of differences. Differences were expressed as mean ±SEM; P<0.05 was considered significant. Tumor incidence data was analyzed using Gehan-Breslow-Wilcoxon test. All analyses were performed using the GraphPad Software package.Student fl/flAr mice to obtain MMTV-cre, MMTV-NeuNT, flAr/+  MARKO mice and MMTV-NeuNT, flAr/+ female  control littermates , receptor activator of nuclear factor kappa-B ligand (Rankl), and wingless-type MMTV integration site family, member 4 (Wnt4) were also unchanged in non-tumor bearing mammary glands of MARKO mice compared to controls . Increased ERBB2 protein levels were detected in tumors  (Similarly to y glands . We obse 0.4385) . Correla 0.4385)  and AR ( 0.4385)  in MMTV-Areg, RankL and Wnt4) were examined. As was observed for the receptors, expression of these target genes was significantly reduced in MMTV-NeuNT tumors compared to normal mammary glands . Pr is an ERα target gene and therefore the reduced expression of Pr and downstream genes may be the consequence of reduced ERα expression. This data further confirms that steroid receptor signaling is reduced in MMTV-NeuNT tumors. No significant difference in expression of Areg, RankL, or Wnt4 was detected between the tumors of control and MARKO mice . Expression of the rat Erbb2 transgene was detected only in MMTV-NeuNT mice  compared with the incidence reported for the original parent colony ((FVB-Tg(MMTV-ErbB2)NK1Mul/J), which reported 50% incidence in female mice by 25 weeks of age fl/flAr mice are on a C57BL/6 background. C57BL/6 mice are reported to be more resistant to multiple types of tumors In the current study, we noticed a considerable delay in the onset of tumors in control mice  (n = 12) and MARKO (B) (n = 8) tumors stained for Ki67. (C) Staining in A and B were quantified and are shown as the percentage of Ki67 positive cells. Scale bar = 50 µm.(TIF)Click here for additional data file.Figure S2Erα (A), Pr (B), Areg (C), RankL (D), Wnt4 (E). Expression of each gene was normalized to 18S.Steroid receptor signaling is intact in normal MMTV-NeuNT expressing mammary glands. MMTV-NeuNT expressing non-tumor bearing mammary glands from Control (n = 24) and MARKO (n = 15) mice were dissociated from the fat pad. RNA was analyzed for the expression of (TIF)Click here for additional data file.Table S1Sequences of the forward and reverse primers used for the corresponding gene expression analysis by qPCR.(XLSX)Click here for additional data file.


The optimal setting and content of primary health care rehabilitation of older people is not known. Our aim was to study independence, institutionalization, death and treatment costs 18 months after primary care rehabilitation of older people in two different settings.2, ANCOVA, Regression and Kaplan-Meier analyses.Eighteen months follow-up of an open, prospective study comparing the outcome of multi-disciplinary rehabilitation of older people, in a structured and intensive Primary care dedicated inpatient rehabilitation  versus a less structured and less intensive Primary care nursing home rehabilitation . Participants: 302 patients, disabled from stroke, hip-fracture, osteoarthritis and other chronic diseases, aged ≥65years, assessed to have a rehabilitation potential and being referred from general hospital or own residence. Outcome measures: Primary: Independence, assessed by Sunnaas ADL Index(SI). Secondary: Hospital and short-term nursing home length of stay (LOS); institutionalization, measured by institutional residence rate; death; and costs of rehabilitation and care. Statistical tests: T-tests, Correlation tests, Pearson’s χOverall SI scores were 26.1 (SD 7.2) compared to 27.0 (SD 5.7) at the end of rehabilitation, a statistically, but not clinically significant reduction (p=0.003 95%CI(0.3-1.5)). The PCDIR patients scored 2.2points higher in SI than the PCNHR patients, adjusted for age, gender, baseline MMSE and SI scores ). Out of 49 patients staying >28 days in short-term nursing homes, PCNHR-patients stayed significantly longer than PCDIR-patients , p=0.05). The institutionalization increased in PCNHR , but not in PCDIR . The overall one year mortality rate was 9.6%. Average costs were substantially higher for PCNHR versus PCDIR. The difference per patient was 3528€ for rehabilitation ), and 10134€ for the at-home care ). The total costs of rehabilitation and care were 18702€ (=1.6 times) higher for PCNHR than for PCDIR.At 18 months follow-up the PCDIR-patients maintained higher levels of independence, spent fewer days in short-term nursing homes, and did not increase the institutionalization compared to PCNHR. The costs of rehabilitation and care were substantially lower for PCDIR. More communities should consider adopting the PCDIR model.Clinicaltrials.gov ID NCT01457300 The main goal of rehabilitation is to achieve optimal functioning in interaction with the environment. Older pRehabilitation of older people is provided at the specialized-, intermediate- and primary health care