UCHL1 to predict early progression in newly diagnosed patients; a finding reversed by the inclusion of bortezomib. We also found high UCHL1 levels to be a critical factor in the superiority of bortezomib over high-dose dexamethasone in relapsed patients. High UCHL1 partially overlaps with, but is distinct from, known genetic risks including 4p16 rearrangement and 1q21 amplification. Using an orthotopic mouse model, we found UCH-L1 depletion delays myeloma dissemination and causes regression of established disease. We conclude that UCH-L1 is a biomarker of aggressive myeloma that may be an important marker of bortezomib response, and may itself be an effective target in disseminated disease.The success of proteasome inhibition in multiple myeloma highlights the critical role for the ubiquitin-proteasome system (UPS) in this disease. However, there has been little progress in finding more specific targets within the UPS involved in myeloma pathogenesis. We previously found the ubiquitin hydrolase UCH-L1 to be frequently over-expressed in B-cell malignancies, including myeloma, and showed it to be a potent oncogene in mice. Here we show that UCH-L1 is a poor prognostic factor that is essential for the progression of myeloma. We found high levels of  Multiple myeloma (MM) is an incurable plasma cell neoplasm that affects over 20,000 people per year in the US alone. A series of novel therapeutic agents, coupled with improved care for patients undergoing autologous stem cell transplantation has led to dramatic improvements in the progression free survival for individuals with MM , 2. Whilin vitro [in vivo development and/or progression of MM is unknown. Here we show that UCH-L1 is required for the in vivo dissemination and progression of MM and is an important determinant for prognosis in patients with the disease.Using an unbiased approach to identify de-ubiquitinating enzymes deregulated in cancer, we previously found UCH-L1, a deubiquitinating enzyme largely restricted to neuro-endocrine tissues in mice and humans, to be frequently over-expressed in mature B-cell malignancies, including MM , 16–18. in vitro . UCH-L1 in vitro , β-catenin vitro , HIF-1α in vitro , NOXA [2in vitro , and tubin vitro , all of n = 351) of newly diagnosed patients who were treated on the Total Therapy 2 (TT2) protocol that added thalidomide onto a background of tandem autologous stem cell transplants [UCHL1 (UCHL1hi = 75–100%) disease compared with UCHL1lo (0–74%) , a regimen that incorporated bortezomib onto this backbone  trial that compared outcomes in relapsed patients treated with either high-dose dexamethasone or bortezomib [UCHL1hi disease had a significantly worse outcome compared with those with UCHL1lo with a median time to progression of only 83 days compared with 147 days respectively  Figure . The mage Figure . To furtrtezomib . In the y Figure . In cont1 Figure . Consiste Figure . whereas. Figure . We concUCHL1 targeting shRNAs and found them to similarly induce cell death in three different UCH-L1 expressing myeloma cell lines, but not in a UCH-L1 negative line [in vivo.To better understand the impact of UCH-L1 on the response to bortezomib and dexamethasone, we made use of doxycycline-inducible shRNAs. In two prior reports we characterized a series of ive line , 21. To UCHL1hi myeloma cases, we plotted UCHL1 levels across 596 myeloma primary specimens and 50 human myeloma cell lines according to TC grouping. There was a striking association between cases of the 4p16 translocation subtype and increased UCHL1 levels  in the 4p16 group suggesting a worse outcome for those with high levels of UCHL1  groups that reflect the presence of numerical or structural chromosome aberrations, and deregulated expression of D-type cyclins , 28. To s Figure . This isof UCHL1 .UCHL1. Those patients with UCHL1hi disease exhibited significantly increased levels of cyclin D2, FGFR3, and MMSET (WHSC1), and significant reductions in cyclin D1 compared with UCHL1lo cases  cells lacking MMSET [UCHL1 gene between KMS-11 parent and TKO cells . We therefore conclude that high UCHL1 levels correlate with the gene signature seen in patients with chromosome 4p16 rearrangements, but that increased UCHL1 is not directly related to the over-activity of MMSET.We next compared the expression of the key classifying genes between cases with either low or high levels of s Figure . FurtherT Figure . The t to allow non-invasive bioluminescent imaging (BLI). The cells were further stably transduced to encode an UCHL1 targeting shRNA or a control non-silencing shRNA under the control of a constitutive promoter. In animals injected with cells encoding non-silencing shRNA, there was a brisk increase in BLI signal with mice going on to develop hind-limb paralysis  expressing high levels of UCH-L1, whereas there was no impact on the growth of KMS-12 cells that express low levels [s Figure . CellulaLUC cells stably transduced with shRNA (UCH-L1 targeting or non-silencing) under the control of a doxycycline-inducible promoter. Mice injected with these cells were monitored by BLI and doxycycline was added to the drinking water once disseminated myeloma was established . Moreover, this impact on disease translated into a significant extension in endpoint free survival  and there are relatively few events (15 events in 208 patients = 7%). To further examine the impact of UCHL1 on drug response, however, results were reinforced in the head-to-head comparison between bortezomib and dexamethasone. In fact, if one looks only at UCHL1lo cases on the APEX trial, there is no advantage to bortezomib over dexamethasone, suggesting that UCHL1 is an important biomarker for patients who will most benefit from this treatment.We find that UCH-L1 is a marker of MM patients with inferior prognosis at initial diagnosis, and at relapse. While the data need to be verified with primary samples in a prospective manner, these results are significant in that they may allow for more individualized therapy, with more aggressive treatment reserved for those at higher risk of early relapse. As these analyses utilize gene expression profiling cut-off values for defining high UCHL1hi cases are relatively resistant to the other therapies included in TT2 and APEX studies. This is an important distinction as previous cell biology work in fact predicted that high levels of UCH-L1 might facilitate proteasome inhibitor resistance [MYC. In the myeloma datasets included here, there was no relationship between UCHL1 and MYC levels (data not shown). Several signaling networks have been implicated in drug resistance in myeloma [Our data do not, however, suggest that cases with high levels of UCH-L1 are more sensitive to bortezomib compared with those having low levels. Rather, the data suggest that the sistance , though  myeloma . While o myeloma  stronglyUCHL1 and the expression signature of t and del1q21 may contribute to the poor prognosis in these patients, as these structural defects is known to afford a high-risk of early recurrence [UCHL1 levels is unknown. While UCHL1 lies on chromosome 4, it is separated from the FGFR3/WHSC1 locus by nearly 40 megabases, making a direct effect of the translocation on UCHL1 transcription unlikely. The t deregulates the expression of two genes with relevance to cancer FGFR3 and the histone methyltransferase MMSET [UCHL1 expression were observed upon inhibition or depletion of either gene [Our previous work revealed that transgenic mice over-expressing UCH-L1 developed B-cell lymphomas