In this review we focus on the cardiovascular β-adrenergic changes involvement in the aging process and on similarities and differences between aging heart and heart failure.Elderly healthy individuals have a reduced exercise tolerance and a decreased left ventricle inotropic reserve related to increased vascular afterload, arterial-ventricular load mismatching, physical deconditioning and impaired autonomic regulation . Adrenergic responsiveness is altered with aging and the age-related changes are limited to the β-adrenergic receptor density reduction and to the β-adrenoceptor-G-protein(s)-adenylyl cyclase system abnormalities, while the type and level of abnormalities change with species and tissues. Epidemiological studies have shown an high incidence and prevalence of heart failure in the elderly and a great body of evidence correlate the changes of β-adrenergic system with heart failure pathogenesis. In particular it is well known that: (a) levels of cathecolamines are directly correlated with mortality and functional status in heart failure, (b) β Some studies have also reported an increase in Giα activity as a possible additional mechanism in “β-adrenergic desensitization.” Moreover, the reduction in the efficacy of cardiac β-adrenoceptor stimulation with aging could be also related to other mechanisms, such as the upregulation of G protein–coupled receptor kinases  heart failure-dependent cardiac adrenergic responsiveness reduction is related to adrenoreceptor kinases and Giα increased activities.Epidemiological studies reveal an high incidence and prevalence of heart failure in the elderly  the development of knowledge on aging heart over the years, (b) the changes involving the sympathetic system in relationship to the cardiovascular aging in different species, (c) the clinical implications of changes in β-adrenergic mechanisms in the aging heart and d) the similarity between aging and failing heart.1-AR, β2-AR, β3-AR have been recognized. A fourth subtype has been proposed and investigations have been recently clarified its functioning and localization  was described in 1948 by Alquist . Gs protein is a heterotrimetric protein made up of α, β, and γ subunits. The presence of β-AR agonists induces the dissociation of Gs protein in two subunits: α subunit and β−γ subunit. The primary effect of this dissociation is the activation of AC that catalyzes the conversion of ATP to c-AMP, a second intracellular messenger, and induces the activation of c-AMP dependent PKA. Serine and threonine residues of many regulatory proteins are phosphorylated by PKA. These regulatory proteins include: β-AR themselves, myofilament proteins (troponine I and C protein), membrane proteins . The stimulation of β-AR modifies not only the cardiac excitation and contraction but also other cellular functions such as gene transcription and growth, and can induce death. An important role for the above mentioned functions has played by the activation of Mitogenic-Activated Protein Kinase (MAPK). Moreover, these kinases are thought to be implicated in the regulation of several vital cellular processes, including differentiation, proliferation, growth, and death  MAPK. One major pathway of GPCRs-mediated activation of MAPKs is dependent on “transactivation” of a group of receptor tyrosine kinases, such as epidermal growth factor and insulin-like growth factor. Additionally the activation of p38 MAPK, also called “stress-activated protein kinase,” is associated with the initial signs of cardiac hypertrophy in response to “in vivo” pressure overload or ischemic/reperfusion injury . Then, intracellular Ca++ is removed from the cytoplasm by the SR-Ca++/ATPase pump and the Na+/Ca++ exchange. The further acceleration of Ca++ removing leads to the muscle relaxation. The maximum velocity of relaxation is defined as positive lusitropic effect. As a result of PKA activation, the β-AR stimulation triggers the G-Protein Coupled Receptor Kinase family, like GRK2. GRKs are a family of serine/threonine protein kinases that phosphorylates GPCRs only when the receptors are in the activated (agonist-bound) state. When β-ARs are stimulated by agonists, the β−γ subunits G interact with GRK2 bringing the kinase from the intracellular to the transmembranic localization, phosphorylating the β-ARs, becoming target for binding of β-Arrestin proteins. The β-Arrestin bounds to these receptors and prevents their further coupling to the G-protein, reducing the level of functional receptors, inducing the internalization of receptors and, as final result, their decreased density and desensitization -system and the lipolysis.“2-AR, one of the first receptors identified, belongs to the GPCRs family and plays an important role in the cardiovascular and respiratory physiology. Its main effects are related to vasodilatation and bronchodilatation . There is also evidence that β2-AR signaling is coupled to an independent pathway like the Na+/H+ exchanged regulatory factor  as β1-AR does , an important downstream messenger that triggers the antiapoptotic factor Akt and seems to have a cardioprotective role. , is not well clarified. Several evidence indicate that β2-AR–Gi signaling compartmentalizes the β2-AR-Gs–c-AMP signaling. Disrupting the Gi functioning by a potent Gi inhibitor like Pertussis Toxine (PTX) induces an enhance in the phosphorylation of PLB and an increased inotropic effect after β2-AR stimulation. In this occasion the β2-AR signaling is comparative to the β1-AR signaling31  , and phospholamban in the SR. Deficiencies in this pathway have been linked to cardiomyopathy in humans, due to reduced phosphorylation of downstream targets such as cardiac troponin  and non-failing hearts , the contractile response to β-adrenoceptor stimulation has been found to be strongly reduced in single myocytes from failing human ventricle, but a part of this reduction was statistically related to the patient's age  in the decline of contractility related to β-stimulated activity. In these experiments they found that β-AR number was decreased by 27% in senescent animals and the Giα activity, detected by PTX-catalyzed ADP ribosylation, was significantly increased in aged animals, while immunodetectable level of Giα was not modified. The authors concluded that increased Giα activity contributes significantly to the decreased response to β-AR stimulation in myocytes in aged guinea-pigs.Ferrara et al. , in a st1-AR or β2-AR stimulation. Moreover, the activity of β-AR kinase GRK5, and of Gi did not significantly change with aging, suggesting that the marked decreased response to β-ARs stimulation with aging in rat ventricular myocytes was linked to a decrease in both β-AR subtypes densities and a reduction in membrane AC activity, while neither GRKs nor Gi proteins appear to play a role in this mechanism. This apparent contrast with the results from Ferrara et al. (In different species (rats) Xiao et al.  found a a et al.  could beAs in the failing heart, the role of chronic sympathetic drive in the β-AR desensitization in the aging heart can be hypothesized. Because the role of GRKs in the aging heart could be defined controversial same studies have been planned to focus on the role of GRKs in the aged cardiovascular system.Schutzer et al.  examinedFor the first time Leinweber et al.  studied Heart failure and associated clinical implication induced by alterations in β-adrenoceptor mechanisms are a central problem for elderly population  in subjects free from coronary artery disease and hypertension. In fact, these studies have demonstrated no significant changes in cardiac mass in women and a decrease in left ventricular mass in men. Based on these and other studies it has been shown that there is no significant changes in left ventricular mass in the elderly women, but there is a reduction of cardiac mass in men (cardiac sarcopenia). Regarding to this Lin et al.  and calsequestrin protein. In this model a treatment with β-blockers led to an improvement of cardiac contractile functioning and an longer survival.The genetic engineering and the need to establish a quite similar model of human disease have driven the scientific research to develop transgenic animal models. These model are produced by using DNA microinjections in animals or by using another methodology like the gene knock-out models. In 1996 Rockman et al. (g et al.  develope2 and β-arrestin1 are primary regulators in the endothelial function in the heart failure, while the GRK3 and GRK5 play a very important role in the cardiac myocyte functioning. The increased levels of GRK2was related to the infarction induction suggesting that this protein may precipitate the development of acute heart failure. Iaccarino et al. (2. This inverse correlation can be explained by the phsophorylation of β-ARs by GRKs and furthermore their internalization and desensitization process. Interestingly the level of GRK2 is associated to the degree of heart failure, suggesting an importance of GRKs as a heart failure progression biomarker. In 2007, for the first time, Lymperopoulos et al. (2-AR down-regulation independent of species and a GRK2 up-regulation. In 2012, in a post-infarction heart failure rat model Rengo et al. (2 and α2-AR adrenal medulla dysregulation. It has been confirmed that there is an up-regulation of adrenal medulla GRK2 and a down-regulation of α2-AR and that their blockade with β-blockers normalizes the level of GRK2. The same author (Rengo et al., 2-AR in a post-myocardial infarction heart failure rat model. This model underwent an adenoviral-mediated overexpression of β2-AR after 4 weeks of surgery procedure. It was observed that the overexpression of β2-AR improves the angiogenesis process and enhanced the coronary reserve and myocardial blood flow. This pro-angiogenic characteristic of β2-AR was associated to the activation of pro-angiogenic vascular endothelial growth factor, protein kinase B, endothelial nitric oxide synthase VEGF/PKB/eNOS pathway.The development of animal surgery techniques also helped to obtain similar rat models of heart failure. Vinge et al.  studied o et al.  found ano et al.  studied Recently returning to the well-known method of detection GRKs levels from the lymphocytes, Rengo et al.  performeCardiovascular diseases are the most common cause of death in elderly population. Increased longevity is associated to an increased heart failure morbidity with poor prognosis among elderly. For this reasons it is very important to understand and clarify the pathophysiological mechanisms that underlay the aging heart process. From the present literature it seems that “β-adrenoceptor desensitization/down-regulation” is a general and common mechanism which explains age- and heart failure-related decrease in β-adrenoceptor response to agonists. In particular, in both aging and failing hearts the decrease in β-adrenoceptor responsiveness is related to changes in G-proteins and kinases activity, although there are differences in the level and the extent of these changes among different studied species. These molecular alterations are responsible for the most structural and functional changes in aging heart. The improved understanding of this mechanism could help in the future to develop new therapy approach and ameliorate life quality among elderly population.The 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.