level. At each level there are different rehabilitation programmes. Specialized- and intermediate rehabilitation can be inpatient, outpatient or home based and adapted for older patients with specific or different diagnoses. However, it is not clear if any of these programmes are cost-effective.At the specialized level it has been shown that rehabilitation of older patients with different diagnoses in geriatric hospital departments improves function and reduces institutionalization and mortality, to a higher degree than in usual care. GeriatrHowever, there is little information about short- and long-term outcomes and costs of comprehensive primary health care rehabilitation of older people.Due to the increased proportion of older people in the society, the shortage of hospital beds and a limited number of specialists in geriatric and rehabilitation medicine, it is important to study if a proportion of rehabilitation of older people can be managed successfully at the primary health care level. In a previous study we demonstrated that older patients disabled due to different conditions who received multi-disciplinary, structured and intensive rehabilitation in a primary health care inpatient dedicated rehabilitation centre (PCDIR) resulted in a higher level of independence within a shorter rehabilitation period as compared to standard primary health care rehabilitation in short-term beds in nursing homes (PCNHR). This difference sustained at three months follow-up,17. In tThe primary aim of the study was to compare the level of independence of older patients 18 months after PCDIR and PCNHR and to study how this was influenced by patient characteristics, baseline diagnosis, cognitive and emotional status, and the duration and method of rehabilitation.A secondary aim was to analyse hospital and short-term nursing home LOS, institutional residence rate and mortality during 18 months after the rehabilitation, and to examine how these variables were influenced by patient characteristics, baseline diagnosis, cognitive and emotional status, and the rehabilitation method.A tertiary aim was to study the costs of rehabilitation and care in the two rehabilitation models.In Norway the health care is mainly public and is divided into the specialized and the primary levels. Specialized rehabilitation services are provided both by the public and private health care, mainly in inpatient settings. From 2006 the private rehabilitation institutions have been partly funded through the public specialized health care system by a national agreement. The primary level rehabilitation services for older people are mainly provided in short-term beds in nursing homes, beds which are also intended to serve the relief-, palliative- and sub-acute care needs. Some municipalities have Home based rehabilitation served by multi-disciplinary ambulatory teams and some have dedicated inpatient facilities, as in the present study, but these services are in a minority. Like in other countries Norway has also through the last two decades developed some intermediate care rehabilitation services based on a shared care between the specialized and primary health care.This was an 18 months follow-up of an open, prospective comparative observational study.The study was carried out in two districts in the county of Vestfold, Norway. The number of inhabitants and the demographic, rural and urban distribution of people in the two districts were similar. In one district the multi-disciplinary primary care based rehabilitation of older patients was provided in a dedicated inpatient centre (PCDIR ), and in the other district in short-term beds in nursing homes (PCNHR). The key features of the setting and content of the two rehabilitation models are shown in TableThe study population was disabled older people living in the two districts described above. They were admitted to rehabilitation either post-acute from the district general hospital or from their own residences. Inclusion criteria were both genders and age ≥ 65years. The referral diagnoses were disability due to stroke, osteoarthritis, hip fracture and “others” . Only patients considered to have a rehabilitation potential were included. Rehabilitation potential was defined as the physiological and psychological possibilities of a disabled patient to restore, improve or maintain an optimal level of function and quality of life.The validated scale Sunnaas ADL Index, SI was the MMSE, Mini Mental Status Evaluation[aluation, measurealuation,21. In oSCL-10, Symptom Check List-10[ List-10 is a valOther secondary outcome variables were hospital and short-term nursing home LOS, institutionalization as measured by institutional residence rate, and mortality during 18 months after the rehabilitation. The source of this information was the GP- and nursing care files of the patients and the official Norwegian Death Registry.Age, gender, marital status and diagnostic group were recorded at baseline. Type of residence was recorded at baseline