and plasmacytomas, demonstrating that UCH-L1 is not simply another gene that associates with prognosis – but rather is a mechanistic biomarker involved in disease pathogenesis. In addition to the impact of UCH-L1 in mTOR-Akt signaling, the association between high levels of se MMSET . While iher gene , 38. Usiher gene .in vitro and in vivo [There have been many conflicting reports regarding the impact of UCH-L1 expression in human malignancy, with some citing oncogenic effects while others demonstrating a tumor suppressive effect. A potential explanation behind the varied impact of UCH-L1 on tumor cell survival may be its ability to inhibit the activity of mTOR complex 1 (mTORC1) and stimulate mTORC2. We previously reported an extensive biochemical analysis of the impact of UCH-L1 on the mTOR-Akt signaling pathway and found its expression to promote mTORC2 phosphorylation of Akt in three independent myeloma cell lines, in primary B-cells from mice, in HeLa and 293T cells, and in the brain – a mammalian tissue with high levels of endogenous UCH-L1 . Given t in vivo –45. Bort in vivo . It is p in vivo  suggestsSCID-Beige mice (8–10 weeks) were purchased from Charles Rivers Laboratories, Inc. . All mice were housed in a pathogen-free barrier facility. All protocols involving mice were reviewed and approved by the Mayo Clinic Institutional Animal Care and Use Committee.LUC luciferase-expressing cells were generated by lentiviral transduction of PHR-SiN-Luciferase prepared as described [In vitro expression of shRNA was induced by the addition of 1 mg/ml doxycycline (dox) to complete medium. Where indicated, bortezomib or dexamethasone  was included at the indicated concentrations. Where indicated, WP1130 (Selleckchem) was added to a final concentration of 5 μM. Cell viability was monitored using the CellTiter AQueous One Solution Cell Proliferation Assay . Caspase activity was determined with the Caspase-glo (3/7) Assay System (Promega). In vivo shRNA expression was induced with 2 mg/ml−1 dox in drinking water supplemented with 5% sucrose.KMS-11, KMS-18, and KMS-28 cells were cultured and transduced with shRNAs and characterized as previously described , 21. KMSescribed . In vitr7) were suspended in 200 μl PBS and injected via tail vein. The development and progression of disease was monitored at regular intervals using the IVIS 200 imaging system  through the Mayo Clinic Animal Imaging Shared Resource. Prior to imaging each mouse received 150 mg/kg XenoLight D-Luciferin potassium salt  via intra peritoneal injection. Histological analysis was performed on decalcified and formalin-fixed mouse femur sections. The antibodies used include goat anti-rabbit UCH-L1 , and mouse anti-human CD138 . Biotin-conjugated secondary antibodies were purchased from Vector Labs.Orthotopic mouse model of MM was established as described . Brieflyhttp://www.ncbi.nlm.nih.gov/gds) or the R2: microarray analysis and visualization platform (http://r2.amc.nl). In GSE9782, only those patients identified as “study code 39” were analyzed . The designation between UCHL1 LO/HI was set at the 75% percentile unless otherwise noted. T+C classifications were made according to published criteria [http://dldcc-web.brc.bcm.edu/lilab/NSD2/). Graphs were generated using GraphPad Prism software. All statistical calculations were performed using GraphPad Prism software with the exception of the Cox proportional hazards analysis, which was performed using the JMP software package. Survival data were analyzed with the Log-rank (Mantel-Cox) test as indicated. Histograms were analyzed as indicated using the 2-tailed unpaired t test. Correlation was assessed using the Pearson's r test. Statistical analysis was performed with the assistance of the Mayo Clinic Center for Clinical and Translational Science.Data were extracted from GSE2658, GSE9782, GSE24746 and GSE29148 from Gene Expression Omnibus (GEO; criteria . Histone


R2 of the models varied from 0.6978 to 0.9045. Support vector machine (SVM) was then utilized to verify the feasibility of the fluorescence parameters for monitoring LNC. Comparison of the fluorescence parameters indicated that F740 is the most sensitive  to the changes in LNC among all fluorescence parameters. Experimental results demonstrated that fluorescence parameters based on LIF technology combined with SVM is a potential method for realizing real-time, non-destructive monitoring of paddy rice LNC, which can provide guidance for the decision-making of farmers in their N fertilization strategies.Leaf nitrogen content (LNC) is a significant factor which can be utilized to monitor the status of paddy rice and it requires a reliable approach for fast and precise quantification. This investigation aims to quantitatively analyze the correlation between fluorescence parameters and LNC based on laser-induced fluorescence (LIF) technology. The fluorescence parameters exhibited a consistent positive linear correlation with LNC in different growing years (2014 and 2015) and different rice cultivars. The  The results of a recent meta-analysis showed that CH4 emissions from paddy rice systems account for almost 90% of the total global warming potential146Paddy rice plays an important role in the structure of the ecosystem. China grows paddy rice on approximately 30 million hectares per year and is the world’s leading producer of paddy rice2In previous studies, many models have been proposed to analyze the LNC of plants based on passive remote sensing technology78812A few decades ago, the laser induced fluorescence (LIF) technique was proposed to study the growth status of plants1718192021222425o), maximum fluorescence intensity after short dark adaptation (Fm), and quantum efficiency of photosystem II9These investigations mainly utilized fluorescence kinetics, minimum fluorescence intensity after short dark adaptation  and fluorescence ratio (F740/F685: fluorescence intensity at 740 nm divided by that at 685 nm) were extracted to analyze the LNC of paddy rice.To analyze the optimal fluorescence emission wavelength for predicting paddy rice LNC, further discussion was conducted on the correlation between fluorescence ratios and LNC by using datasets from 2014 and 2015 . Figure As shown in 162629R2 of the regression models of F685, F740, and F740/F685 areis 0.847, 0.8871, and 0.7981, respectively  displayed a closely positive linear correlation with LNC for different paddy rice cultivars and different growing seasons. For the two growing seasons (2014 and 2015), the ectively . They alectively . To compR2 of F685 ranges from 0.7811 to 0.8658, the R2 of F740 ranges from 0.8362 to 0.9045, and the R2 of F740/F685 ranges from 0.6978 to 0.842. 