In turn, these properties have the potential to be developed for therapeutic use to promote immune tolerance. Here we describe the nature and functions of Nrp1, including its potential use as a therapeutic target in transplantation tolerance.In the immune system, Neuropilin-1 (Nrp1) is a molecule that plays an important role in establishing the immunological synapse between dendritic cells (DCs) and T cells. Recently, Nrp1 has been identified as a marker that seems to distinguish natural T regulatory (nTreg) cells, generated in the thymus, from inducible T regulatory (iTreg) cells raised in the periphery. Given the crucial role of both nTreg and iTreg cells in the generation and maintenance of immune tolerance, the ability to phenotypically identify each of these cell populations  Neuropilin-1 (Nrp1) is a 120–130 kDa type-I transmembrane glycoprotein with a multi-domain extracellular region, a single transmembrane helix, and a cytoplasmic domain. The extracellular region consists of two CUB domains (denoted a1/a2), two coagulation factor V/VIII homology domains (denoted b1/b2), and a MAM domain (denoted c) . The intNeuropilin-1 was first described to be involved in the development of neurons –11, wherIn addition to its expression in different cell types, an important feature of Nrp1 is its ability to bind many different ligands; these ligands control a variety of biological processes through binding to Nrp1 –19. In tXenopus laevis through screening candidate molecules implicated in retinotectal projection development  receptor (discussed later), confirming an important role for Nrp1 in axonal guidance –27. ThesNeuropilin-1 has a homologous protein called Neuropilin-2 (Nrp2), which shares 44% peptide sequence homology , 29. BotA notable feature of Nrp1 and Nrp2 is their ability to bind with relatively high affinity to several families of molecules, which involves them in a variety of physiological processes in a manner that is not yet fully understood. These molecules include Sema3 and heparin-binding members of the VEGF family that bind to Nrp1. VEGF family members are potent angiogenic molecules with chemotactic, survival, and proliferating effects in endothelial cells . Nrp1 isNeuropilin-1 is expressed in epithelial cells from different tissues , neurons, melanocytes, and keratinocytes , 41. In Neuropilin-1 is expressed by a variety of human tumor cell types , such asSema domain , which is expressed on activated lymphocytes and thymocytes. Its receptor is VESPR/CD232/plexin-C1, present in monocytes and macrophages . Sema7A integrin . Furtherintegrin .Sema6D is expressed on T cells, B cells, and NK cells. Its receptor is plexin-A1, known for being expressed specifically on DCs. Studies have demonstrated that recombinant Sema6D recognizes and binds to plexin-A1 on activated DCs, increasing IL-12 production. Additionally, Plexin-A1-knockout mice exhibit impaired T-cell responses, being resistant to experimental autoimmune encephalomyelitis (EAE). Specifically, it has been shown that Sema6D expression increases belatedly during CD4+ T-cell activation, and its blockade causes an impaired late-phase T-cell proliferation and the inhibition of CD127 expression . Also, iSema4d-knockout mice have impaired humoral responses against T-cell-dependent antigens   . Given tis (CIA) , 76.in vitro , but since there was not activation of CD4+ T-cell signaling pathways upon Nrp1 binding, the mechanism and relevant in vivo impact of this phenomenon are still unknown  and performed gene array analysis. In these experiments, Tregs could be distinguished from the CD4+CD25− T-cell counterparts by a defined group of differentially expressed genes, which contained Foxp3, KLRG1, and Nrp1. Nrp1 expression on Tregs at the plasma membrane was confirmed using antibody surface staining. Polyclonal and antigen-specific activation of Tregs in vitro demonstrated that the level of Nrp1 remains unchanged on Tregs, while Nrp1 expression on activated CD4+ T cells is almost lost  receptor . They dein vivo importance of Nrp1 expression on CD4+ T cells was determined by Solomon et al. through analysis of Nrp-1flox/floxCD4Cre conditional knockout mice where both CD4+ and CD8+ T cells lack Nrp1  in which the offspring of myelin-binding protein (MBP)-TCR-Tg mice crossed with RAG-KO mice lack nTregs, but iTregs can be detected in the periphery. In addition to this, they also tested the low dose-antigen immunization model to induce iTregs. In their experiments, they could identify and isolate iTregs to compare differentially expressed genes by gene arrays analysis, with the nTregs counterpart as control. Results from these experiments demonstrated that Nrp1 is preferentially expressed by nTregs. Nrp1− T cells express Foxp3 and have suppressive function in vitro and in vivo  and induced-Tregs (iTregs) , 86. Usi in vivo .in vivo models of iTreg generation in mucosa (lung and intestine), Weiss et al. observed the same phenomena but additionally, they tested for possible factors controlling Nrp1 expression. In this attempt, the group identified TGF-β and IL-6 to induce and inhibit Nrp1 expression, respectively  is an intracellular protein, thus it is not a useful tool to identify and isolate viable Tregs. Furthermore, Foxp3 expression in human Tregs is not characteristic of these suppressor cells compared to murine Tregs . Yadav ain vitro through treatments with anti-CD4, TGF-β, and Retinoic acid (RA) or anti-CD4 plus rapamycin, and showed that the majority of Foxp3-expressing Tregs generated under anti-CD4, TGF-β, and RA condition express Nrp1, mainly due to the expansion of nTregs instead of de novo iTregs (in vitro and in a skin transplantation setting, highlighting the role of Nrp1 in Treg cells.In murine studies, the contribution of Nrp1 biology in transplantation tolerance indicates that CD4+Nrp1+ T cells may play a suppressive role in allograft rejection. One important report shows that CD4+Nrp1+ T cells transferred into heart allograft-recipient mice extend the survival of the transplant, mainly by inhibiting the production of inflammatory cytokines, such as IFN-γ and IL-17, enriching for Tregs and inducing anergy on effector T cells . The supo iTregs . They alin vitro. Interestingly, the frequencies of CD4+Nrp1+CD25high T cells were reduced in the tumor-draining lymph nodes of cervical cancer patients after chemoradiation therapy, which was associated with a decrease in the tumor mass. Based on this data, one possibility is that the presence of human CD4+Nrp1+CD25high T in lymph nodes cooperates with the tolerogenic microenvironment of tumor burden areas (In humans, Nrp1 may be a useful indicator of the immunological status of patients. For example, Battaglia et al. analyzed the frequencies of CD4+Nrp1+ T cells in peripheral blood and lymph nodes of patients with benign diseases undergoing lymphadenectomy. Their results show that Foxp3 expression is detected in the CD25high fraction of CD4+Nrp1+ T cells from lymph nodes and not from blood, suggesting an anatomical influence in the presence of Nrp1-expressing CD4+ T cells . Importaen areas .Finally, a small study proposed the expression of Nrp1 on T cells as a putative predictor of allograft rejection, since the presence of Nrp1 on lymphocytes residing in kidney transplant biopsies decreased during acute rejection, as compared with biopsies from non-rejecting individuals , suggestImportant advances have been made in the study of Nrp1 on immune cells. While most of the available information indicates a role for Nrp1 in the formation of the immunological synapse, Nrp1 may also play a key role as a Treg marker, permitting the discrimination between nTregs and periphery-induced-Tregs. More investigation is required to clearly demonstrate that Nrp1 will be a feasible predictor of tolerance in the transplantation field since murine and human data are not compatible. Hence, the question of whether Nrp1 is a new marker for transplantation tolerance remains to be answered.Mauricio Campos-Mora wrote the manuscript and designed the figures, Rodrigo A. Morales wrote the manuscript and edited the figures, Tania Gajardo wrote the manuscript and designed the figures, Diego Catalán wrote the manuscript, and Karina Pino-Lagos wrote the manuscript and designed the figures.The 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.


Arabidopsis CPR5 in the ABA signaling and LOX pathways. The cpr5 mutant was hypersensitive to ABA in the seed germination, cotyledon greening and root growth, whereas transgenic plants overexpressing CPR5 were insensitive. Genetic analysis demonstrated that CPR5 gene may be located downstream of the ABI1 in the ABA signaling pathway. However, the cpr5 mutant showed an ABA independent drought-resistant phenotype. It was also found that the cpr5 mutant was hypersensitive to NDGA and NDGA treatment aggravated the ABA-induced delay in the seed germination and cotyledon greening. Taken together, these results suggest that the CPR5 plays a regulatory role in the regulation of seed germination and early seedling growth through ABA and LOX pathways independently.The phytohormone abscisic acid (ABA) and the lipoxygenases (LOXs) pathway play important roles in seed germination and seedling growth and development. Here, we reported on the functional characterization of  Arabidopsis thaliana. For example, ABA helps maintain seed dormancy to ensure that seed germinate under favorable conditions. Immediately after germination, ABA may inhibit the establishment and subsequent development of young seedlings, with this post-germinative arrest representing an early developmental checkpoint to slow seedling growth until better conditions arise Seed germination is the first adaptive decision in the developments of many land plants. Advances in genetics and molecular physiology have taught us much about the control of germination by the phytohormone abscisic acid (ABA) using the model plant Arabidopsis thalianaThe phytohormone abscisic acid (ABA) is well known for its regulatory roles in integrating environmental constraints with the developmental programs of plant Z,Z)-1,4-pentadiene system to produce an unsaturated fatty acid hydroperoxide, and initiates the synthesis of a group of acyclic or cyclic compounds collectively called oxylipins, which are products of fatty acid oxidation, with diverse functions in the cell. Feussner and Kindl reported that LOX is the main lipid body protein in cucumber cotyledons during the early stages of seed germination In plants, products of the lipoxygenases (LOXs) pathway have showed diverse functions involved in abiotic stress Larrea tridentate, which grows in some desert areas of southwest United States and northern Mexico Nordihydroguaiaretic acid (NDGA) is a phenolic compound that is present in high concentration in the leaves of the evergreen desert shrub Creosote bush, CPR5 gene has recently been isolated and shown to encode a protein with 5 potential transmembrane regions at the carboxy terminus, a bipartite nuclear localization signal at the amino terminus, and no sequence similarity to other known proteins CPR5 appears to act just downstream of pathogen recognition and upstream of salicylic acid in a resistance pathway dependent on NPR1 (NONEXPRESSOR OF PATHOGENESIS RELATED GENES 1) CPR5 activates the PR gene expression in the RPS2-mediated pathway cpr5 mutants exhibit defects in cell proliferation and expansion cpr5 (hys1) mutants are hyperresponsive to glucose and sucrose and prematurely accumulate senescence upregulated transcripts cpr5 alleles isolated so far exhibit early cotyledon senescence, have areas of localized cell death on the rosette leaves, and have trichomes that are glassy and reduced in size and branching CPR5 is a master regulator of cellular ROS status and/or signaling CPR5 also plays important roles in the pathway controlling postgermination arrest of