and at 18 months follow-up.Cost calculations were based on average costs per patient according to the 2009 price level. (8 Norwegian kroner (NOK)=1 Euro(€)). The per patient PCDIR costs (2750NOK=343€/day) and the hospital costs (4000NOK=500€/day) were given from the official accounts of the specific institutions. The per patient nursing home costs (2280NOK=285€/day) were given from Statistics Norway. The perA two points difference in SI between the two models was judged to be clinically significant. Power calculation estimated a need for including 100 patients in each model, based on a beta of 0.90, an alpha of < 0.05 and SD=4.3 in SI. We decided to include 200 patients in PCDIR to ensure enough patients for subgroup analyses.2 test. Differences in SI gain between the groups were analysed by ANCOVA  to correct for SI imbalance at baseline[Data were analyzed in SPSS version 19.0 for Windows. Two groups of continuous, symmetrically distributed variables were compared by T-tests, and several groups by one way ANOVA (posthoc test if p<0.05). Asymmetrically continuous variables were compared by Mann–Whitney Wilcoxon-test. Correlations between continuous variables were analysed by Pearson’s  or Spearman’s  correlation coefficient. Categorical variables were compared by Pearson’s χbaseline. PossiblThe study was approved by the Regional Ethics Committee for Medical Research and by the Norwegian Social Science Data Services.The study clinicaltrials.gov ID is NCT01457300.In total 302 patients were recruited into the study at baseline, 202 into PCDIR and 100 into PCNHR. Eligible patients were recruited consecutively throughout the recruitment period. All eligible patients were asked and all but one accepted and gave informed consent to participate in the study. Consent was given on admission to the rehabilitation. Two of the patients in PCDIR were excluded shortly after inclusion due to a serious stroke and a leg amputation, respectively. Totally 43 patients died during the 18 months follow-up period, and two patients were lost to follow-up, which left 255 patients for follow-up assessment at 18 months.TableThe patients scored 26.1 (SD 7.2) points in SI at 18 months compared to 27.0 (SD 5.7) points at end of the rehabilitation period, a statistically, but not clinically significant reduction of 0.9 point (p=0.003 95%CI(0.3-1.5), Paired Samples T-T).The predictor analyses showed that SI at 18 months follow-up was independent of gender, marital status, diagnoses, emotional status and the duration of the rehabilitation and was predicted by age, cognitive status and the rehabilitation method. The exact results were that if other variables were kept constant, a one year higher age meant a 0.1 point lower level of SI, a one point higher MMSE score meant a 0.5 point higher level of SI and a change in rehabilitation method from PCNHR to PCDIR meant a 2.2 points higher level of SI Table.Ninety-four (37%) of the patients had short-term nursing home stays, and the patients in PCNHR had longer LOS compared to PCDIR Table. Sixty sSixteen (11.8%) of the patients aged ≥80years resided in a nursing home at 18 months follow-up (9 (9.8%) in PCDIR and 7(15.6%) in PCNHR), compared to no patients at baseline. The proportion of patients residing in a care-flat or nursing home increased significantly in PCNHR, (from 12(12.0%) to 25(28.1%) ), but not in PCDIR (from 28(16.9%) to 32(19.3%) (McNemar p=0.45)).Forty-three of the 298 patients  died during the study period, giving a one year mortality of 9.6%. The patients who died were older than the surviving patients and had lower SI at the beginning and end of the rehabilitation period, , SI at beginning: 20.9 versus 23.3, p=0.04 95%CI(0.1-4.7), SI at end: 24.4 versus 27.0, p=0.03 95%CI(0.2-5.0) - Independent Samples T-test). The difference in survival curves for the patients in the two rehabilitation models was not statistically significant ) and a one point higher SI at end of rehabilitation meant 5.4 fewer days in nursing homes (p<0.001 95%CI(−3.1—7.7)).The average rehabilitation costs were 3 528€ higher per patient in PCNHR compared to PCDIR and the at-home care costs were 10 134€ higher. Both differences were statistically significant  outcomes compared to standard community or general hospital care,29,33,34Cost-saving effects of different rehabilitation strategies are unclear, and it is difficult to compare costs across countries since both the reimbursement systems, delivery agreements and the price levels differ. Norwegian community hospitals are likely to provide health care at lower costs than alternative models of care, like general hospitals, nursing homes and at-home care. A commuThe PCDIR model includes the main features of the WHO rehabilitation cycle. We beliThis study shows that disabled older people who receive multi-disciplinary PCDIR, maintain higher levels of independence, spend fewer days in short-term nursing homes and do not have increased institutionalization during 18 months follow-up, compared