9The results demonstrated that the relationship between fluorescence parameters and LNC displayed high consistency in all the samples for different growing years and different paddy rice cultivars. For the three cultivars and the two growing years, the To verify the possibilities that the fluorescence parameters  can be used to precisely estimate LNC, SVM was applied in this study. The fluorescence spectra of each cultivar and each growing season were randomly divided into two parts: 70% was employed to train the SVM model on the basis of the different fluorescence parameters and 30% was utilized as a validation set to predict the LNC of paddy rice. For different cultivars and different growing years, the relationships between the predicted and observed paddy rice LNC are illustrated in As shown in 2 (ranges from 0.8529 to 0.9226) of the relationship between the predicted LNC on the basis of the F740 and measured LNC is the highest among these fluorescence parameters. The R2 (ranges from 0.7842 to 0.8524) of F740/F685 is lower than that of the other fluorescence indices. The corresponding RMSEs are converse. These results demonstrate that fluorescence parameter F685 is more sensitive to the changes of LNC than F740/F685 and is lower than that F740. However, these fluorescence parameters are useful for inversing the LNC of paddy rice .et al.At present, LIF spectral data have already been employed to monitor the status of plants but only a few articles focused on paddy rice, despite its significance in environmental problems and its role as an aliment source17R2 ranges from 0.8729 to 0.9426 for the three cultivars and two growing years) to the changes of LNC than the other two fluorescence parameters (F685 and F740/F685) in this study , non-photochemical quenching (NPQ) and demonstrated that F740/F685 is sensitive also to changes in photosystem I to photosystem II ratio and the difference of LNC will result in the change of F740/F685 ratio.10is study . A probaMost fluorescence kinetics and variable fluorescence parameters were proposed to monitor pigment content and were conductive to the monitoring of the photosynthetic efficiency of plants19394029Although this investigation preformed with two growing years and three cultivars of paddy rice, the monitoring models of LNC based on LIF technology were established and verified by using SVM in an ecological region with a typical subtropical monsoon climate. However, other significant fluorescence parameters and the regression analysis models still need to be analyzed comprehensively in other ecological locations, including different growing years and production systems. To obtain the optimal algorithm and fluorescence parameters, LIF technology combined with other multivariate analyses also needs to be tested and used to estimate the LNC of other crops in the future research.R2 of linear regression analysis obtained by using the fluorescence parameters ranged from 0.6378 to 0.9145. SVM was utilized to verify if LNC can be evaluated on the basis of the fluorescence parameters . The inversion results revealed that the N content of paddy rice can be accurately evaluated by utilizing the fluorescence parameters combined with SVM. In addition, F740 is the most sensitive to the changes of the LNC of paddy rice among these fluorescence parameters. Therefore, these fluorescence parameters combined with SVM will be very helpful in realizing real-time, non-destructive monitoring of paddy rice leaf N status based on LIF technology. They can also be employed to guide farmers in the application of reasonable doses of N fertilizers, which may result in remarkable environmental and economic interests because of reduced environmental pollution and increased N utilization efficiency.In this research, linear regression models between different fluorescence parameters  and LNC were established. A close linear positive correlation was observed between fluorescence parameters and LNC for different paddy rice cultivars  and different growing years (2014 and 2015). The All experiments were conducted at Junchuan County, Suizhou City, in the province of Hubei, and in the experimental station of Huazhong Agricultural University (HAU) in Wuhan City in the Jianghan China Plain during the paddy rice cultivating seasons of 2014 and 2015. The experimental area is characterized by a typical subtropical monsoon climate with abundant rainfall; it is sunny and hot in summer and cold in winter. The longitude of this area ranges from 113°41′ E to 115°05′ E and the latitude varies from 29°58′ N to 31°22′ N. The sunshine duration and rainfall are above 1800 hours and over 1200 mm per year, respectively. Thus, the area is suitable for growing paddy rice and is also known as one of the largest agricultural production installations for providing food security in ChinaThe paddy rice varieties cultivated were Japonica rice and Hsien rice. They were cultivated in Junchuan County, Suizhou City, in the province of Hubei, China. There cultivars were seeded on April 27 and then transplanted on June 1, 2014. During the entire growth period, six N fertilization levels of urea were used  in the experimental fields. N fertilization was divided into four splits: 30% at seeding, 20% at tillering, 25% at shooting, and 25% at booting. The experimental field had an absolute block design with three replications for each treatment under the same cultivation conditions. Other managements were advised by the local farm extension service in rice production. Paddy rice samples were collected on July 15 and August 1, 2014.Yangliangyou 6 was cultivated in the experimental station of HAU in Wuhan City in 2015. It was seeded on April 30 and transplanted on May 27. During the entire growth period, four N fertilization levels of urea were used  in the experimental fields. N fertilization was divided into three splits: 60% at seeding, 20% at tillering, and 20% at shooting. The experimental field had an absolute block design with three replications for each treatment. Paddy rice samples were gathered on four dates . mJ and 5 ns respectively, and the omitted wavelength was 355 nm with a pulse repetition frequency of 20 Hz. A single-mode optical fiber with a diameter of 200 μm and 25o angular field of view was utilized to transmit the fluorescence signal which was placed at the position of the focus of the Maksutov-Cassegrain telescope. In addition, a long-pass filter of 355 nm (the edge of 360 nm) which was used to eliminate the reflected light from the laser entering the optical fiber was positioned behind the telescope. The fluorescence induced by the ultraviolet laser then entered the spectrometer. The fluorescence signal was measured by utilizing the ICCD, and the data was stored in a personal computer. In this study, the fluorescence spectra ranged from 360 to 800 nm with sampling interval of 0.5 nm. Normalized fluorescence intensity  varying with the wavelength was showed in The system of LIF consisted with three parts: the excitation assembly, optical receiver system, and the data collection and treatment part. The laser source was an Nd: YAG that emitted pulses with the output energy and the pulse duration time being 1.5Paddy rice leaves were destructively sampled by stochastically cutting six leaves with three replicates for each experimental field. All samples were fully expanded the second leaves from the top. These samples were sealed in plastic bags, kept in an ice chest, and then immediately transported to the laboratory for fluorescence spectra measurements442), root mean square error (RMSE), and relative error (RE) in prediction. RMSE and RE can be presented as follows:SVM displays excellent generalization performance in practical application with a solid theoretical foundation in statistical learning theory46Mi denotes measured values, Pi represents predicted values, and n corresponds to the number of samples. R2 and low RMSE and RE denote the high precision and accuracy of the model in predicting LNC. The prediction is recognized as excellent if RE is less than 10%, good if RE ranges from 10% to 20%, fair if RE ranges from 20% to 30%, and poor if RE >30%1048where How to cite this article: Yang, J. et al. Analyzing the performance of fluorescence parameters in the monitoring of leaf nitrogen content of paddy rice. Sci. Rep.6, 28787; doi: 10.1038/srep28787 (2016).