development through LOX pathway and ABA signaling pathway.The Columbia, Landsberg erecta were used throughout this study. The cpr5 mutant allele used in this paper was cpr5-1−2 s−1). After one week, seedlings were potted in soil and placed in a growth room at 22°C. The ABA-insensitive mutant abi1-1 was used to generate double mutants with cpr5-1. The double mutant lines were created by cross-pollination between the relevant mutants, and putative double mutant plants were screened from the phenotype segregation ratio of the F2 progeny.The Arabidopsis thaliana ecotypes 35S-CPR5 plants, a 1695 bp KpnI-SpeI fragment containing the CPR5 (The Arabidopsis Information Resource locus At5g64930) cDNA was cloned into the vector pCanG vector and verified by sequencing, in which transgene expression is under the control of the CaMV 35S promoter. For the CPR5 promoter and GUS fusion constructure, a 741 bp promoter region just upstream of the ATG start codon of CPR5 was amplified from genomic DNA by PCR. The PCR fragment was cloned into the SalI -BamHI site of binary vector pBI101.1 to obtain a transcriptional fusion of the CPR5 promoter and the GUS coding sequence. For the functional analysis of the transmembrane domains predicted in CPR5, a truncated form, CPR5ΔTM, with last transmembrane domains deleted (residues 525 to564) (ΔTM) was also cloned into the vector pCanG. To prepare the 35S-CPR5-GFP fusion construct, the entire coding region of CPR5 was inserted directly upstream of the EGFP coding region in pBEGFP (pBEGFP is reconstructed based on pBin19). Plants were transformed with Agrobacterium tumefaciens by the floral dipping method Transgenic plants carrying constitutively expressing transgenes were generated. To produce The plants include the CPR5: GUS was assayed for the GUS color reactivity. The plant material was immersed in GUS staining solution  and incubated overnight at 37°C. The material was washed repeatedly in 95% ethanol until the tissue was bleached. The stained tissue was then observed and photographed using an Olympus BX51 Microscope .http://www.zeiss.com).35S-CPR5-GFP plants were used for GFP subcellular localization analysis. Roots of 7-day-old transgenic seedlings were used for the green fluorescence analysis  by a Carl Zeiss laser scanning system LSM 510 (http://www.digimizer.com).Plants of different genotypes were grown in the same conditions and seeds were collected at the same time. For each comparison, seeds were planted in the same plate containing MS medium  without or with different concentrations of ABA, NDGA and tea polyphenols. Plates were chilled at 4°C in the dark for 2 d (stratified) and moved to 22°C with a 16-h light/8-h dark cycle. The percentage of seed germination was scored at indicated times. Germination was defined as an obvious emergence of the radicle through the seed coat. Cotyledon greening is defined as obvious cotyledon expansion and turning green. As primary root length measurements, plates were placed vertically in growth chamber. At indicated times, the plates were scanned by an Epson perfection V200 photo scanner, and the primary root length was measured by the tool DIGIMIZER 3.2.1.0  from 3-week-old soil-grown plants were used.http://www.olympus-global.com). Measurements were performed as described by Ichida et al http://www.digimizer.com).Epidermal peels were stripped from fully expanded leaves of two week-old plants, and were floated in a solution of 30 mM KCl and 10 mM 2-(N-morpholine)-ethanesulphonic acid  in Petri dishes. ABA was added to the solution, and stomatal apertures were recorded under a light microscope  with SYBR Premix Ex Taq . Total RNA was extracted as described above. cDNAs were synthesized from 0.5 µg of total RNA using PrimeScript™ RT reagent Kit  . Each PCR reaction contained 1× SYBR Premix Ex Taq, 0.2 µM of each primer, and 2 µl of a 1∶10 dilution of the cDNA in a final volume of 20 µl. The following PCR program was used: initial denaturation, 95°C, 15 s; 40 cycles of 95°C for 4 s, 60°C for15 s and 72°C for 31 s. In melting curve analysis, PCR reactions were denatured at 95°C, re-annealed at 60°C, then a monitored release of intercalator from PCR products or primer dimmers by an increase to 95°C. cDNA quantities were calculated by ABI Prism 7300 SDS Software Ver.1.3 (Applied Biosystems Inc.), and transcript data were normalized using CPR5, the expression pattern of this gene was examined. RT-PCR analysis detected CPR5 in all tissues of Arabidopsis, including root, leaf, siliques and seeding (CPR5 promoter–β-glucuronidase (GUS) fusion was analyzed. Histochemical staining revealed GUS activity mainly at the cotyledons in seedlings  35S promoter was used to transform the cpr5-1 mutant and wild type plants. The complementation and overexpression lines had clearly increased expression of CPR5 confirmed by RT-PCR , whereas the corresponding survival rate was 47% for wild type, and 27% for the 35S-CPR5 plants  observed in germination assay scored by radicle emergence as well as in assays measuring the emergence of green cotyledons  with EGFP and permanently expressed in cpr5-1 mutant under the control of the CaMV 35S promoter. The 35S-CPR5-EGFP fusion transgenic plant can complement the phenotype of cpr5-1 mutant , it has also been proposed that the protein may be involved in a signaling cascade in which the cytoplasmic domain is proteolytically cleaved and transported into the nucleus CPR5 transcript  rha2a and ocp3 mutants are hypersensitive to ABA, while ABA induced marker genes were unaltered in both mutants.In addition, our findings that mutation or overexpression of ept ABI1 , which icpr5-1 plants are hypersensitive to ABA; this phenotype is opposite to abi1-1 mutant which is a dominant-negative type mutation. To test the genetic relationship between CPR5 and ABI1, double mutant cpr5-1 abi1-1 was generated by crossing cpr5-1 with abi1-1, and its response to ABA was compared with that of the two single mutants. Given that cpr5-1 is sensitive to ABA while abi1-1 is insensitive to ABA, we reasoned that if CPR5 act in the same pathway to ABI1, the double mutant should be exhibited either the cpr5-1 or the abi1 sensitivity to ABA. In our ABA response assays, double mutant cpr5-1 abi1-1 plants were also sensitive to ABA . Initial conversion of PUFAs by lipoxygenases (LOXs) ic lines , which sCPR5 plays a regulatory role in regulation of seed germination and early seedling growth through the ABA and LOX pathways, both pathways appear to work independently, but are both regulated by CPR5.In summary, our observations indicate that Figure S1The influence of fluridone on ABA sensitivity of cpr5-1 and 35S-CPR5 plants. Matched seed lots were pretreated with deionized water or 100 mM fluridone for 24 h at 4°C before being placed at 22°C for germination. Seeds were germinated on MS and MS medium containing 0.4 µM ABA, and grown for 5 days.(TIF)Click here for additional data file.Figure S2MeJA response analysis of cpr5-1 and 35S-CPR5 plants. Seeds of wild type, cpr5, 35S-CPR5 plants were germinated and growth for 12 days on MS medium containing 0 µM and 50 µM MeJA.(TIF)Click here for additional data file.Figure S335S-CPR5-EGFP fusion transgenic plant complements the phenotype of cpr5-1 mutant. Two-week-old seedlings of 35S-CPR5-EGFP in cpr5-1 showing the phenotype of the wild-type.(TIF)Click here for additional data file.Table S1Primers used in this study.(DOC)Click here for additional data file.


N,N-Diisopropyl-2-propylbenzamide 6-H undergoes lateral deprotonation by t-BuLi in the presence of the Lewis base PMDTA  to give a benzyllithium 6-Lil·PMDTA that incorporates a trigonal planar secondary carbanion. In the solid state, the amide directing group and the PMDTA additive work together to abstract the metal ion from the deprotonated α-C of the propyl group (4.107(4) Å). A short distance of 1.376(3) Å is observed between the deprotonated carbon centre and a planar aromatic system that shows a pattern of bond lengths which contrasts with that reported for related tertiary carbanion systems. Analogous benzylic deprotonation is seen if 6-H is treated with t-BuLi in the presence of diglyme to give 6-Lil·DGME. X-ray crystallography now shows that the metal ion more closely approaches the tertiary carbanion (2.418(6) Å) but that the planarity of the deprotonated carbon centre and the bonding pattern in the organic anion seen in the PMDTA complex are retained. DFT analysis corroborates both the short distance between aromatic ring and carbanion centre and the unperturbed nature of aromaticity in 6-Lil·L (L = Lewis base). The observation of two structure-types for the carbanion in solution is explained theoretically and by NMR spectroscopy in terms of cis and trans isomerism imparted by partial double bond character in the arene–(α-C) bond. The following abbreviations are used for NMR spectra: s = singlet, d = doublet, t = triplet, sept = septet, (v) br = (very) broad and m = multiplet.Reactions and manipulations were carried out under dry NF2, by full-matrix least squares /Σ[w(Fo2)2]}1/2 = 0.1261, conventional R = 0. 0493 for F values of 2591 reflections with Fo2 > 2σ(Fo2), GoF = 0.976 for 310 parameters. Residual electron density extrema 0.159 and −0.141 eÅ−3.6-Lil·DGME: t-BuLi  was added to a solution of 6-H  in toluene/hexane (0.3:0.1 mL) containing diglyme  under nitrogen at −78 °C. The resulting purple solution was warmed to room temperature and stored at −30 °C for 3 days to yield red crystals of 6-Lil·DGME. Yield 44 mg (45%); mp <25 °C; 1H NMR  δ 7.13–7.00 , 6.88  = 7, 8 Hz, 1H, Ar#1), 6.74  = 8 Hz, 0.15H, Ar#2), 6.63 , 6.46  = 7 Hz, 1H, Ar#1), 6.38  = 9 Hz, 1H, Ar#1), 5.69 , 5.63  = 6, 7 Hz, 1H, Ar#1), 4.4–3.6 , 3.54 , 3.39  = 4 Hz, 16H, CH2-DGME), 3.27  = 5 Hz, 16H, CH2-DGME), 3.15 , 3.08 , 3.05 , 2.69–2.58 , 2.35 , 1.79 , 1.62 , 1.61  = 7 Hz, 9H, NCMe#3), 1.60  = 7 Hz, 9H, NCMe#3), 1.36  = 7 Hz, 3H, ArCHCH2Me#1), 1.24 , 1.13 , 0.97  = 7 Hz, 9H, ArCH2CH2Me#3), 0.73  = 7 Hz, 18H, NCMe#3) ppm; {1H}13C NMR  δ 179.5 (C=O#1), 177.2 (C=O#2), 169.6 (C=O#3), 145.9 (C-Ar#1), 138.7 (C-Ar#3), 138.6 (C-Ar#3), 137.5 (C-Ar#1), 130.8 (CH-Ar#2), 130.1 (CH-Ar#1), 129.4 (CH-Ar#3), 127.8 (CH-Ar#3), 127.1 (CH-Ar#1), 126.5 (CH-Ar#2), 125.6 (CH-Ar#3), 125.0 (CH-Ar#3), 122.6 (CH-Ar#2), 112.4 (CH-Ar#1), 99.2 (C-Ar#2), 97.6 (CH-Ar#1), 71.5 (CH2-DGME), 70.1 (CH2-DGME), 64.6 (ArCH#1), 58.4 (Me-DGME), 50.1 (NCH#3), 45.4 (NCH#2), 35.3 (ArCH2#3), 24.4 (ArCH2CH2#3), 22.3 (ArCHCH2#1), 20.4 (NCMe#3), 20.2 (NCMe#3), 20.0 (NCMe2#3), 16.6 (ArCHCH2Me#1), 14.2 (ArCH2CH2Me#3) ppm; 7Li NMR spectroscopy  δ 0.62 , 0.26  ppm; anal. calcd for C22H38LiNO4: C, 68.19; H, 9.88; N, 3.61; found: C, 68.00; H, 9.62; N, 4.12; crystal data for 6-Lil·DGME: C22H38LiNO4, M = 387.47, monoclinic, space group P21/n, a = 7.8821(16), b = 18.975(4), c = 15.737(3) Å, β = 92.84(3)°, V = 2350.8(8) Å3, Z = 4, ρcalcd= 1.095 g cm−3; Mo Kα radiation, λ = 0.71073 Å, μ = 0.073 mm−1, T = 173 K. 13765 data  were collected on a Nonius Kappa CCD diffractometer. The structure was solved by direct methods and refined by full-matrix least-squares on F2 values of all data to give wR2 = {Σ[w(Fo2−Fc2)2]/Σ[w(Fo2)2]}1/2 = 0.2729, conventional R = 0. 0938 for F values of 2961 reflections with Fo2 > 2σ(Fo2), GoF = 1.040 for 253 parameters. Residual electron density extrema 0.729 and −0.616 eÅ−3.cis/trans-6-Lil·L  was carried out using the B3LYP density functional and a 6-311++G basis and Gaussian 09 .NMR spectroscopic data and DFT calculations for