to disabled older people who receive multi-disciplinary PCNHR. The PCDIR model is shown to be both more effective and less expensive. To sustain independence and reduce institutionalization and treatment costs among older people, more communities should consider adopting the PCDIR model, which includes the main features of the WHO rehabilitation cycle, into the primary health care.PCDIR: Primary Care Dedicated Inpatient Rehabilitation; PCNHR: Primary Care Nursing Home Rehabilitation; SI: Sunnaas ADL Index; MMSE: Mini Mental Status Evaluation; SCL10: Symptom Checklist 10; LOS: Length of stay.The authors declare that they have no competing interests.IJ conceived the study and contributed substantially to its design and the collection of data. She performed the statistical analyses, interpreted the data and drafted the manuscript in collaboration with MB, who was the main supervisor. IJ wrote the paper. MB, ML and JKS contributed substantially to the design of the study. ML and JKS participated in the interpretation of the data and revised the manuscript critically for important intellectual content. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6963/12/400/prepub


Spectroscopic and X-ray single-crystal diffraction analyses determined the structures of the four β-diketiminates. While thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) showed two distinct endothermic peaks for each β-diketiminate at temperatures of 92.55°C and 221.50°C (4a), 93.51°C and 238.82°C (4b), 109.60°C and 329.22°C (4c), 115.43°C and 243.25°C (4d), respectively, corresponding to their melting and boiling points.Four novel unsymmetrical β-diketiminates 2-amino-4-(phenyl)imino-2-pentene ( In addition, the solid state structures of the compounds 4a-d have been analyzed by single crystal X-ray diffraction.The availability of straightforward, multigram syntheses for many classes of β-diketiminates has generated widespread popularity of the ligand for coordination and organometallic chemistry. Prototypical symmetrical β-diketimines with N-aryl substituents can be synthesized in one step from commercially available anilines and diones through simple condensation reactions (McGeachin 4a-d were synthesized in two steps: (1) condensation of acetylacetone with one equiv. of primary aromatic amine 2,6-diisopropylaniline in methanol, with formic acid as catalyst, afforded the (Z)-4-pent-3-en-2-one intermediate 3; (2) another equiv. of the other aromatic amine  was pre-treated with para-toluenesulfonic acid in a 1 : 1 ratio for 3 h to afford para-toluenesulfonate, which was then reacted with the (Z)-4-pent-3-en-2-one intermediate 3 to give unsymmetrical β-diketimines 4a-d in good yields. In the second step, the one-pot reaction of (Z)-4-pent-3-en-2-one 3, aromatic amine and para-toluenesulfonic acid failed to give any target product; the reaction of (Z)-4-pent-3-en-2-one with the para-toluenesulfonic acid took place instead, preventing further reaction with the aromatic amine.The synthetic pathway for unsymmetrical β-diketiminates described in this work is outlined in Scheme 1H NMR spectra of all the unsymmetrical β-diketimines show a characteristic downfield shift in the range δ = 12.87-13.14 ppm for the NH proton and high field shift in the range δ = 4.79-4.84 ppm for the methyne proton attributable to the formation of unsymmetrical β-diketimines from (Z)-4-pent-3-en-2-one and amines. Two sharp singlets observed at the range 1.69-1.83 and 1.56-1.65 ppm are assigned to the protons of the two methyl groups (CH3C=NAr and CH3CNHAr) of the unsymmetrical β-diketiminates. The resonance due to the four CH3 protons (CH(CH3)2) were observed as a two doublets at the range 1.15-1.19 and 1.09-1.14 ppm while that of CH appeared as a septet at the range 3.12-3.18 ppm.The 13C NMR is in good agreement with the proposed unsymmetrical β-diketiminate structures as well. The 13C-NMR spectra of unsymmetrical β-diketiminates showed a peak at the range 161.8-164.1 ppm which is assigned to carbon of the C=N group. The methyne carbon appears at the range 95.9-96.6 ppm. Four peaks at the range 24.3-28.7, 20.3-20.6, 21–22.8 and 20.7-21.2 ppm are due to carbons of the six methyl groups 2) respectively. In addition, their identities have also been confirmed by a molecular ion peak [M+] from GC-MS mass spectra.The 4a-d are recorded in the solid state using the KBr disc technique at the region from 400 to 4000 cm-1. The (C=N) bands are observed at 1645–1557 (ν) cm-1; the position of these bands varies with the molecular structure, though no regularity can be pointed out. The bands at the range 3055–3072 (ν) cm-1 is typical of the NH group. Weak to medium absorptions around 3100–3000 cm-1 observed corresponding to the =C–H stretch of aromatic ring.The FT-IR spectra for the compounds 4a-d were grown by the slow evaporation method using methanol as the solvent at room temperature. The solid state structures of 4a-d with an atom-numbering scheme are shown in Figures 4a-d are displaced