Motor rehabilitation of stroke survivors may include functional and/or nonfunctional strategy. The present study aimed to compare the effect of these two rehabilitation strategies by means of clinical scales and functional Magnetic Resonance Imaging (fMRI). Twelve hemiparetic chronic stroke patients were selected. Patients were randomly assigned a nonfunctional (NFS) or functional (FS) rehabilitation scheme. Clinical scales  and fMRI were applied at four moments: before rehabilitation (P1) and immediately after (P2), 1 month after (P3), and three months after (P4) the end of rehabilitation. The NFS group improved significantly and exclusively their Fugl-Meyer scores at P2, P3, and P4, when compared to P1. On the other hand, the FS group increased significantly in Fugl-Meyer at P2, when compared to P1, and also in their ARA and Barthel scores. fMRI inspection at the individual level revealed that both rehabilitation schemes most often led to decreased activation sparseness, decreased activity of contralesional M1, increased asymmetry of M1 activity to the ipsilesional side, decreased perilesional activity, and decreased SMA activity. Increased M1 asymmetry with rehabilitation was also confirmed by Lateralization Indexes. Our clinical analysis revealed subtle differences between FS and NFS. Stroke is the leading cause of disability and the second cause of death in the world . Very ofAmong all motor rehabilitation strategies, physical therapy is still the most frequently used. Rehabilitation strategies in physical therapy are often based on active, active-assisted, or passive exercises and bilateral repetitive movements and often require strength. The exercises can be movements of articulation in a specific direction and have no functional purpose, for example, isolated motion of shoulder flexion. On the other hand, there are exercises that aimed at stimulating functional motor tasks, for example, picking up an object. Some functional exercises reproduce everyday motor functions and moreover there are functional approaches, such as neurodevelopmental techniques (NT) , which eFunctional approaches are largely used in clinical practice, although there are still not enough evidences of their eventual higher efficacy when compared to nonfunctional exercises –14. FurtIncreasingly, noninvasive functional neuroimaging, such as functional Magnetic Resonance Imaging (fMRI), is becoming an important tool to evaluate poststroke functional reorganization. Overall, fMRI studies of poststroke motor reorganization have consistently reported increased activity of the primary motor cortex (M1) of the unaffected hemisphere –19. FurtThe present study aimed at using fMRI and three clinical scales  to perform a longitudinal evaluation and comparison between two rehabilitation strategies: one based on NT, named functional strategy (FS) which simulates daily life activities, and the other based on a conventional nonfunctional strategy (NFS). We hypothesize that FS group will present a broader clinical improvement, accompanied by consistent patterns of cortical reorganization, as increased fMRI signal of the affected hemisphere, particularly of M1.This study was approved by the Ethics and Research Committee of the University of Sao Paulo and individual written informed consent was obtained from all subjects.Twelve chronic ischemic stroke survivors (aged between 38 and 71 years) were enrolled in this study . The timAll patients met the following inclusion criteria: middle cerebral artery stroke confirmed either by Computed Tomography (CT) and/or Magnetic Resonance Imaging (MRI); ability to understand and perform the fMRI motor task; National Institute of Health Stroke Scale (NIHSS) between 1 and 5 ; and modhttp://dx.doi.org/10.1155/2016/6353218). FS was based on Bobath, PNF, and movements simulating daily life activities involving upper limbs. Based on Bobath, we selected exercises to normalize muscle tone, such as rolling. Furthermore, the exercises evolved from simpler postures to positions that require greater motor control. The exercises were initiated in lying position and ended in a stand position. Based on PNF, we selected to our study movements that are functional and that are performed on the diagonal, such as playing with a tennis racquet, diagonal movement with a stick, and diagonal arm movement to pick up a ball. Furthermore, we selected movements that reproduce everyday motor functions like brushing hair, opening a door, and writing. FS used (i) rolling, performing abduction of the shoulder, extension of the elbow, extension of the fingers, and supination of the forearm to both sides; (ii) lying prone with elbow support, flexion of the elbows, pronation of the forearm, and abduction of the fingers ; (iii) changing from prone position to cat position and then from cat position to sitting position; (iv) in a sitting position, patients raising a stick on the diagonal using both hands on both sides; (v) in a sitting position, patient performing movement of pinch with the fingers holding small objects, writing or drawing, playing cards, buttoning, and unbuttoning; (vi) in a sitting position, combing the hair; (vii) in standing position, playing ball with a tennis racket; (viii) in standing position, opening and closing a lock and performing pronation and supination of forearm. The number of repetitions was established with the same criterion of nonfunctional exercises. At the beginning of rehabilitation, patients spent an average of about 2 minutes and 50 seconds to perform each activity and 21 minutes to perform the entire sequence .Selected patients were randomized with respect to the rehabilitation strategy: functional (FS) versus nonfunctional (NFS). Both approaches were applied five times a week, for 30 sessions, 90 minutes each. Nonfunctional exercises were initiated by the proximal articulations and finalized in the distal articulations. These exercises did not reproduce motor functions similar to everyday use. Patients performed the movements in sitting, lying, and standing positions. The sequence was performed bilaterally and with repetitions. The number of repetitions was established in the first session based on each patient capacity to perform the exercises for 90 minutes without fatigue. From this moment on, as long as the patient presented an improvement, the number of repetitions increased gradually. That is, the patient performed faster the same sequence of exercises. At the beginning of treatment, patients spent about 40 seconds to perform each activity and 6 minutes to perform the entire sequence. NFS consisted of active or, when appropriate, assisted-active or passive movements of all upper limbs articulations in all directions   test, and the modified Barthel index , 27. FugWe used fMRI and all clinical scales to evaluate the patients at four instants: before treatment (P1), immediately after rehabilitation (P2), at one month (P3), and at three months (P4) after the end of rehabilitation.p < 0.05.To evaluate the effect of time (P1 to P4) and group (FS versus NFS) in all scales, we used a repeated measures Generalized Linear Mixed Model (GLMM) in a 2 (group) × 4 (time) design, with dependent variables standardized by ranks prior to statistical analysis (SPSS v 18). Tukey post hoc comparisons were also performed. Data were presented with respect to median and interquartile range, and significance was set at MRI were acquired in a 1.5 T scanner  with a TX/RX head coil. fMRI consisted of 66 contiguous echo-planar (EPI) volumes, each with 16 axial slices . High-resolution anatomical images were also acquired using a T1-weighed GRE sequence with the following parameters: TR = 9.7 ms; TE = 4 ms; flip angle = 12°; matrix = 256 × 256; FOV = 256 mm; slice thickness = 1 mm; voxel dimension = 1 mm × 1 mm × 1 mm.Prior to the fMRI session, subjects trained for the motor task. It consisted of opening and closing one of their hands at self-pace. fMRI paradigm followed a block design, alternating six blocks of rest (27 seconds each), with five blocks of unilateral hand movement (27 seconds each). There were two runs in each session, one for each hand. Patients were monitored to ascertain correct task execution, to count the number of repetitions in each task period, and to inspect for synkinesis. The duration of each fMRI session was 25 minutes.q[FDR] < 0.05. fMRI evaluation was based on careful visual inspection, made by two experienced fMRI researchers (Draulio B. de Araujo and Antonio C. Santos)  < 0.05) in the contralateral M1 (to the moving hand) and Ni in the ipsilateral hemisphere. Positive LI indicates asymmetric activity to the contralateral hemisphere, while negative LI values indicate asymmetric activity to the ipsilateral side. LI ~ 0 indicates symmetrical M1 activity. These values were extracted only for the paretic hand. For the control group, ipsilateral and contralateral M1 were defined with