Mental disorders are the main reasons for rising proportions of premature pension in most high-income countries. Although inpatient medical rehabilitation has increasingly targeted work-related stress, there is still a lack of studies on the transfer of work-specific interventions into work contexts. Therefore, we plan to evaluate an online aftercare program aiming to improve vocational reintegration after medical rehabilitation.Vocationally strained patients (n = 800) aged between 18 and 59 years with private internet access are recruited in psychosomatic, orthopedic and cardiovascular rehabilitation clinics in Germany. During inpatient rehabilitation, participants in stress management group training are cluster-randomized to the intervention or control group. The intervention group (n = 400) is offered an internet-based aftercare with weekly writing tasks and therapeutic feedback, a patient forum, a self-test and relaxation exercises. The control group (n = 400) obtains regular e-mail reminders with links to publicly accessible information about stress management and coping. Assessments are conducted at the beginning of inpatient rehabilitation, the end of inpatient rehabilitation, the end of aftercare, and 9 months later. The primary outcome is a risk score for premature pension, measured by a screening questionnaire at follow-up. Secondary outcome measures include level of vocational stress, physical and mental health, and work capacity at follow-up.We expect the intervention group to stabilize the improvements achieved during inpatient rehabilitation concerning stress management and coping, resulting in an improved vocational reintegration. The study protocol demonstrates the features of internet-based aftercare in rehabilitation.ISRCTN33957202)International Standard Randomised Controlled Trial Number Register (ISRCTN: Sick leave and premature pension are rising constantly in most high-income countries because of mental disorders -3. Job set al.) and by the ethics committees responsible for the cooperating rehabilitation clinics.The procedures set out in this protocol pertaining to the conduct, evaluation and documentation of this trial were designed to ensure that all persons involved in the trial abide by Good Clinical Practice and the ethical principles described in the current revision of the Declaration of Helsinki. The trial will be carried out in keeping with local legal and regulatory requirements.Before being admitted to the clinical trial, patients must consent to participate after the nature, scope and possible consequences of the clinical trial have been explained in a form understandable to them. The patients must give written informed consent to participate in the study, including their consent to publish.We expect a very low risk for adverse events. Nevertheless, participants have the possibility to contact the staff at the study center via e-mail or on a mobile help number with the guarantee of a response during working hours (8.00 a.m. to 5.00 p.m.) on weekdays. On weekends and at all other times, patients are referred to a Germany-wide crisis telephone number, which is indicated on our internet platform. All adverse events reported by the participants or detected by the online therapist or respective study staff will be collected during the trial and must be documented in the case report form. The clinical course of the adverse event will be followed by the principal investigators , who will contact the participants in case of a crisis for further diagnostics via telephone or for necessary referrals to practitioners near the participant.Work-related stress often leads to serious somatic and psychosomatic complaints and, vice versa, chronic diseases of different indications have a negative impact on job strain. For this complex interdependency, the German rehabilitation system has developed a focus on work-related interventions during inpatient medical rehabilitation . High quBased on previous studies, we expect good acceptance of internet-based data ascertainment and diagnostics in inpatient medical rehabilitation  and a hiOne of the other challenges of the study seems to be the lack of immediate interaction, which presumably requires more incentives for the participants than in face-to-face interactions . It remaOne restriction is that not all rehabilitation centers have the resources for online assessments during inpatient rehabilitation, specifically for an introduction to the internet platform. Following the review of Wantland and colleagues , we assuBy evaluating our online intervention comprehensively, we aim to estimate the short-term and especially the long-term effects of the program on stress management and coping concerning job strain and, finally, on successful occupational reintegration.With the cluster randomization, patients are enrolled in the study as cohorts and therefore we are confident that they are more motivated to take part and less likely to drop out of the study than in an individually randomized design. With the, for the most part, blinded inpatient training we try to enable that both intervention group and control group are treated the same during inpatient rehabilitation. Unfortunately, a completely blinded assessment of the outcome was not possible, because patients needed to be informed about the group and intervention they are randomized to, during inpatient rehabilitation. However, our main outcome is assessed by study participants, independent from the online therapist. The fact that the intervention group and control group use the same medium after inpatient rehabilitation for the follow-up assessments will help us to reduce possible confounding factors when interpreting the results of the comparative analyses. Overall, and from a practical point of view, we think that not only because of the close connection between the design of our online aftercare program and the inpatient GSA training during rehabilitation but also because of the interdisciplinary approach, combining cognitive, behavioral and psychodynamic elements in training and aftercare, our internet-based aftercare program could be a very promising supplement for inpatient rehabilitation.The first patients were enrolled to the study on 1 July 2011. Follow-up assessments for the last included patients are expected to be completed by September 2013.AVEM-44: Arbeitsbezogenes Verhaltens und Erlebensmuster Kurzform 44 ; GSA: Gesundheitstraining Stressbewältigung am Arbeitsplatz ; SIBAR: Screening Instrument zur Messung des Bedarfs an berufsbezogenen Behandlungsangeboten in der medizinischen Rehabilitation (Short Screening Instrument for the Assessment of Need for Occupation Related Treatment in Medical Rehabilitation.The authors declare that they have no competing interests.RZ wrote the first draft of the manuscript. RZ, KG, JE and MEB wrote the final draft of the manuscript and critically revised it for its intellectual content. RZ, KG, JE, MH, RJK, SSD, UK and MEB substantially contributed to the conception and the design of the study. All authors read and approved the final manuscript.


NPR1 is a gene of Arabidopsis thaliana required for the perception of salicylic acid. This perception triggers a defense response and negatively regulates the perception of jasmonates. Surprisingly, the application of methyl jasmonate also induces resistance, and NPR1 is also suspected to be relevant. Since an allelic series of npr1 was recently described, the behavior of these alleles was tested in response to methyl jasmonate.npr1s alleles and NPR1 paralogs null mutants was measured by the growth of a pathogen. We have also tested the subcellular localization of some npr1s, along with the protein-protein interactions that can be measured in yeast. The localization of the protein in npr1 alleles does not affect the response to methyl jasmonate. In fact, NPR1 is not required. The genes that are required in a redundant fashion are the BOPs. The BOPs are paralogs of NPR1, and they physically interact with the TGA family of transcription factors.The response to methyl jasmonate of different npr1 alleles have a phenotype in this response likely because they are affecting the interaction between BOPs and TGAs, and these two families of proteins are responsible for the resistance induced by methyl jasmonate in wild type plants.Some  Arabidopsis thaliana (Arabidopsis) to biotrophic pathogens like Pseudomonas spp[in planta is JA-Ile (reviewed by [Plants are constantly defending themselves against pathogens by means of a wide array of mechanisms. Some of them are pre-existing (or non inducible) and others are induced in response to the pathogen attack. Salicylic acid  is a plmonas spp. When a monas spp). SA hasmonas spp). In theiewed by ), while NON-EXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1) is the main gene required for SA perception [NPR1 in Arabidopsis [BLADE-ON-PETIOLE1 (BOP1) and BOP2 have an important role in development [NPR3 and NPR4 have a role in defense [NPR2 has been described, besides a secondary role in SA perception [TGA transcription factors whose products interact with NPR1 [rception . There abidopsis , BLADE-Oelopment , NPR3 an defense , probabl defense , and no rception . There aith NPR1 , but theNPR1 has been described as having more than one role in defense, since it is also important in the Induced Systemic Resistance . ISR iseudomonas. It has of MeJA .BOP1 was first identified by its mutant phenotype of ectopic blades along the petioles, as well as some alterations in the flowers [bop1 did not reproduce the phenotypes of ectopic blades [BOP1 was identified as paralog to NPR1[BOP2, functions redundantly with BOP1[bop1 bop2 reproduced all the developmental phenotypes of ectopic blades, but it was wild type when inoculated with Pseudomonas[ flowers . The firc blades . Once BOg to NPR1, it was with BOP1. The doueudomonas, and it eudomonas.npr1 alleles has recently been available [NPR1 in RIM is cytosolic. In this work, we show that NPR1 has no role in RIM in wild type conditions, since the genes responsible for RIM are BOP1 and BOP2, with an important part being played by the TGAs. Therefore, two genes required for the normal development of the leaf, are also required for plant defense.Since a collection of vailable , we testNPR1 has been characterized as a result of observing the response to SA of the great number of alleles described for it [NPR1 has also been described as essential for RIM, but there are differences between alleles, since npr1-1 and npr1-3 have different RIM . npr. nprNPR1n npr1-3 . Other dnt genes . These dresponse .NPR1 in RIM, and to asses the role of the cytosolic vs. nuclear localization, we tested an allelic series of 43 npr1 alleles, defined by their inability to perceive SA [npr1 alleles produced a mixture of phenotypes in RIM . The rest of alleles showed no response .In order to determine the precise role of ceive SA . The nprnpr1-22, -35 and −44, Additional file npr1-1, -40, and −56, Additional file NPR1. Then, these seven cDNAs were transiently expressed in N. benthamiana under the control of the 35S promoter and with the GFP marker. The free GFP was detectable in the nucleus and in the cytoplasm. But when the wild type and the six alleles of npr1 were expressed in the same conditions, GFP was detected mainly in the nucleus, with no difference existing between the two classes of alleles , and therefore the transgene was transferred to an npr1-1 background (RIM-) to check for complementation. Treatments with BTH and with and without glucocorticoid dexamethasone (DEX) showed that NPR1-HBD was functional . NPR1-H1 Figure b. When Dnpr1 alleles RIM+ or RIM- did not share any obvious feature, so it would be difficult to assign a precise role to the wild type gene. A critical genetic resource to discern the role of a gene is the null allele. Therefore, the response of two null alleles of npr1 to RIM was measured .The d Figure a. Both n control . These tt Figure b. Since npr1 alleles produced more SA when infected with Pto[The role of NPR1 in this response might be indirect. Thus, one scenario would be a reinforcement of the negative crosstalking between SA and MeJA.  with Pto and seem with Pto. RIM- alnpr1-1 and NahG , eds5 , and sinthesis, ), were cw Figure c, so theNPR1 