in Figures 4a-d is shown in Table 4a-d shows that they are coplanar. Compounds 4a-d have two aromatic rings and a central linkage. Compounds 4a-d crystallized in the triclinic space group P4a-d is planar; the bond lengths  259.39, found: 259.21. Anal. Calcd for C17H25NO: C, 78.72; H, 9.71; N, 5.40; Found: C, 78.61; H, 9.59; N, 5.36. IR  ν: 3418 (m), 2972 (w), 16015 (s), 1573 (m), 1515 (m), 1467 (w), 1411 (m), 1397 (m), 1344 (m), 1299 (w), 1203 (w), 1137 (m), 1076 (w), 983 (m), 842 (w), 722 (w), 601 (m), 513 (m), 484 (w).2-amino-4-(phenyl)imino-2-pentene (4a). Aniline , and para-toluenesulfonic acid monohydrate  in 100 mL of toluene were stirred for approximately 3 h at room temperature. To the obtained yellow suspension, 4-amino-3-penten-2-one  was added. A Dean-Stark apparatus was attached and the mixture was heated at reflux for 24 h to remove the water. The reaction mixture was cooled to room temperature and all the volatiles were removed under reduced pressure to give a yellow solid. The solid was treated with diethyl ether (100 mL), water (100 mL) and sodium carbonate , and the obtained mixture was kept stirring. After complete dissolution, the aqueous phase was separated and extracted with diethyl ether. The combined organic phase was dried over MgSO4 and rotary evaporated to dryness under reduced pressure to afford a brownish yellow solid. Yellow crystals  were obtained after recrystallization from methanol. 1H NMR  δ (ppm): 13.14 , 7.16-7.14 , 7.10-7.09 , 6.97-6.94 , 6.88-6.85 , 4.84 N), 3.18 2), 1.82 , 1.63 , 1.19 2), 1.14 2). 13C NMR  δ (ppm): 163.1 (NCCH3), 157.3 (NCCH3), 144.9 (Ar-C), 142.8 (Ar-C), 141.1 (Ar-C), 129.2 (Ar-C), 125.2 (Ar-C), 123.4 (Ar-C), 123.3 (Ar-C), 122.7 (Ar-C), 96.6 (CH), 28.7 (CH3C=NAr), 24.3 (CH(CH3)2), 22.7 (CH(CH3)2), 21.0 (CH(CH3)2), 20.6 (CH3CNHAr). Melting point: 93.0°C. Mass data (TOF MS EI+): Calcd for C23H30N2 [M+] 334.50, found: 334.49. Anal. Calcd for C23H30N2: C, 82.59; H, 9.04; N, 8.37; Found: C, 82.55; H, 9.01; N, 8.41. IR  ν: 3055 (w), 2960 (w), 2920 (w), 2875 (w), 1925 (w), 1873 (w), 1800 (w),1740 (w), 1630 (s), 1547 (s), 1489 (w), 1360 (m), 1275 (m), 1260 (m), 1155 (s), 1100 (w), 1030 (w), 800 (s), 798 (s), 750 (s), 699 (m), 595 (w), 501 (w), 425 (m).2-amino-4-(4-methylphenyl)imino-2-pentene (4b). β-Diketiminate 4b was synthesized by the same procedure as 4a. Yield . 1H NMR  δ (ppm): 13.11 , 7.13-7.11 , 6.89-6.85 , 4.83 N), 3.16 2), 2.05 ,1.83 , 1.63 , 1.17 2), 1.13 2). 13C NMR  δ (ppm): 163.8 (NCCH3), 156.6 (NCCH3), 143.7 (Ar-C), 141.6 (Ar-C), 140.5 (Ar-C), 132.8 (Ar-C), 129.8 (Ar-C), 124.8 (Ar-C), 123.4 (Ar-C), 123.1 (Ar-C), 96.4 (CH), 28.7 (CH3Ar), 24.3 (CH3C=NAr), 22.7 (CH(CH3)2), 21.1 (CH(CH3)2), 20.7 (CH(CH3)2), 20.5 (CH3CNHAr). Melting point: 94.0°C Mass data (TOF MS EI+): Calcd for C24H32N2 [M+] 348.52, found: 348.47. Anal. Calcd for C24H32N2: C, 82.71; H, 9.25; N, 8.04; Found: C, 82.69; H, 9.17; N, 8.03. IR  ν: 3149 (w), 3072 (w), 3015 (w), 2960 (m), 1635 (w), 1598 (s), 1542 (m), 1468 (m), 1439 (m), 1388 (w), 1255 (m), 1236 (s), 1180 (w), 1120 (m), 1105 (m), 1037 (m), 760 (m), 741(m), 664 (w), 615 (w), 602 (w), 557 (w).2-amino-4-(4-methoxyphenyl)imino-2-pentene (4c). β-Diketiminate 4c was synthesized by the same procedure as 4a. Yield . 1H NMR  δ (ppm): 13.04 , 7.14-7.11 , 6.87-6.84 , 6.65-6.63 , 4.84 N), 3.26 , 3.17 2), 1.81 , 1.65 , 1.19 2), 1.14 2). 13C NMR  δ (ppm): 164.1 (NCCH3), 156.8 (NCCH3), 156.6 (Ar-C), 144.2 (Ar-C), 140.2 (Ar-C), 136.6 (Ar-C), 125.0 (Ar-C), 124.6 (Ar-C), 123.4 (Ar-C), 114.5 (Ar-C), 95.9 (CH), 54.9 (OCH3Ar), 28.7 (CH3C=NAr), 24.2 (CH(CH3)2), 22.8 (CH(CH3)2), 21.2 (CH(CH3)2), 20.3 (CH3CNHAr). Melting point: 110.0°C. Mass data (TOF MS EI+): Calcd for C24H32N2O [M+] 364.52, found: 364.36. Anal. Calcd for C24H32N2O: C, 79.08; H, 8.85; N, 7.68; Found: C, 79.10; H, 8.72; N, 7.57. IR  ν: 3057 (m), 2971 (m), 2925 (w), 2878 (m), 2832 (w), 1631 (m), 1548 (m), 1520 (s), 1472 (s), 1400 (s), 1320 (m), 1289 (m), 1260 (w), 1251 (m), 1233 (w), 1169 (m), 1115 (m), 930 (w), 795 (m), 759 (m), 753 (w), 740 (m), 679 (w), 641 (w), 597 (m), 578 (w), 415 (m).2-amino-4-(4-methoxyphenyl)imino-2-pentene (4c). β-Diketiminate 4d was synthesized by the same procedure as 4a. Yield . 1H NMR  δ (ppm): 12.87 , 7.14-7.08 , 7.00-6.98 , 6.63-6.60 , 4.79 N), 3.12 2), 1.69 , 1.56 , 1.15 2), 1.09 2). 13C NMR  δ (ppm): 161.8 (NCCH3), 159.2 (NCCH3), 144.9 (Ar-C), 142.2 (Ar-C), 141.0 (Ar-C), 129.2 (Ar-C), 128.3 (Ar-C), 125.9 (Ar-C), 123.6 (Ar-C), 123.5 (Ar-C), 96.6 (CH), 28.7 (CH3C=NAr), 24.4 (CH(CH3)2), 22.7 (CH(CH3)2), 20.7 (CH(CH3)2), 20.6 (CH3CNHAr). Melting point: 116.0°C. Mass data (TOF MS EI+): Calcd for C23H29ClN2 [M+] 368.94, found: 368.92. Anal. Calcd for C23H29ClN2: C, 74.88; H, 7.92; Cl, 9.61; N, 7.59; Found: C, 74.89; H, 7.90; Cl, 9.58; N, 7.55. IR  ν: 3062 (s), 2960 (m), 1670 (s), 1558 (m), 1530 (w), 1495 (m), 1450 (m), 1417 (s), 1325 (m), 1271 (w), 1239 (m), 1215 (w), 1182 (m), 1120 (m), 1051 (m), 1030 (m), 930 (w), 864 (m), 837 (w), 800 (w), 745 (m), 677 (w), 635 (w).