respect to the dominant hand.Lateralization Index (LI) was based on two spherical volumes of interest (VOI)  was used to evaluate the between-subjects effect (controls × patients) and also within-subject effects among patients (P1 to P4). Significance level was set at Pearson's correlation analysis was performed between changes in LI and in all scales .The primary outcome measure of the study was the difference of clinical scales scores between at baseline (P1) and immediately after (P2) the rehabilitation program, between groups. Secondary outcome measures included differences of clinical scales scores between at baseline (P1), after 1 month (P3), and after 3 months (P4) of the rehabilitation program. In addition we also aimed to evaluate the patterns of fMRI maps in each group.Baseline characteristics of both groups were compatible regarding gender, time of stroke, NIHSS, mRS, and Ashworth. There were no significant differences of clinical scales between groups, before rehabilitation.An interaction effect group × time was found (Supplementary Table 1). For the NFS group, we observed differences only on the Fugl-Meyer scale, where scores at P1 were significantly smaller than at all other periods. For the FS group, differences on all three scales were found over time. In Six patients presented uncorrelated head movement or claustrophobia in at least one fMRI session, and data was analyzed only for the remaining periods. The other seven patients completed all fMRI evaluation successfully, in all sessions .At least one of five patterns was consistently observed as a result of rehabilitation, independently of the technique (FS × NFS): (i) decreased fMRI map sparseness, (ii) decreased activity of contralesional M1 (intact hemisphere), (iii) increased M1 activity in the ipsilesional side (damaged hemisphere), (iv) decreased perilesional activity, and (v) decreased SMA activity.An example is presented in Another consistent finding was the maladaptive increased activity of contralesional M1  found before rehabilitation (P1), which was related to motor performance. Patient #3, for instance, presented increased activity of contralesional M1, at P1, and subtle activity of ipsilesional M1. Increased ARAT score after training was related to a decrease of activity of both contralesional M1 and SMA .Increased perilesional activity was also found prior to rehabilitation, for instance, patient #5 . After rChanges in SMA were also observed. For instance, decreased activity of this region was related to clinical improvement. Patients #1 and #12 improved their ARAT scores with a reduced activity of SMA , except for P4. The Tukey post hoc test showed a significant difference only between P1 and P2, with respect to P1. LI values increased significantly at P2  and decreased back again at P3 (p = 0.008).Quantitative inspection of the contralateral and ipsilateral motor pathways was further based on the obtained LI values . We founr = −0.6652 and p = 0.1031 (LI × Fugl-Meyer), r = 0.0971 and p = 0.8359 (LI × ARAT), and r = 0.0933 and p = 0.8422 (LI × Barthel) .No significant Pearson's correlation was found between LI and the three scales used: This study aimed at evaluating and comparing functional and nonfunctional rehabilitation strategies in ischemic stroke. Assessment was made with clinical scales and fMRI. Independent of the rehabilitation used, our results indicate that patients improve significantly at P2 (right after rehabilitation), in at least one clinical scale. Furthermore, the observed improvement persisted even without rehabilitation in the FS group, observed by both Barthel and ARAT, which evaluate fine movements. On the other hand, Fugl-Meyer scores decreased significantly after rehabilitation (both at P3 and at P4), particularly in the FS group .As already pointed out in previous studies, a number of different rehabilitation techniques may improve motor functions of stroke survivors –32. Our The impact of rehabilitation on fMRI was marked by at least one of the following: decreased sparseness, particularly in the infarcted hemisphere, decreased activity of M1 of the intact hemisphere, and decreased SMA activity.Consistent with our results Figures  and 3, iThere are increasing evidences that the observed activity of contralesional M1 is related to worse stroke recovery , 37–39. Reduced sparseness also appears as a marker of clinical improvement in stroke survivors. For instance, longitudinal studies have shown that long-term training of specific tasks reduces the area of activity as detected by fMRI, for instance, following motor training , 42.Additionally, our study found reduced activity of SMA, which was coincident with clinical improvement. Some studies support the idea that SMA activity is important for recovery , 44, andAlthough our study found consistent clinical and fMRI changes related to rehabilitation, it is important to point out some of its limitations and caveats. First of all, the limited number of patients (6 in each group) hampers broader conclusion to the general population of stroke survivors. Furthermore, fMRI methods are based on unaltered cerebrovascular coupling, which is not the case in stroke –47. MoreThe search for new physical therapy techniques for patients with neurological deficits has been constant. The clinical outcome after rehabilitation can be measured by specific clinical scales and evaluates specific variables after treatment. In fact, our analysis revealed subtle differences between FS and NFS, indicating that the strategy of choice depends ultimately on the main goal to be achieved with rehabilitation. Furthermore, our fMRI results indicate some specific patterns that are best associated with the observed clinical improvements, which can be the focus of further investigation.Additional supporting information was added to the online version of article. Supplementary table 1 contains detailed information related to the analysis of clinical scales . Figure S1 shows the fMRI results of a patient with perilesional increased activity, together with improved ARA-t and Fugl-Meyer scores at P2, with respect to P1. Figures S2 and S3 were added to clarify the movements used during the Functional Strategy , and Non-Functional Strategy . Figure S4 brings the results of the correlation analysis between LI and the clinical scales .


FOXO transcription factors (FOXOs) are central regulators of lifespan across species, yet they also have cell‐specific functions, including adult stem cell homeostasis and immune function. Direct targets of FOXOs have been identified genome‐wide in several species and cell types. However, whether FOXO targets are specific to cell types and species or conserved across cell types and throughout evolution remains uncharacterized. Here, we perform a meta‐analysis of direct FOXO targets across tissues and organisms, using data from mammals as well as Caenorhabditis elegans and Drosophila. We show that FOXOs bind cell type‐specific targets, which have functions related to that particular cell. Interestingly, FOXOs also share targets across different tissues in mammals, and the function and even the identity of these shared mammalian targets are conserved in invertebrates. Evolutionarily conserved targets show enrichment for growth factor signaling, metabolism, stress resistance, and proteostasis, suggesting an ancestral, conserved role in the regulation of these processes. We also identify candidate cofactors at conserved FOXO targets that change in expression with age, including CREB and ETS family factors. This meta‐analysis provides insight into the evolution of the FOXO network and highlights downstream genes and cofactors that may be particularly important for FOXO's conserved function in adult homeostasis and longevity. In wormet al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., In mammals, FOXO factors are expressed broadly, including in the brain, immune system, liver and muscle, and are implicated in several tissue‐specific processes   . Interestingly, a significant number of genes, 461 , are bound by FOXO in all cell types, representing the ‘core FOXO’ direct targets  under these more restrictive conditions. To independently examine the correlation between FOXO binding enrichments at target genes in each cell type, we used Spearman's rank correlation. There was a significant correlation between the datasets . For direct comparison, we processed the available raw sequencing data using a uniform and stringent pipeline . Of the available datasets, endogenous FOXO binding datasets from four different cell types passed our quality control thresholds : FOXO3 binding in neural progenitor cells (NPCs)  , redox regulation/oxidative stress , and p53/DNA damage repair  . Shared and core FOXO‐bound genes were also enriched for pathways involved in several additional aging‐related processes, including chromatin regulation, immune function, and neurodegenerative