was not necessary for RIM. Perhaps NPR1 and other gene(s) would be redundantly responsible of RIM, and while null npr1 alleles would have a RIM+ phenotype, some npr1 alleles could be RIM- by interacting with other protein(s) negatively. The most likely candidates for these interactions were the NPR1 paralogs, since their proteins shared the same overall structure. There are five additional paralogs of NPR1 in the Arabidopsis genome [npr1-70 with different paralogs  were analyzed for RIM  to deplM Figure b. Five oM Figure c; five oBOP1 and BOP2 were acting redundantly in RIM . Another effect of MeJA is the increase of senescence in detached leaves, measured as chlorophyll production [bop1 bop2 responded as the wild type in this particular system . If bop1 bop2 was not a MeJA signaling mutant, but specifically in RIM, it could be defective in the other signaling required for ISR; ET. It was not; when 1 mM of 1-aminocyclopropane-1-carboxylic acid  was sprayed to bop1 bop2, the resistance triggered was similar to that triggered in the wild type controls , or a si1,[jin1,). When bs Figure a. Other oduction . bop1 bom Figure b. Corona planta, ), therefronatine  grows lee Figure c. Inocule plants , did nots Figure d. etr1  was incnpr1 RIM- alleles could have a dominant negative effect on BOP activity, either directly or indirectly. To first test whether NPR1 had an effect in the interaction between BOP proteins, we used a yeast two-hybrid assay. As reported, BOP1 and BOP2 interacted with each other [NPR1 or various mutant npr1 alleles presented in Figure npr1 on RIM were a direct interaction between NPR1 and the two BOPs, the alleles that diverge in their RIM phenotype would diverge in their ability to interfere in the interaction of BOP1 and BOP2. The two classes of npr1 alleles did not have a distinct behavior , therefore the dominant negative effect did not seem to be direct.Once it was clear that both BOP genes are required for RIM, we tested the model that ch other . Next, wTGA1 and TGA7 produced a significant RIM , there BOP1 and each of the mentioned TGAs were introduced in the yeast two-hybrid system with the npr1 alleles mentioned above in a third plasmid. TGA2 and TGA6 interact differentially with BOP1 depending on the npr1 protein present  might have a functional interaction with the BOPs, which might be affected by the RIM- alleles. To test this hypothesis t Figure c. There NPR1 is an essential gene for SAR and SA perception [npr1-1, the most widely used allele, is also impaired in RIM [npr1-3 is wild type for RIM .rception . npr1-1,d in RIM . We specfor RIM ,25), and, andNPR1 npr1-3 ,19), the, theNPR1npr1 alleles are RIM+ regardless of the background . In devs  rs Figure . But theenotypes . Regardienotypes . It seemnpr1 alleles were the majority of the alleles found (32 RIM- vs. 11 RIM+). How is this compatible with the fact that the null npr1 alleles are RIM+? A possible explanation was the selection used in the screening. Since the selection was made for complete loss of SA perception, perhaps most of the RIM+ alleles had a phenotype of partial SA perception, as the null alleles. Then, the prediction would be that a good number of random alleles of npr1 would be RIM+ and partially receptive to SA. We previously showed that for SA perception, there are genetic interactions between the npr1 alleles and the NPR1 paralogs [npr1 alleles on one side and the BOPs on the other. Thus, the RIM- alleles were a phenocopy of the bop1 bop2 mutant in defense but not in development. This discrimination was a consequence of the different thresholds for the phenotype in development and defense ; NPR1-HBD in the cytoplasm did not complement npr1-1 in the RIM phenotype, but when DEX was applied there was complementation of the phenotype. Likely, when no DEX was present npr1-1 would somehow interfere with the function of the BOPs. When DEX was present, the presence of NPR1-HBD in the nucleus would trigger the degradation of both NPR1-HBD and npr1-1. If npr1-1 was degraded, the BOPs would function normally.Mechanistically, the levels of expression of the in planta, all the RIM- alleles enhance the interaction between the BOPs and the TGAs, titering out the TGAs and thus rendering them unable to fulfill their function of triggering defense. On the other hand, the RIM+ alleles , and NPR1 would not affect the interaction either way. Since in the yeast assays two out of three alleles worked as proposed in either way, it may be possible that a factor(s) is present in the plant that is not in yeast, or perhaps the fact that there are ten TGAs[NPR1 is expressed between 3 and 18 times more than BOP1 + BOP2  are required for the resistance triggered by methyl jasmonate against Pto. We propose that the phenotype of the npr1 RIM- alleles is caused by their interference between BOPs and TGAs.In sum, we have shown that, in wild type conditions, the Arabidopsis thaliana (L.) Heynh. was sown and grown as described [-2 s-1, and nights of 16 h at 19°C. Treatments, inoculations, and sampling started 30 minutes after the initiation of the artificial day to ensure reproducibility. The following genotypes were used: npr1-1 and npr1-3 [npr2, npr1-20 to npr1-71, and combinations of npr1-70 with other genotypes [35SCaMVp:NPR1HBD[sid2[eds5[NahG[npr3 and npr4[bop1-3 and bop2-1 [coi1-40 ; kant6, pan1-1 to pan1-3, tga1, and tga7[35S:BOP1 and 35S:BOP2[etr1-3 [tga2 tga5 tga6[Pseudomonas syringae pv. tomato DC3000 (Pto) was grown, inoculated and measured as described [Pto at OD600=0.1 with 0.02% Silwet L-77 . Three days later, the amount of colony forming units (cfu) per plant was quantified and represented in a logarithmic scale. When indicated, a strain of Pto lacking coronatine was used (Pto(cfa–),[escribed  in contrd npr1-3 ; npr2, nenotypes ; 35SCaMVp:NPR1HBD; sid2[30sid2[eds5; NahG[28d bop2-1 ; coi1-40 and tga7; 35S:BOP 35S:BOP2; etr1-3 2[etr1-3 ; tga2 tgtga5 tga6. Pseudomescribed . Brieflyto(cfa–),). For alNPR1 and six alleles of this gene were cloned in pDONR222 or pDONR221  and then transferred to pMDC43 [in planta with GFP and to pARC352 [, BOP1, BOP2, TGA2, TGA5, and TGA6, were cloned and then transferred to pDEST22 and pDEST32 (Invitrogen) for expression in yeast. Yeast n-hybrid analyses were done as described [N. benthamiana leaf tissue was mounted in water under a coverslip 4 days after infiltration with Agrobacterium tumefaciens containing the constructs. All imaging was conducted with a Leica TCS SL confocal laser scanning microscope  using an HCX PL APO CS 40X/1.25 oil objective to study the subcellular localization of the fluorescence-tagged proteins. Green fluorescent protein was visualized by 488-nm excitation with an Ar laser, and its emissions were examined with a band-pass filter for 500 to 530 nm. The primers used are included as Additional file amiRNA(BOP1-BOP2), the plasmid pRS300 was modified [o pMDC43  for expr pARC352  for exprescribed , and theescribed . N. bentmodified , and plamodified .Pto growth 100 μM methyl jasmonate (MeJA) in 0.1% DMSO and 0.02% Silwet L-77 (Crompton Europe Ltd) was applied by spray one day before the pathogen inoculation [To measure the effect in culation . Dexametin vitro culture, plants were grown in Johnson’s media [2PO4. When indicated, the plates were supplemented with 50 μM MeJA. The length of the roots was measured with ImageJ software [For ’s media  with 1 msoftware . Senescesoftware .Total RNA from 3-week-old Figure c or 6-weThe authors declare that they do not have any competing interest.JVC, AD, and JF performed the experiments, analyzed the data and revised the paper. PT designed the research, analyzed the data and wrote the paper. All authors read and approved the final manuscript.Figure S1. Localization of cloned npr1 alleles mentioned in the text. Figure S2. NPR1HBD treated with DEX is more sensitive to BTH. Figure S3. Expression levels of NPR1 paralogs. Figure S4. Response of bop1 bop2 to SA and BTH. Table S1. List of primers used.Click here for file


Bacterial growth kinetics and the percent of arsenite removal (PAR) were performed simultaneously with the detection of an arsenite oxidase enzyme through Dot Blot analysis.The purpose of this study was to identify arsenite-oxidizing halobacteria in samples obtained from Salar de Punta Negra, II Region of Chile. Seven bacterial isolates, numbered as isolates I to VII, grown in a culture medium with 100 ppm as NaAsOAn arsenite oxidase enzyme was detected in all isolates, expressed constitutively after 10 generations grown in the absence of As (III). Bacterial growth kinetics and corresponding PAR values showed significant fluctuations over time. PARs close to 100% were shown by isolates V, VI, and VII, at different times of the bacterial growth phase; while isolate II showed PAR values around 40%, remaining constant over time.Halobacteria from Salar de Punta Negra showed promising properties as arsenite removers under control conditions, incubation time being a critical parameter. It isIn Chile, arsenic is found in most ecosystems of the northern and central area. Geology, volcanic activity, and mining operations are responsible for the presence of arsenic concentrations in surface waters and groundwater sources. The most extensive arsenic enrichment is located between 17° 30' - 26° 05' south latitude and between 67° 00' east longitude in the Andean Highlands . The Atacama Desert is located in this vast region, where concentrations of total arsenic have been reported as high as 200 ppm (unpublished report).2O2), which have different toxicity degrees [Different water treatment technologies have been developed for arsenic removal. They are based on diverse processes such as coagulation, filtration, adsorption, and reverse-osmosis . However degrees . Thus, tAlcaligenes faecalis strains [Pseudomonas alcaligenes, Agrobacterium tumefaciens, Alkalilimnicola ehrlichii, Wautersia solanacearum, Acinetobacter calcoaceticus, and Enterobacter cloacae). Some bacteria gain energy from arsenite oxidation [in-situ bioremediation, which involves growth stimulation of native bacterial flora having metabolic capabilities to biotransform arsenite. Therefore, it is desirable to search for autochthonous organisms leading to in-situ bioremediation strategies, by making site-specific studies.The importance of microorganisms in arsenic transformation and its biogeochemical cycle has recently received much attention -12. Most strains ,7. Subsexidation -10,12; hxidation . Though in-situ bioremediation strategies were evaluated. The presence of the enzyme arsenite oxidase was analyzed by using molecular techniques.A high functional diversity of extremophiles microorganisms is developed in Highland Andean ecosystems . These mCharacterization of Bacteria Isolates: After 18 h of culture, different colonies grew on IM and CIM media plates. There were no significant differences in number and/or shape among colonies grown in either culture media , showing arsenite reductions for all isolates as compared to the control, at the same incubation time ; isolate V showed reductions up to t27; and isolate II from t4 to t27. Some of these measurements were close to the detection limit of the method (0.001 ppm), showing values close to zero, revealed at 6 h (t6) for isolate VI and at 27 h (t27) for isolate VII . It is also observed that for isolates II, IV, V, VI, and VII, bacterial growth and arsenite variation follow parallel trends between t22 and t27 , seem to indicate an adequate adaptation of isolated bacterial populations to a medium containing arsenite, despite the high toxicity induced in the medium. This adaptation could be facilitated by the fact that the isolation media was prepared with steril water from Salar de Punta Negra in order to keep the complex salt balance of the collection site . Thus, tConsidering bacterial adaptation to As III, the presence of elongated bacteroids