However, these receptors regulate transcription programs that are largely receptor-specific. To address the role of the DBD in and of itself in ensuring specificity of androgen receptor (AR) binding to chromatin in vivo, we used SPARKI knock-in mice whose AR DBD has the second zinc finger replaced by that of the glucocorticoid receptor. Comparison of AR-binding events in epididymides and prostates of wild-type (wt) and SPARKI mice revealed that AR achieves selective chromatin binding through a less stringent sequence requirement for the 3′-hexamer. In particular, a T at position 12 in the second hexamer is dispensable for wt AR but mandatory for SPARKI AR binding, and only a G at position 11 is highly conserved among wt AR-preferred response elements. Genome-wide AR-binding events agree with the respective transcriptome profiles, in that attenuated AR binding in SPARKI mouse epididymis correlates with blunted androgen response in vivo. Collectively, AR-selective actions in vivo rely on relaxed rather than increased stringency of cis-elements on chromatin. These elements are, in turn, poorly recognized by other class I steroid receptors.The DNA-binding domains (DBDs) of class I steroid receptors—androgen, glucocorticoid, progesterone and mineralocorticoid receptors—recognize a similar  The DNA-binding domain (DBD) of class I steroid receptors—AR, glucocorticoid receptor (GR), progesterone receptor (PR) and mineralocorticoid receptor (MR)—is highly conserved. They all recognize a similar palindromic response element that comprises an inverted repeat of the 5′-AGAACA-3′ hexamer with a 3-nt spacer, usually termed as a canonical androgen/glucocorticoid response element (ARE/GRE)  that regulates expression of androgen-dependent genes by binding to ARE/GRE) . The firo assays . In trano assays . One-halcis-elements that receptor DBDs recognize on chromatin. Selective AREs resembling direct repeats of the 5′-AGAACA-3′ hexamer are not recognized by GR or MR in vitro  AR  AR . A trans (wt) AR . SPARKI  (wt) AR .in vivo model system to investigate the importance of DBD structure in and of itself in setting apart genome-wide androgen-specific responses from those potentially regulated by glucocorticoids as well. Here, we compared AR-binding events and transcription programs in epididymides and prostates of wt and SPARKI male mice to delineate potential genome-wide rules behind androgen-selective responses. Our results show that, counter-intuitively, AR selectivity is achieved by relaxed rather than tightened cis-element stringency at chromatin binding sites which, in turn, attenuates binding of other class I steroid receptors to these cis-elements.SPARKI mice provide an excellent All the experiments were carried out using 12-week-old adult male mice of C57BL/6 strain. SPARKI knock-in mice have been described previously . Wt litt2, 5 mM ethylene glycol tetraacetic acid, 1 mM PIPES, 2 mM dithiothreitol, 1 × proteinase inhibitor cocktail; Roche Diagnostics, Indianapolis, IN, USA) using Ultra Turrax , filtered through nylon net (20 µm), and nuclei isolated by low-speed centrifugation. The crude nuclear pellet was resuspended in radioimmunoprecipitation assay buffer and sonicated using a micro-tip sonicator  to yield chromatin fragments of 100–500 bp in size. Anti-AR (Supplementary Table S1), and the results are shown as percentage of input values.ChIP assays were performed as described previously  with mod Anti-AR  and normChIP samples were processed according to Illumina’s library preparation protocol by pooling three immunoprecipitates for each library . In briede novo motif analysis was performed using MEME Suite  were labeled with [α-32P]dCTP by a filling-in with the Klenow fragment of DNA polymerase. Slp-MUT and Slp-HRE sequences have been described , 1 mM dithiothreitol and 0.05% Triton X-100 for 20 min on ice. DBD-bound and unbound probes were separated by electrophoresis on a 4% polyacrylamide gel. Radioactive bands were visualized with STORM 840 PhosphorImager (Molecular Dynamics).AR and GR DBD fragments of ∼11 kDa in size, corresponding to residues 533–637 of rat AR DBD and residues 432–533 of rat GR DBD, were expressed as glutathione S-transferase fusion proteins in the 1 strain . The GSTl-glutamine, penicillin (100 IU/ml)–streptomycin (100 μg/ml)  and 10% fetal calf serum (Invitrogen). For transactivation experiments, cells were seeded onto 96-well plates (8000 cells/well) in Dulbecco's modified Eagle's medium supplemented with 5% charcoal-stripped serum (Sigma-Aldrich) and transfected using GeneJuice  with 10 ng of AR or GR expression plasmid (Supplementary Table S1) and 5 ng of β-galactosidase expression plasmid . Reporter plasmids