disease (Parkinson's disease in the shared FOXO targets and Alzheimer's disease in NPC‐specific targets) . These results indicate that mammalian FOXO targets involve a large number of aging‐related genes. Notably, FOXOs bind both ‘pro‐longevity’ and ‘antilongevity’ GenAge functional regulators of aging. Thus, while FOXO is a central hub in regulating a network of genes involved in aging, some targets may actually antagonize FOXO's role in longevity.To test whether the FOXO targets (bound in any cell type) included pathways and processes involved in aging or longevity, we overlapped the mouse FOXO targets with aging‐related genes from the GenAge database  . There was significant overlap between FOXO/DAF‐16 targets identified by ChIP‐seq and those identified by DamID  . PANTHER analysis showed that FOXO/DAF‐16 direct targets are enriched for genes in the ubiquitin–proteasome pathway, growth factor signaling pathways, and processes such as metabolism , as previously shown  . This reanalysis of published data confirms and extends previous findings, and serves as a foundation to probe the evolution of the shared FOXO network between invertebrates and mammals.The enrichment for aging‐related genes in FOXO targets in the mouse raises the question of whether the mammalian FOXO targets include components from an ancestral network that regulates aging. In C. elegans to mouse, we performed a cross‐species analysis using orthologs (see C. elegans (bound in wild‐type or daf‐2)  (P = 1.06 × 10−97 in Fisher's exact test). Together, these results indicate target conservation for FOXO transcription factors across species. As C. elegans and mouse diverged at least 500 million years ago, this result also raises the possibility that the FOXO network in mammals and worms is very similar to the ancient regulatory network in their common ancestor.To determine whether direct FOXO binding is conserved from logs see . InteresC. elegans and mouse , and cell cycle . This analysis also uncovered novel pathways downstream of FOXO/DAF‐16 such as transcriptional regulation by bZIP transcription factors (14 genes) and tRNA metabolic processes  , FOXO/DAF‐16 targets conserved between C. elegans and mouse were enriched for DNA repair, regulation of oxidative stress, insulin/IGF/TOR signaling, translational regulation, and metabolism , we observed that DAF‐16 binding is enriched at the genes most upregulated in conditions that activate DAF‐16 . We observed that 50.9% of Drosophila dFOXO‐bound genes were also bound by mouse FOXO  . Together, these results highlight the strong cross‐species conservation among FOXO direct target genes and their cellular function.We next asked whether FOXO targets are also conserved in other species. Increased activity of the et al., C. elegans through human. It will be important to compare FOXO binding across species as additional human datasets become available to gain additional insight into the conservation and differences of FOXO targets between mouse and human.Similarly, we compared FOXO binding in mouse cells to genome‐wide binding in human cells Fig. B . This overlap identified 46 targets conserved across these four species , although FOXO direct targets in mouse also exhibited change in expression with age . While we did identify several conserved FOXO targets that changed with age , the overlap was not statistically significant in either case, suggesting that most genes that change in expression with age are actually not conserved direct targets of FOXO/DAF‐16. These results are consistent with the notion that changes in the highly conserved activity of FOXO transcription factors are not directly responsible for the age‐related changes to global gene expression networks during aging. Instead, the conserved gene expression network downstream of FOXOs may be crucial for preserving cells and tissues even in older animals.Are FOXO target genes conserved across species affected, or on the contrary preserved, transcriptionally, during aging? Using aging time course data from DNA microarrays from mouse (brain and thymus) and C. elegans, FOXO/DAF‐16 targets that change in expression with age are clustered into several functional groups: collagen, metabolism , Notch signaling, and the heat shock response . Interestingly, the network signature among the mouse FOXO targets that change with age is different and includes immune function, translation, and cell cycle genes . Collagen remodeling, lipid and fatty acid metabolism, and Notch have been implicated in aging . In all cases, the Forkhead consensus motif (TGTTTAC) was the top enriched motif , and E2F motifs. The enrichment for GATA family factors is notable as this family includes ELT factors and PQM‐1, and these factors have been previously implicated in aging in C. elegans  knockout in C. elegans extends lifespan in a daf‐16‐dependent manner  and AP‐1 (activator protein 1). CREB has been implicated in the regulation of longevity and tissue function during aging in et al., Only a subset of the FOXO/DAF‐16 targets change in expression with age, consistent with previous studies , GSE15567 (C. elegans wild‐type), GSE35486 (human DLD‐1 cells), GSE44686 (Drosophila chico heterozygotes and adults with ablated IPCs).ChIP‐seq datasets: GSE48336 (mouse NPCs), GSE35024 (mouse PreB cells), GSE46944 (mouse CD8+ T cells), GSE40657 (mouse Treg cells), GSE15567 (Microarray data: GSE18326 (mouse NPCs), GSE10273 (mouse PreB cells), GSE46942 (mouse CD8+ T cells), GSE40655 (mouse Treg cells).http://bowtie-bio.sourceforge.net/index.shtml; mm9, ce10, dm3, or hg19) . Briefly, normalized strand and relative strand cross‐correlation scores (NSC and RSC) were calculated   , using the dm3 and ce10 Refseq annotations, respectively. C. elegans ‐ and Drosophila‐associated gene names were obtained from BioMart. Binding enrichment around TSSs was performed using NGS plot (https://code.google.com/p/ngsplot/) using the k‐means clustering argument: ngs.plot.r –G mm9 –R tss –E UnionGeneList –GO km –KNC NumberOfClusters –C ConfigurationFile –O OutputName  for the ChIP‐seq datasets  were downloaded from the GEO database and processed with the following pipeline; reads were mapped to the appropriate genome using Bowtie 1  (Szklarczyk http://pantherdb.org/) .Functional annotation of gene sets was performed using the PANTHER database . Human–mouse orthologs were obtained from the Mouse Genome Informatics (MGI) Vertebrate Homology Database (http://www.informatics.jax.org/homology.shtml). Cross‐species comparisons were performed using one‐to‐one mouse– C. elegans, mouse–Drosophila, and mouse–human relationships.http://homer.salk.edu/homer/index.html)  were used in all analyses, and P‐values from the Homer Known Motif Enrichment Results are reported. In cases where a motif appeared multiple times, only the top  P‐value is reported.Motif analysis was performed using the Homer findGenomeMotifs.pl tool (https://r-forge.r-project.org/projects/vennerable/). Statistical analysis for the significance of overlaps was performed using Fisher's exact test. When calculating significance, we limited the ‘background dataset’ to only the relevant background genes. For the calculation within a species , all unique genes within the species were used as ‘background’. For the cross‐species analysis, only the genes with orthologs  were used as ‘background’. For the aging analysis, the genes included as probes in the microarrays were used as ‘background’, and the genes on the microarray and with orthologs (mouse–C. elegans) for the comparisons of the conserved genes.Venn diagrams were created in R using the ‘Vennerable’ package  or FOXO/DAF‐16 responsiveness in C. elegans , followed by Benjamini–Hochberg correction. ggplot2 was used to plot aging time course expression data.Microarrays (Affymetrix) were analyzed using RMA  from the ‘affy’ package and the RankProd implementation of the Rank Products method ‘RankProd’ package , cell type‐specific targets, targets that are shared between at least two cell types, or bound in all cell types (Core).Click here for additional data file.Table S2 FOXO/DAF‐16 target genes in Caenorhabditis elegans .Click here for additional data file.Table S3 FOXO/DAF‐16 target genes that are mouse specific, Caenorhabditis elegans specific, or conserved between C. elegans and mouse.Click here for additional data file.Table S4 FOXO/dFOXO targets that are mouse specific, Drosophila specific, or conserved between mouse and Drosophila.Click here for additional data file.Table S5 FOXO targets that are mouse specific, human specific (mouse names listed), or conserved between mouse and human.Click here for additional data file.Table S6 FOXO targets that are conserved across all species .Click here for additional data file.Table S7 FOXO targets that change in expression with age in the mouse and Caenorhabditis elegans.Click here for additional data file. Click here for additional data file.