could indicate a cellular response to metabolic stress generated by exposure to arsenic. This result is consistent with those described by different authors -19, rela2 values. Furthermore, bacteria growth was associated with a decrease of As (III) in the medium in some cases, even though this reduction did not remain over time, as revealed by the bacterial growth kinetics examined in this study. In addition, no reductions of arsenite levels in free-bacterial controls indicate that bacteria produced real decrements of this element in the media. It is important to highlight that the neutral pH conditions of the media  . Here, tThiomonas sp. str. 3As; Agrobacterium tumefaciens str. 5As and the microbial biofilm GM1 (isolated from arsenite-containing acid mined drainage waters of Giant Mine in Canada) also express the arsenite oxidase when the organisms are growing in the absence of arsenite, but in all of these cases, the expression does not occur before the logarithmic phase [A. tumefaciens strains involving As (III) sensing, components of signal transduction, and quorum sensing [Arsenite Oxidase Detection Essays: Dot blot analysis showed the presence of arsenite oxidase in both arsenic-baited bacteria and arsenic non-baited bacteria. Indeed, kinetic essays showed arsenite reductions starting early, during the lag phase of bacterial growth, which is an indicator that arsenite oxidase is constitutively expressed by Salar de Punta Negra bacterial isolates. This was observed in 5 out of 7 cases . The stin-situ arsenite removal is needed.Studies show that halobacteria from Salar de Punta Negra exhibit promising properties as arsenite removers under control conditions, where incubation time is a critical parameter and, particularly, in those situations where 2 athalassohaline wetland located in the Atacama Desert, northern Chile . It consists of several temporary saline lagoons covering about 3 Km2. Mining activities occurring around this place obtain most of the water for their processes from the groundwater below the salt flat through a network of extraction wells. The geographic and climatic characteristics of the Salar together with their chemical composition provide ideal conditions for the growth of extreme microorganisms, such as bacteria, archaea, and microalgae [Description of Sampling Site: Salar de Punta Negra is a 250 Kmcroalgae , which a2) in order to isolate and grow microorganisms. These media and their composition were named as Isolation Media (IM), Control Isolation Media (CIM), Growth Media Supplemented with As III (GM/S), and Growth Media Non Supplemented with As III (GM/NS), respectively, as shown in Table Agrobacterium tumefaciens C58/ATCC 33970 was cultured in Luria broth and employed as positive control for arsenite oxidase detection. It was named Luria Media (LM). In order to promote the adaptation of isolated microorganisms and obtain a representative fraction of the microbial biodiversity in each sample, the isolation medium was prepared with sterile-filtered water from Salar de Punta Negra (SWS). This was done so as to make bacterial growth possible and maintain the chemical equilibrium present in the water from Salar de Punta Negra in these media. Thus, the nutritive agar (DIFCO) was dissolved in a minimal milli-Q water volume sterilized at 121°C/1 atm. Then, the volume was completed with SWS at 60°C. Table 2.H2O (Merk), respectively. For GM/S preparation a sterile-filtered NaAsO2.H2O solution was added at 60°C, after autoclaving the media. The resulting concentration in the GM/S was 100 ppm in As III, prepared from a stock solution of NaAsO2.H2O 200 ppm.Growth Media and Bacterial Isolation: Samples from the Salar were incubated in different media with and without sodium meta-arsenite NaAsO in order3 (9.3) and the neutral pH of culture media, this compound dissociates according to equation 1, with an oxidation state 3+:Given the pKa of As(OH)0), was verified. Then, colonies from this generation, with different morphology , were transferred to plates with GM/S and incubated at 30°C for 18 to 24 h. The colonies obtained in this step were named as G1 and compared with the colonies of G0, through a Gram test. To increase the biomass required for the next experiments, colonies from G1 were transferred to flasks with 200 mL of GM/S until obtaining a new bacteria generation(G2).A mixture of surface sediments and water from Salar de Punta Negra was diluted in SWS in a 1:1000 ratio. This diluted mixture was named as dilute sample. Ten plates containing IM were inoculated with 1 mL of the previous dilute sample and incubated at 30°C until the presence of colonies, named here as “generation zero” (G′ AGAGTTTGATCCTGGCTCAG 3′) and 1392R (5′ ACGGGCGGTGTGTAC 3′), using 10 ng of DNA as a template in each amplification reaction. PCR conditions were selected as previously described by Norton et al. (2008) [HhaI and MspI restriction enzymes in separated reactions for 3 h at 37°C. Restriction patterns were compared by electrophoresis in 2% agarose gel and stained with etidium bromide.Molecular Identification of Isolates: In order to determine the genomic variability between bacterial colonies, a restriction fragment length polymorphism analysis (RFLP) was conducted. Genomic DNA from isolates were obtained by Power Soil DNA isolation Kit  and amplified by PCR. 16S rRNA genes were amplified using the primer pairs specific for Bacteria 8F (5. (2008) . About ~2. Thus, bacterial pellets of each of the seven isolates selected from G2 (10 μL) were inoculated in 200 mL of GM/S and GM/NS media (Table 3). This procedure was repeated 10 times for each isolate to reach generation number 13th or G13. At the same time, the reference strain of Agrobacterium tumefaciens C58/ATCC 33970 was grown in LM, according to the protocol described above , clones selected by RFLP analysis were cultured for several generations in media with and without NaAsO13 was performed in separate experiments. Thus, 10 μL of pellets from each bacterial isolate were inoculated in screw cap tubes with 15 mL of GM/S and incubated at 30°C for 18 h. Once bacterial growth was obtained, 100 μL of bacterial inoculation were transferred to Erlenmeyer flasks containing 200 mL of GM/S and incubated at 30°C, while stirring at 300 rpm for 36 h. Medium controls consisting of 200 mL non-inoculated GM/S were incubated together with the previous experimental treatment flasks under the experimental conditions above to monitor the concentration of As (III) in the supernatants of the treatment flasks, relative to the total As. The latter is represented by arsenite concentration in the control medium since it does not suffer any significant change during the experiments.Arsenic Kinetic Experiments: In order to determine the correlation between bacterial growth and As (III) removal from the culture media, the kinetics of 7 isolates from G0, t2, t4, t6, t8, t18, t22, t27, t30, and t36. To do this, 10 mL from each culture flask were filtered through sterile disposable filters of 0.22 μm pore. The filtered-recovered medium was used for arsenic measurements. The same procedure was used for the control culture flasks. Both, treatment and control flasks, were analyzed in triplicate. Simultaneously with arsenic measurements, non-filtered samples from the treatment flasks were used to estimate bacterial density. For this purpose, the samples were fixed in buffered glutaraldehyde  and cell counts were determined by the Neubauer chambers technique.As (III) in the culture medium of each treatment flask and the medium control were measured after bacteria removal, at specific incubation times: t0) and their corresponding values at each incubation time.The arsenite removal capabilities of bacteria were expressed as Percentage of Arsenite Removal (PAR) and calculated from the initial values of As (III) present in the culture media  measurements were carried out by hydride-generation flow-injection atomic absorption spectrophotometry (FIAS-HG-AAS) with a detection limit of 0.001 ppm. This is a suitable method for the analysis of arsenic in waters, and it is used in most laboratories because of its high sensitivity, speed of analysis, and comparatively low cost ,39. A ba13 at exponential growth phase were re-suspended in 4 mL of sterilized Milli-Q Water and lysed by sonication (140 Hz) with 3 30-s cycles on ice. Once the bacterial pellets were lysated, suspensions were transferred to 10 mL sterilized tubes. Then, 20 μL of 3 different protease inhibitors were added: aprotin (1 mg/mL); leupeptine (1 mg/mL); and PMSF/DMSO (100 mM). Five μL of each lysated were put on a nitrate-cellulose membrane (pore size of 0.47 mm Millipore). To check the specificity of antibodies and prevent cross reactivity, another 5 μL of bovine xantine oxidase (another molibdopterin protein) from bovine milk, Sigma – Aldrich diluted in 1:50, were dotted on the same membrane. This enzyme was chosen because it exhibits a high structural similarity with arsenite oxydase [A. faecalis NCBI 8687  [A. tumefaciens C58/ATCC 33970 were used as positive controls of AOX expression. Sterilized GM/S was used as negative control for the same purpose. The membrane was blocked with 6% non-fat milk in TBS (0.1% Tween 20) (TBS-T) for 2 h at room temperature. To detect the arsenite oxidase, the membrane was incubated overnight at 4°C with diluted 1:25 in PBS-T polyclonal rabbit anti A. faecalis AOX primary antibody , also provided by Dr. Gretchen Anderson. Then, the membrane was washed 3 times with TBS-T and incubated with 0.2 μg/mL HRP-conjugated goat anti-rabbit IgG for 1.5 h. They it was washed again and revealed with Pierce ECL Substrate and GE Healthcare ECL Reagent prepared according to the manufacturer’s instructions.Arsenite Oxidase Dot Blot Analysis: A dot blot analysis was conducted for arsenite oxidase detection. Bacterial pellets of Gnication 0 Hz withBoth microbiological and physicochemical assays of the present study were carried out following security protocols normalized in the Laboratorio de Servicios Analíticos (LSA) at the Universidad Católica del Norte wich has the ISO/IEC 17025 accreditation .PAR: Percentage of arsenite removal; IM: Isolation medium; CIM: Control isolation medium; GM/S: Growth media supplemented with arsenite; GM/NS: Growth media non supplemented with arsenite; LM: Luria medium; Gn: Generation n; tn: Time n; SWS: Sterile-filtered water of salar de punta negraNo competing interests are involved in this work.D-PP: Conceived the study and participated in its design and coordination. She also wrote the core of the manuscript. GA; PS, and GR: They conducted the microbiological analysis and the As kinetics. They participated as undergraduate students. MH: She conducted the molecular genetic studies for clone identification, RFLP analysis and helped write the manuscript draft. PP: She coordinated the dot blot analysis and helped write the manuscript draft. SS, FQ, and CC: She coordinated the As Analysis and validation methods for As data. DA and KG: They conducted the Dot Blot analysis. All authors read and approved the final manuscript.The main author, D-PP, has always worked in environmental microbiology concerning saline environments of arid zones in Chile and Spain. In the last few years, she has been part of a research group including analytical chemists , physical chemists (CC), and biochemists (PP and MH), who have been studying stress factors affecting the biological communities of Chilean and Peruvian Highlands . Together with Doctors SS, FQ, and DC, she is part of the research group at UCN Analytical Services Laboratory, the only testing lab accredited by ISO 17,025 in northern Chile. In addition, Dr. FQ is the president of the Analytical Chemistry Division of the Chilean Chemical Society. All students, DA, KG, GR, and PS, who participated in this study, prepared their undergraduate and graduate theses.