were generated as previously described  were cultured in Dulbecco's modified Eagle's medium containing 4.5 g/l glucose, 4 mM  plasmid , 100 ng escribed . Cells wescribed .n = 5 in both cases) were harvested in RNALater (Qiagen) after a 7-day treatment with testosterone or vehicle. Total RNA was isolated using RNeasy Mini Kit (Qiagen). cDNA synthesis was carried out from 2 µg of total RNA with Transcriptor High Fidelity cDNA synthesis kit using random hexamers (Roche). Quantitative reverse transcription PCR (qRT-PCR) was performed using SYBR Green mastermix (Roche) and normalized to 18 S rRNA levels. Student’s t-test (P < 0.001) was used to calculate the statistical significance of differences in gene expression in wt and SPARKI mice between testosterone- and vehicle-treated groups and between testosterone-treated wt and SPARKI groups. The primer sequences are provided in Supplementary Table S1.Caput epididymides of wt and SPARKI mice (in vivo AR-binding sites (ARBs) on epididymal chromatin were performed in biological duplicate samples. Examples of AR ChIP-seq tracks of wt and SPARKI mice are shown in Fkbp5 (FK506 binding protein 5) encoding a chaperone protein and Acox3 (acyl-coenzyme A oxidase 3) involved in fatty acid metabolism in peroxisomes. AR occupancy is indistinguishable at the Fkbp5 regulatory region between wt and SPARKI mice, which agrees with the result that Fkbp5 mRNA accumulation is induced by androgen to the same extent in epididymides of these mice (Supplementary Data sets S1 and S2).ChIP-seq analyses of ese mice . Sequencese mice ,14. Impoese mice A and by ese mice B. To incese mice . The peain vivo AR-binding events that are favored by the SPARKI receptor . Interestingly, a similar cis-element is also highly enriched among the ARBs preferred by SPARKI AR (E = 3.2e-437). In both instances, there is a high degree of sequence conservation. The best conserved nucleotides are a G at position 2 and a C at position 5, followed by an A at positions 4 and 6, and their complementary partners T, G, T and C at positions 10, 11, 12 and 14, respectively , in which the 5′ hexamer is almost identical with that described above but separated by a 3-nt gap from a hexamer with weak sequence conservation , and 21% of the SPARKI AR-preferred peaks have >4-fold enrichment over those of wt AR   B. Import set S4) B. To chaches cf.A and C. PARKI AR D.in vitro conditions. Classical and selective AREs, characterized in previous work (Electrophoretic mobility shift assay (EMSA) with AR DBD and GR DBD was used to examine the specificity of a few ARE sequences in the three categories under ous work , were usous work A and B. ous work C. Thus, via all nine AREs, confirming their functional enhancer nature . Even though intact male mice have physiological androgen levels, these differentially expressed transcripts may not all be androgen-regulated. To address this issue, we compared the Kerkhofs et al. data set (Supplementary Data set S5). Unsupervised hierarchical clustering of the 219 transcripts revealed that most of the wt-preferred genes are upregulated by androgen, whereas most of the SPARKI-preferred genes are downregulated by androgen exposure  that are stably expressed in both wt and SPARKI epididymides are shown in Fkbp5 and Acox3 . SPARKI AR has low affinity to wt AR-preferred sites and wt AR to SPARKI AR-preferred sites, whereas both receptors bind to shared sites with high affinity (Supplementary Data set S8), and 609 SPARKI AR-preferred sites have >4-fold enrichment over wt AR (Supplementary Data set S9). Importantly and similar to epididymis, the cis-element identified by de novo motif search among the wt AR-preferred ARBs in prostate shows weak conservation of the second hexamer sequence, highlighting the relaxed cis-element stringency, with the G at position 11 being an important determinant for selective AR binding in vivo  classical AREs corresponding to the canonical ARE/GRE and recognized by other class I steroid receptors as well, and (ii) selective AREs recognized primarily by AR and showing features that resemble direct repeats of the 5'-AGAACA-3' hexamer .We examined in this work the role of DBD in and of itself in assuring the selectivity of AR signaling by comparing wt and chimeric receptors. However, specific transcriptional outcome  factors . Pioneerequences ,11. Pionequences ,11,30, ain vivo binding of SPARKI AR to a subset of ARBs remain elusive. The DNA sequence in itself may act as an allosteric ligand for nuclear receptors, thereby affecting receptor conformation and activity  (to V.D.). Funding for open access charge: Sigrid Jusélius Foundation and Biocentrum Helsinki.Conflict of interest statement. None declared.