Phalaenopsis species) is an ornamental crop that is highly commercialized worldwide. Over 30,000 cultivars of moth orchids have been registered at the Royal Horticultural Society (RHS). These cultivars were obtained by artificial pollination of interspecific hybridization. Therefore, the identification of different cultivars is highly important in the worldwide market.The moth orchid -simple sequence repeat (SSR) markers. After de novo assembly, the obtained sequence covered 29.1 Mb, approximately 2.2% of the P. aphrodite subsp. formosana genome , and a total of 1,439 EST-SSR loci were detected. SSR occurs in the exon region, including the 5’ untranslated region (UTR), coding region (CDS), and 3’UTR, on average every 20.22 kb. The di- and tri-nucleotide motifs  were the two most frequent motifs in the P. aphrodite subsp. formosana. To validate the developed EST-SSR loci and to evaluate the transferability to the genus Phalaenopsis, thirty tri-nucleotide motifs of the EST-SSR loci were randomly selected to design EST-SSR primers and to evaluate the polymorphism and transferability across 22 native Phalaenopsis species that are usually used as parents for moth orchid breeding. Of the 30 EST-SSR loci, ten polymorphic and transferable SSR loci across the 22 native taxa can be obtained. The validated EST-SSR markers were further proven to discriminate 12 closely related Phalaenopsis cultivars. The results show that it is not difficult to obtain universal SSR markers by transcriptome deep sequencing in Phalaenopsis species.We used Illumina sequencing technology to analyze an important species for breeding, Phalaenopsis breeding germplasm after preliminary validation. The potential universal EST-SSR markers are highly valuable for identifying all of Phalaenopsis cultivars.This study supported that transcriptome analysis based on deep sequencing is a powerful tool to develop SSR loci in non-model species. A large number of EST-SSR loci can be isolated, and about 33.33% EST-SSR loci are universal markers across the  Phalaenopsis spp.) are among the most graceful and popular plants. They consist of approximately 66 natural species worldwide, fifty-six of which are extant [Phalaenopsis is divided into five subgenera, Proboscidioides, Aphyllae, Parishianae, Polychilos, and Phalaenopsis that are determined mainly by plant size and floral morphology . The subgenus Polychilos was further subdivided into four sections, including Polychilos, Fuscatae, Amboinenses, and Zebrinae. Additionally, the subgenus Phalaenopsis was also subdivided into four sections, Phalaenopsis, Deliciosae, Esmeralda, and Stauroglottis. Phalaenopsis species are found throughout tropical Asia and the larger islands of the Pacific Ocean. All Phalaenopsis species, excluding the natural tetraploid species P. buyssoniana Rchb.f., have 38 (2n = 38) chromosomes [P. aphrodite has been completely sequenced [Phalaenopsis species also have been constructed based on the internal transcribed spacer (ITS) of the ribosomal (rDNA) and plastid DNA [P. x intermedia, showing that P. aphrodite was the maternal parent and P. equestris was the paternal parent [P. equestris [Moth orchids  molecular markers. The results showed that these two molecular techniques could offer informative markers to separate those from samples that are closely related. Another RAPD analysis was conducted by Goh et al. [Phalaenopsis, and four Paraphalaenopsis species were used as outgroups. Six out of twenty random primers were selected for analysis and 123 polymorphic bands have been obtained. Cluster analysis derived from the RAPD molecular markers showed that Phalaenopsis form seven groups and are basically congruent with previous studies derived from morphological charactersRandom amplified polymorphic DNA (RAPD) has been conducted to reveal the phylogenetic relationship of 16  species . Three-h species  examinedh et al. . They exPhalaenopsis amabilis based on microsatellite DNA . Three SSR loci were cloned and evaluated from P. amabilis accessions. The results indicated that these loci are good molecular markers to identify intraspecific variation of Phalaenopsis. EST-SSRs separately developed from the Phalaenopsis ESTs database have obtained 42 [Phalaenopsis equestris were discovered by large-scale BAC end sequencing [Generally, the high repeated motifs of microsatellites are prone to mutation through slipped-strand mispairing . The relained 42  and 261 quencing . Deep sequencing ,30,31,32de novo transcriptome deep sequencing of P. aphrodite subsp. formosana to analyze EST-SSR, develop molecular markers, and test the transferability between most members of Phalaenopsis that are used as parents for moth orchid breeding. To our knowledge, this is the first study to develop EST-SSRs by deep sequencing of transcriptomes in Phalaenopsis species. Furthermore, the developed EST-SSR markers in the present study can be applied for genetic diversity analysis, gene mapping, linkage map development, marker-assisted selection breeding, and cultivar identification in Phalaenopsis species/cultivars.Here, we performed P. aphrodite subsp. formosona. These short sequence reads have been deposited at NCBI as SRA accession number SRX1253908 and SRX1253909. The reads with high quality bases above Q20 were more than 90% indicated a very good quality calls bases (Q20 means 1 error per 100 sequenced bases) and with per sequence quality score above 38  mean a good quality. The high-quality PE reads were used for de novo assembly to join into scaffolds step-by-step, based on paired-end information. Finally, 22,598 unigenes (≥ 100 bp) were generated, with a final unigene N50 length of 2,047 bp and a total length of 29,062,410 bp  high-quality PE reads were generated from Sanger/Illumina 1.9 sequencing, approximately 4Gb of sequence data was obtained from leaves of 2,410 bp  Fig 1)..P. aphroThe 22,598 unigenes generated in this study were used to search potential microsatellites that were defined as perfect di-, tri-, tetra-, penta-, and hexa-nucleotide motifs with a minimum of 9, 6, 5, 5, and 4 repeats, respectively. There are potentially 1,439 EST-SSRs that can be found after SSR mining. The potential 1,439 EST-SSRs were further classified and shown in Phalaenopsis species that are representative germplasms for breeding most Phalaenopsis cultivars. Of these, ten EST-SSR loci were stably amplified, polymorphic, and transferrable SSR loci across 22 native Phalaenopsis species  value across 22 native Phalaenopsis species ranged from 0.163 to 0.889, with an average of 0.588  or tri-nucleotide motifs (35.23%). The average number of potential EST-SSRs per unigene is 0.064. Of the detected EST-SSRs, 1,051 EST-SSRs were obtained for suitable primer designation by BatchPrimer3 . The inf species . In totaof 0.588 . The ampPap-3222  and Pap-Pap-3222 . GeneticPap-3222 . The resPap-3222  by 10 pooglottis .Phalaenopsis amabilis (Taxon abb. 15) belong to Section Phalaenopsis, Phalaenopsis equestris (Taxon abb. 21), and Phalaenopsis lindenii (Taxon abb. 22) belong to Section Stauroglottis, revealed an admixture genetic components. Based on the best fit number of grouping, 22 taxa of Phalaenopsis were divided into two groups  in the Bayesian