Overall, 147 patients had TKA . The fast-track rehabilitation patients received a group therapy, early mobilization (same day as surgery) and 1:1 physiotherapy (2 h/day). Patient monitoring occurred over 3 months (1 pre- and 4 post-operative visits). The standard rehabilitation group received individual postoperative care according to the existing protocol, with 1:1 physiotherapy (1 h/day). The cumulative American Knee Society Score (AKSS) was the primary evaluation variable, used to detect changes in joint function and perception of pain. The secondary evaluation variables were WOMAC index score, analgesic drug consumption, length of stay (LOS), and safety.This was an open, randomized, prospective clinical study, comparing the fast-track rehabilitation group showed enhanced recovery compared with the standardrehabilitation group, as based on the differences between the groups for the cumulative AKSS (p = 0.0003), WOMAC index score (<0.0001), reduced intake of concomitant analgesic drugs, reduced LOS , and lower number of adverse events.After TKA, patients in the fast-track rehabilitation is achievable and beneficial as based on the AKSS and WOMAC score, reduced intake of analgesic drugs, and reduced LOS.For TKA, implementation of pathway-controlled  Total knee arthroplasty (TKA) is a standard procedure in orthopedic surgery , 2. The Over the last decade, the concept of clinical pathways has been introduced in various specialized clinical and applied to surgical procedures, including TKA. The aim of clinical pathways is to use streamlined procedures and protocols to improve medical the quality of the treatment, minimize unnecessary variation in care, and reduce costs –22. Protfast-track approaches such as those reported from Denmark , assessed by the investigator as not related to the procedure.Overall, 23 AEs were observed for 147 patients. There were 20 AEs assessed as related to the procedure and 3 AEs [cerebral stroke (N = 2) and minor (N = 18). The two severe AEs were: deep infection (fast-track rehabilitation group) and humerus fracture (standard rehabilitation group). Minor AEs  were: stiffness (N = 13), urinary tract infection (N = 2), subluxations of the patella (N = 2), tibial fissure (N = 1). None of the patients died during the study.The intensity of the procedure-related AEs was assessed as severe , with a significantly higher cumulative AUC score  than in the standard rehabilitation group   , whereas for the standard rehabilitation group at this visit, a decrease in AKSS was seen, with a statistically significantly lower values, i.e., 122.25 versus 80.52  .The results of our study show that after a TKA, the e 4,089.6 than in roup 2,41.61 . To exclude such a potential bias, the time of analgesic drug intake, frequency, and amount of the analgesic drug were monitored and analyzed in our study. The validated Oxford league of pain table was not appropriate for our study because it does not include the standard epidural application of clonidine/ropivacaine/sufentanil, which was administered for patient-controlled anesthesia within the first 24 h postoperatively. Therefore, we used an in-house, pharmacologist-approved, classification of the analgesic drugs  was considerably lower for the fast-track rehabilitation group throughout the rest of the observation period than for the standardrehabilitation group (Fig. fast-track rehabilitation group than in the standard rehabilitation group (Fig. The perception of pain level plays an important role for both AKSS and WOMAC index. The outcomes for both scores therefore might be biased by different medications used to control pain (e.g., higher dosage of analgesic drugs in the oup Fig. . Additiooup Fig. . Such a fast-track rehabilitation after TKA in an orthopedic hospital unit in Germany. Conclusions drawn from our data are similar to other studies such as those performed in Denmark, where the concept of fast-track surgery in TKA has been widely implemented for more than 10 years [This study shows data on the feasibility of 10 years . Our dat10 years , who con10 years . Positiv10 years .fast-track rehabilitation concept, a high AKSS together with a reduction in LOS, and fewer AEs were safely achievable; the patients’ daily living parameters, as assessed by the WOMAC index score, were favorable and statistically highly significant. Furthermore, the fast-track rehabilitation concept used in the present study is patient-focused, is feasible, transferable to other centers, and may have economic implications through reduced hospital costs and health-care benefit contributions. Indeed, the results of our study endorse and contribute some answers to the recently posed question “Why still in hospital after fast-track hip and knee arthroplasty?” [In the present study, the target for discharge after TKA was 6 days, which was in compliance with the German DRG system in 2005 . At thatplasty?” .


Androgen signalling is essential both for male development and function of the male reproductive system in adulthood. Within the adult testis, Germ cells (GC) do not express androgen receptor (AR) suggesting androgen-mediated promotion of spermatogenesis must act via AR-expressing somatic cell-types. Several recent studies have exploited the Cre/lox system of conditional gene-targeting to ablate AR function from key somatic cell-types in order to establish the cell-specific role of AR in promotion of male fertility. In this study, we have used a similar approach to specifically ablate AR-signalling from Vascular Endothelial (VE) cells, with a view to defining the significance of androgen signalling within this cell-type on spermatogenesis.AR expression in VE cells of the testicular vasculature was confirmed using an antibody against AR. A Cre-inducible fluorescent reporter line was used to empirically establish the utility of a mouse line expressing Cre Recombinase driven by the Tie2-Promoter, for targeting VE cells. Immunofluorescent detection revealed expression of YFP (and therefore Cre Recombinase function) limited to VE cells and an interstitial population of cells, believed to be macrophages, that did not express AR. Mating of Tie2-Cre males to females carrying a floxed AR gene produced Vascular Endothelial Androgen Receptor Knockout (VEARKO) mice and littermate controls. Ablation of AR from all VE cells was confirmed; however, no significant differences in bodyweight or reproductive tissue weights could be detected in VEARKO animals and spermatogenesis and fertility was unaffected.We demonstrate the successful generation and empirical validation of a cell-specific knockout of AR from VE cells, and conclude that AR expression in VE cells is not essential for spermatogenesis or male fertility. Androgens, produced by the testicular Leydig cells (LC), act as essential regulators of both the development of the male reproductive system and the initiation and maintenance of spermatogenesis in postnatal life . AndrTunica Intima) of all vessels of the circulatory system. VE cells possess an anti-thrombotic surface which facilitates laminar blood flow, whilst permitting nutrients and macromolecules to flow out of the bloodstream through intercellular spaces, the result of endothelial cell contraction or active transport through the cells themselves by transcytosis. The endothelium also regulates angiogenesis, vasoregulation, and leucocyte trafficking into sites of injury during inflammatory responses  cells . VE cellview see ).There remains some debate in the literature regarding the presence of AR within testicular VE cells, with studies arguing both for and against AR expression -15. GiveTo confirm that vascular endothelial (VE) cells of the testicular vasculature express AR, immunofluorescent detection of AR was performed on sections of testis taken from control adult animals (n = 3). Nuclear AR expression was confirmed within all smooth muscle cells of the vasculature as previously described . In addiTunica Intima , demonstrating that the presence of the Cre Recombinase transgene did not itself induce a phenotype.In order to specifically target AR in VE cells, a mouse line carrying a Cre Recombinase transgene driven by the promoter of the endothelial-specific angiopoietin receptor Tie2 was exploited . To empia Figure ; howevera Figure  but thata Figure ; we therTo generate Vascular Endothelial Androgen Receptor Knock-Out (VEARKO) mice and littermate controls, heterozygous Tie2-Cre males were mated to homozygous ARflox  females.PCR interrogation of genomic DNA extracted from testes of VEARKO mice confirmed ablation of AR from a subset of testicular cells Figure . No ablaComparison of bodyweight, seminal vesicle (SV) weight, and anogenital distance (a marker of androgen action during the masculinisation programming window ), revealGross phenotypic analysis revealed no significant difference in testis weight between VEARKO mice and controls in adulthood Figure . Blind-sThe testicular vasculature is known to be an important regulator of both testis development ,22 and fEmpirical validation of Cre Recombinase expression in the Tie2-Cre line provides a good example of why such validation is necessary (reviewed in ). WhilstA combination of genomic PCR and immunofluorescent localisation confirmed specific AR ablation from the VE cells of the testis in VEARKO mice. However, unlike our previous study targeting AR in the smooth muscle cells of the testicular vasculature , we obseMice in which the AR has been selectively ablated from VE cells were generated using Cre/loxP technology. Male congenic C57BL/6 J carrying a random insertion of Tie2-Cre  were matEight week old VEARKO male mice (n = 7) and littermate controls (n = 5) were singly housed with 129SV/C57BL/6 J females for a period of five days. Females were culled by inhalation of carbon dioxide and subsequent cervical dislocation, and fetuses counted ten days later.Male mice were culled at post-natal day (d) 57 by inhalation of carbon dioxide and subsequent cervical dislocation. Body weight and anogenital distance were measured and mice were examined for any gross abnormalities of the reproductive system. Testes and SV were removed and weighed. Tissues were fixed in Bouin's fixative  for 6 h. Bouin's-fixed tissues were processed and embedded in paraffin wax, and 5-μm sections were used for histological analysis as reported previously . SectionSingle immunofluorescent staining for AR (Santa Cruz sc-816) was performed on VEARKO and littermate controls and double fluorescent staining for AR/YFP (Abcam ab38689) or YFP/CD31 (Abcam ab28364) was performed on YFP reporter mice. Sections were deparaffinized and rehydrated, and high-pressure antigen retrieval was performed in 0.01 M pH6 citrate buffer for 5 min. Endogenous peroxidase was blocked using 3% hydrogen peroxide in methanol and non-specific antibody binding sites were blocked with a mixture of 20% normal goat serum/80% tris-buffered saline/5% bovine serum albumin (NGS/TBS/BSA). Sections were incubated overnight at 4°C with the primary antibody diluted in NGS/TBS/BSA. Immunostaining was detected using the appropriate secondary antibody (Goat anti-rabbit peroxidase (Dako P0448), or Goat anti-mouse peroxidase (Dako P0447)) diluted 1:200 in NGS/TBS/BSA incubated for one hour at room temperature, then fluorescein Tyramide Signal Amplification system  diluted 1:50 in its own buffer to manufacturers instructions for 10 min at room temperature. If a second primary antibody was used, the process was then repeated from the peroxidase block onwards with the appropriate conditions and detection for the second antibody and use of Cyanine - 5 TSA™ for a different fluorescent wavelength to distinguish between the two antibodies. Slides were counterstained with propidium iodide  and mounted with PermaFluor mounting medium . Images were captured using a LSM 710 confocal microscope (Zeiss) with Zen software. A minimum of three mice of each genotype were used and sections from VEARKO and control littermates were processed in parallel on the same slide, on at least two separate occasions. Appropriate negative controls were included to ensure that any staining observed was specific.Mice were genotyped for inheritance of Cre Recombinase as previously described . PCR amploxP sites and deletion of AR exon 2.DNA was isolated from a piece of frozen testis using a Mouse tail genomic DNA kit  according to the manufacturer's instructions and subjected to PCR amplification using primers GCTGATCATAGGCCTCTCTC and TGCCCTGAAAGCAGTCCTCT and Biomix Red Taq polymerase PCR kit . PCR amplification products were resolved using a QiaXcel capillary system . An amplicon of 1142 bp indicated presence of a floxed AR whilst an amplicon of 612 bp indicated recombination between t test.Statistical analyses of weights, AGD and fertility testing were carried out in GraphPad Prism  using a 2-tailed unpaired The authors declare that they have no competing interests.LBS conceived the study; LO carried out the experiments; LO and LBS wrote the paper. All authors read and approved the final manuscript.