Mutation events were studied at 160 SSR loci in the S1 generation (the first generation after chromosome doubling) of the three SynDH populations. Of the 148260 SSR alleles investigated in S1 generation, only one mutation (changed number of repeats) was confirmed with a mutation rate of 6.74 × 10−6. This mutation most likely occurred in the respective F1 hybrid. In comparison with previously reported data, our results suggested that allohexaploidization of wheat did not increase SSR mutation rate.Simple sequence repeats  are known to be mutational hotspots in genomes. DNA rearrangements have also been reported to accompany allopolyploidization. A study of the effect of allopolyploidization on SSR mutation is therefore important for understanding the origin and evolutionary dynamics of SSRs in allopolyploids. Three synthesized double haploid (SynDH) populations were made from 241 interspecific F Simple sequence repeats  are a class of DNA that is composed of short tandem repeats of a basic motif of one to six nucleotides. SSRs are ubiquitous in genomes. The nature of motif repetition makes SSRs inherently instable and prone to mutation by mechanisms of replication slippage or unequal crossing over . Because−6 in fruit fly [−2 in human [−3~10−4 in crops like durum wheat [in vitro culture etc. will increase SSR mutation rates [Since it is a critical parameter in models of population genetics, the SSR mutation rate has been estimated in numerous studies ,4. The eruit fly , 10−2 inum wheat , common um wheat . ExposedBrassica [Triticale [Arabidopsis [Allopolyploidization is an important evolutionary process that merges two distinct genomes into the nucleus to form an allopolyploid species. This process is particular important to higher plants. The merger of two distinct genomes presents a “genomic shock”, as McClintock  describeBrassica –18, Tritriticale –21 and Abidopsis ,23. Allobidopsis .1 (the first generation after chromosome doubling) lines from three synthesized double haploid (SynDH) populations. Each of these SynDH lines was derived from an independently obtained interspecific F1 haploid hybrid between Triticum turgidum L. and Aegilops tauschii Coss . However, the expected SSR patterns from T. turgidum parents for marker Xcfd4  loci wPt-4658, wPt-5316 and wPt-8016 (data not shown). Since all these flanking loci in SynDH1 line 61 were derived from LDN, this novel SSR was apparently mutated from the LDN allele.SSR gain was only observed in the line 61 of SynDH1 population at locus e assays ,4, suggeXwmc312 alleles cloned from SynDH1 line 61 and its parents. A total of 50 clones were sequenced and analyzed. All of the clones contained the same SSR flanking sequences of 173 bp and differed only in the number of the GA repeats they had. However, we failed to get a clean sequence for each line, which may be caused by PCR-generated stutter [Xwmc312 alleles of AS313, AS60, LDN, and SynDH line 61 were estimated as 197 bp , 197 bp , 229 bp , and 215 bp , respectively  by the total number of possible mutant alleles (n). The n number for S1 generation of each SynDH population was estimated based on the total observed SSR alleles at all of the loci , but high frequent variations for DNA methylation. The methods used to assess DNA variation based on methyl-sensitive or nonsensitive restriction enzymes also revealed a lot of variations in allopolyploid wheat [As mentioned earlier in this communication, allopolyploidization-accompanied DNA sequence rearrangements have been reported in some allopolyploids. However, no evidence for sequence variation was found in re-synthesized cotton  and someet al.  with theid wheat –13. Howeid wheat .Xwmc312 allele observed in this study might result from DNA polymerase slippage, although the unequal recombination model could not be completely excluded [T. turgidum and Ae. tauschii. Comparing these enzymes among different species and their relationships with SSR mutation rates are necessary to further test this hypothesis.The different conclusions from different DNA types may be finally attributed to different mutation mechanisms. Ellegren  proposedexcluded . The higTriticum turgidum ssp. durum line Langdon (abbreviated as LDN), ssp. dicoccon line PI377655, ssp. turgidum lines AS313 and AS2255, Aegilops tauschii accessions AS60, AS66 and AS87, and their synthesizing double haploid populations SynDH1, SynDH2, and SynDH3 . The purified PCR products were cloned into pMD19-T vector using cloning kit from TaKaRa Biotechnology  Co., Ltd. [Mutant alleles and their parent alleles were re-amplified, cloned, and sequenced to reconfirm the mutation and to determine its nature. The re-amplified PCR products were separated by 6% polyacrylamide denaturing gels. The target fragments were cut and boiled in 30 μL ddHo., Ltd. . PositivT. turgidum and the other from Ae. tauschii; (B) only amplifying SSR products at one of the two SSR alleles from T. turgidum and Ae. tauschii.In this study, a SSR amplification pattern from a parent was estimated as an SSR allele. This estimation was supported by our mapping results that most of the 160 markers used in this study were mapped as a single locus on a chromosome based on their genotyping using the three SynDH populations . Based on) of total possible mutation events, which were calculated by summarizing all the alleles from all the plants in a SynDH population. Depending upon the nature of a SSR marker, an S1 plant with AABBDD genome composition could reveal two homologous alleles from T. turgidum and two from Ae. tauschii or only reveal two homologous alleles from one of the parents. Meanwhile, a bulked DNA of five S1 plants for each SynDH line was used. Therefore, for a S1 population, the n was estimated by the following equation:The mutation rate was calculated by dividing the allele number of observed mutants by the allele number  was confirmed with a mutation rate of 6.74 × 10−6. This SSR mutation was homozygous in the analyzed S1 plants of line SynDH1-61. Therefore, this mutation most likely occurred in the respective F1 hybrid and then homologized in the S1 via the union of unreduced gametes. Compared with previously reported data as regards durum wheat or common wheat itself, our results suggested that allohexaploidization of wheat did not increase the SSR mutation rate.In this study, mutation events were studied at 160 SSR loci in S