Introduction. The role of robotics in poststroke patients' rehabilitation has been investigated intensively. This paper presents the state-of-the-art and the possible future role of robotics in poststroke rehabilitation, for both upper and lower limbs. Materials and Methods. We performed a comprehensive search of PubMed, Cochrane, and PeDRO databases using as keywords “robot AND stroke AND rehabilitation.” Results and Discussion. In upper limb robotic rehabilitation, training seems to improve arm function in activities of daily living. In addition, electromechanical gait training after stroke seems to be effective. It is still unclear whether robot-assisted arm training may improve muscle strength, and which electromechanical gait-training device may be the most effective for walking training implementation. Conclusions. In the field of robotic technologies for stroke patients' rehabilitation we identified currently relevant growing points and areas timely for developing research. Among the growing points there is the development of new easily transportable, wearable devices that could improve rehabilitation also after discharge, in an outpatient or home-based setting. For developing research, efforts are being made to establish the ideal type of treatment, the length and amount of training protocol, and the patient's characteristics to be successfully enrolled to this treatment. Stroke from an ischemic or haemorrhagic intracranial vascular event is a leading cause of movement disability in the USA and in Europe . The WorThus, the rehabilitation goal in poststroke subjects is to promote recovery of lost function, to allow independence and early reintegration into social and domestic life. The number of people that require rehabilitation after stroke is growing rapidly , with inNeural Plasticity” indicates the recovery mechanisms and functional adaptation resulting from global changes in neuronal organization. Neural adaptation leads to a more robust recruitment of motor neuron pools, transfer of function from damaged areas to preserved adjacent or correlated areas, strengthening of redundant or parallel synapses, new synapse formation, increased dendritic sprouting, enhanced myelination of remaining neurons, and modification of cortical and noncortical representations. Recently, cerebellum has been demonstrated to play a key role in modulating cortical motor output and in motor learning [Poststroke patients require continuous medical care and intensive rehabilitation often requiring one-on-one manual interaction with the physical therapist. Unfortunately, present demands and budget restrictions do not allow this intensive rehabilitation. Hence, there is an urge for new technologies improving the efficacy and effectiveness of poststroke rehabilitation. The available scientific literature suggests that the most effective rehabilitative interventions are those providing early, intensive, task-specific, and multisensory stimulation. The well-known capacity of the central nervous system to adapt its structural organization after brain lesion is mainly influenced by sensory input, experience, and learning , 6. Nudolearning . Hence, Because of the continuous and rapid evolution of the robotic technology, the aim of the present review is to provide a comprehensive insight into the main robotic devices and their possible use in stroke patients' rehabilitation. The main devices available nowadays for upper and lower limbs rehabilitation are hereby presented. For each class of robots, areas of agreement among researchers, together with controversial aspects about their application in stroke rehabilitation are reported and discussed in the present review. Finally, the current main topics of study and areas timely for developing research are presented.Robotic Devices. The robotic devices currently available can be divided into two main categories: those for the upper limb and those for the lower limb. As we previously indicated [ndicated , the uppThe lower limb devices include exoskeletons and end-effectors and the characteristics of the main automated electromechanical gait machines are illustrated in Search Methods. The authors undertook literature search in February 2013 about robot-assisted upper and lower limbs rehabilitation after stroke, using as keywords “robot AND stroke AND rehabilitation.” The databases were PubMed/Medline, Cochrane Library, and PeDRO. Only papers written in English were considered and the search was extended to the whole database. The authors found about 300 papers, published from 1991 to February 2013.Areas of agreement and areas of controversy among researchers emerging from the analysis of the literature on this growing field are reported.Restoring hand and arm motor function is essential to preserve patient's independence in the performance of the activities of daily living (ADLs). Rehabilitation plays a fundamental role in minimizing the residual motor deficits of stroke patients, both during the acute and subacute phases and in the chronic phase. Physicians are usually prone to prescribe treatments including intense , highly The robot-assisted rehabilitation of the upper limb in the acute and subacute poststroke phases may be successfully used in alternative to conventional mobilization, resulting at least as effective as conventional therapy, especially when used in addition to nonrobotic techniques , 26. MasHowever, robotic rehabilitation is not merely a matter of increasing the amount and intensity of therapy. In fact, robotic systems may be used not only to produce simple and repetitive stereotyped movement patterns, as well as for most of the existing devices, but also to generate a more complex, controlled multisensory stimulation of the patient. It seems that rehabilitation technology impact on functional outcome could be optimized offering more chances to the nervous system to experience “real” activity-related sensorimotor input during training of upper limb movement . In thisThe role of electromechanical devices is that, in contrast with the action of one physical therapist alone, they can provide nonambulatory patients' intensive, high repetitive, practice of complex gait cycles. Compared to treadmill training with partial body weight support, these robotic devices may reduce the effort for therapists; in fact, they no longer need to set the paretic limbs or assist trunk movements . HoweverAn important target of robotic rehabilitation is to increase patients' function, activity, and participation. Although most physicians experienced of robotics consider task-oriented approaches beneficial and promising for future research , most reAnother question is represented by the super specialization of the existing robots, most of which focus on the proximal  or the distal (Master II) part of the upper limb: just ADLER, allows the training of the whole arm . Moreover, at the present time robot training towards the full range of joint motion and the possible degrees of freedom is not possible. This aspect might explain the lack of functional recovery in the daily life, which is based on the use of the whole arm, not just the shoulder or the elbow. As Reinkensmeyer and Boninger  recentlyAn important potential advantage is that robotic systems can measure several kinematic and dynamic parameters during the motion of the patient's limb, allowing for both online and offline evaluation of relevant indicators of the patient performance  . These vThe acceptance of robotic technology by patients and physiotherapists may be an issue itself, although this does not represent a major concern for the devices developed to date. Moreover, the cultural gap between technology providers, rehabilitation professionals and final users is becoming gradually smaller thanks to the widespread diffusion of knowledge in the recent past.Regarding the lower limb, there is evidence of beneficial effects of electromechanical devices for gait training after stroke, but their relatively high cost limited the diffusion of such devices in the clinical practice. Hence, more evidence about the cost effectiveness of such technology is required, in order to warrant their broad clinical application.In the international context there is an open discussion on which kind of electromechanical gait training device is more effective. A recent review study  includinAfter pointing out the areas of agreement and those of controversy in the field of robotic rehabilitation after stroke, we conclude illustrating the currently growing points and the areas timely for development research.Robotic rehabilitation is certainly undergoing a period of rapid growth, although many issues remain open. However, this technique may offer considerable benefits, not only in terms of cost reduction and training program implementation, but also for the scientific community with an approach inspired from evidence-based medicine. Robotic technology, indeed, differently from other physiotherapy options, allows quantifying objectively the amount and quality of multisensory stimuli and measure patient outputs and outcomes .The modality in which the subject performs: brain stimuli and motor gain seem to be greater with intense, active, assistive, and repetitive movements than with nonassistive or passive movements.The graduation of amount and typology of feedback (visual, auditory , haptic The multiplanarity of the exercises that seems to induce more motor cortex excitation.Present researches in this field are trying to improve the efficacy of robotic manipulation, the trajectories, strengths, and multisensorial inputs that robots must provide in order to improve the quality and efficiency of rehabilitation. In the last years, very different robotic systems and approaches were employed for rehabilitation treatment of impaired upper and lower limbs in poststroke patients. The future systems should comply with principles of motor learning mentioned below , 26.The In poststroke patients with upper arm impairment robotic devices can be applied in the acute, subacute, and chronic phases. Robot therapy was introduced only in the chronic phase of the treatment protocol in most of the studies published so far. However, the application of this approach in the acute or subacute phase of stroke may lead to a clinical improvement, mainly due to the increased brain plasticity earlier after stroke , 44. EveAt the present time many scientific efforts are addressed towards the construction of handy and easily transportable devices suitable for a robot assisted training in the acute and subacute phases, such as the wearable technology , 45. In The research in wearable technology is oriented not only to stroke patients, but also to other patients with limited mobility . HopefulGiven the fast developments in rehabilitation technology, it is mandatory to understand the role of robotics in the rehabilitation program. Our review confirms the theory reported in the commentary of Johnson et al.  that tecRobot-mediated neurorehabilitation is a rapidly advancing field based on robotics, virtual reality, and haptic interfaces that could support neuroscience and conventional rehabilitation for a successful treatment of neurological injuries such as stroke, spinal cord injury, and traumatic brain injury. According to the latest research a composite approach including robotics, virtual reality , 52, andMachine-mediated neurorehabilitation is characterized by challenges both in engineering and in clinical practice. From a technical point of view, there is a need for more integrated solutions to perform a therapy in a safe environment and with better compliance from the patients. According to the current practice, new machines patented for rehabilitation use must be tested clinically, and the results published. More research is needed to establish whether ADL tasks can be truly enhanced by robotic training. Moreover, solutions improving the interaction between the therapist and patients in the robotic field are mandatory. As a recent review recommends, methodology in trials including robotic resources should be unified, especially in terms of inclusion criteria according to the poststroke phase, functional scales for outcome measure, intensity, and duration of the interventions . AshfordMoreover, the successful clinical implementation of this promising field meets opposition from the concerns raised about the possibility that robots could “dehumanize” patient rehabilitation or replace the human work force. In conclusion, these machines are intended to represent an adjunctive tool to increase the intensity of therapies, in line with modern principles of motor rehabilitation. A robot can never replace both the multilevel interactions between patient and experienced physical therapist and the manual ability of operators.