clustering analysis. Two genetic components were estimated using assignment test and each taxon was either high percentage of component 1 , which is relatively small compared to other Phalaenopsis species [de novo assembly of deep sequencing data, the obtained sequence covered 29.1 Mb, or approximately 2.2% of the P. aphrodite subsp. formosana genome. Excluding mono-nucleotide repeats that are not nearly useful for molecular markers [P. aphrodite subsp. formosana. Excluding mono-nucleotide repeat motifs, di-nucleotide repeat motifs of EST-SSRs were the most abundant type (51.49%) of microsatellites in the study. This result is consistent with EST-SSR studies in loblolly pine and spruce [Pinus contorta [P. ahrodite subsp. formosana and some plants may result from their high proportion of 5’UTR-SSRs.The genome size of  species ,34. Afte markers , a totald spruce . Pinus ccontorta , bluebercontorta , rubber contorta . In contcontorta ,41,42,43contorta ,45. ThusP. aphrodite subsp. formosana. The average density of EST-SSRs was likely shown to be relatively low frequency in P. aphrodite subsp. formosana compared to most other species; such as one EST-SSR is found every 14 kb in Arabidopsis [P. aphrodite subsp. formosana is higher than that of loblolly pine and spruce, which on average have an EST-SSR every 49.8 kb [SSR occurs in the exon region, including a 5’ UTR, CDS, and 3’UTR, on average every 20.22 kb in bidopsis , every 1bidopsis , every 1bidopsis , and evebidopsis . Althougbidopsis . Using t 49.8 kb . Therefo 49.8 kb , the aveP. aphrodite subsp. formosana, AG motifs were the most frequent SSR motifs, about 29.33% of the total isolated loci and the lowest frequency (0%) in GC repeat di-nucleotide repeat units in the study. Similar results also can be found in SSR loci of other plants derived from either EST-SSR [P. aphrodite subsp. formosana. These results are consistent with those found in SSR loci of other plants derived from either EST-SSRs [Arabidopsis [Arabidopsis [Di-nucleotide repeat units in  EST-SSR ,42,43 or EST-SSR ,49,50,51EST-SSRs ,42,43 orEST-SSRs ,47,50,51bidopsis ,53, and GC repeat units in low frequency of SSR motifs are shown in most SSR transcriptome-wide studies as previously described. GC-rich regions might be relatively stable, resulting in less replication slippage ,55. FurtDeep sequencing clearly offers a rapid strategy of acquiring the sequences required to discover SSRs and to design specific primers to obtain useful SSR markers. Additionally, EST-SSR markers usually have a higher amplification efficiency, and are more likely to be transferable across species than SSR markers derived from non-coding regions of the genome ,62,63,64Phalaenopsis, even though several studies have focused on SSR mining in Phalaenopsis with transferable evaluation across part germplasm of the genus [Phalaenopsis cultivars, the transferability and polymorphisms of SSR loci cloned form EST-SSRs require validation. According to the molecular phylogeny of Phalaenopsis [Phalaenopsis species were selected from the representatives of all subgenera and sections, which include most of the breeding parents for historical Phalaenopsis breeding programs. To develop universal SSR markers for all commercialized Phalaenopsis cultivars, EST-SSR markers were isolated and screened, resulting from having a higher amplification efficiency and are more likely to be transferable across species than those derived from non-coding regions of the genome [To the present, there is no universal SSR markers across genus he genus ,25,27, ohe genus . To deveaenopsis  and the e genome .in vivo [Phalaenopsis cultivars have different ploids, including diploids, triploids and tetraploids [Phalaenopsis cultivars; thus, tri-nucleotide repeat unit microsatellites are considered to be suitable SSR markers to identify Phalaenopsis cultivars.Di-nucleotide repeat unit microsatellites always have larger repeat numbers and high levels of polymorphism in diverse plants ,65,66,67in vivo . SSR marin vivo . Becauseraploids , di-nuclPhalaenopsis species which are usually used as parents for moth orchid breeding. The result showed that the transferability of the EST-SSRs across the 22 native Phalaenopsis species is approximately 33.33% (10/30). According to the PCoA results, the resolution of the first three axes explain 61.91% of the variation between species EST-SSR multilocus genotypes. The distribution of results in the three dimensional plot were clearly scattered between taxa, but can be grouped on cross related species of Sections Zebrinae, Phalaenopsis, Deliciosae, and Stauroglottis. Based on the Bayesian clustering analysis, 22 moth orchid divided into two groups and the Section Stauroglottis was separated out of others. This provides evidence of genetically distinct units in native moth orchids and potential molecular tools to identify commercialized cultivars/lines. The data were sampled from across five subgenera of the genus Phalaenopsis [Phalaenopsis and Doritis [Phalaenopsis cultivars registered in the RHS orchid hybrid database. The results show that the crossing barriers within orchidaceous plant genera are relatively low. Additionally, the amplified products derived from EST-SSR PCR for 22 native Phalaenopsis species are shown to be one or two bands, respectively, showing homozygosity and heterozygosity as described by several studies [Of 507 potential tri-nucleotide microsatellites, 421 were suitable for primer designation. Of these, 30 tri-nucleotide microsatellites were randomly selected to evaluate the transferability and polymorphism across the 22 native  Doritis . In the  Doritis . In moth studies ,77.Phalaenopsis cultivars, more than two bands may exit for an individual, as shown on Phalaenopsis cultivars that are often triploid or tetraploid [Phalaenopsis cultivars can be differentiated from one another based on the analysis of three SSR loci  and are listed in Phalaenopsis cultivars, twelve commercialized varieties were collected for study and are listed in Twenty-two P. aphrodite subsp. formosana using the RNeasy Plant Mini Kit  according to the manufacturer’s protocol. RNA quantity and quality was verified using NanoDrop ND 1000  and 2100 Bioanalyzer (Agilent Technologies), respectively. The cDNA library was constructed according to the manufacturer’s instructions for the mRNA-Seq Sample Preparation Kit . The constructed paired-end library was prepared by using the Genomic Sample Preparation Kit (Illumina) according to the manufacturer’s instructions. After validation on an Agilent Technologies 2100 Bioanalyzer, the library was sequenced using Illumina HiSeq™ 2000  according to the manufacturer’s instructions. De novo transcriptome assembly for the high quality reads (Q < 20) was performed using Trinity software [For Illumina transcriptome deep sequencing, total RNA was extracted from the fresh leaves of software .P. aphrodite subsp. formosana with SciRoKo 3.4 software [SSR loci were isolated in all of the unigenes from software . The seasoftware Phalaenopsis species and 12 commercialized cultivars were the plant materials respectively examined. One hundred milligrams of fresh leaves was ground in liquid nitrogen, and genomic DNA was extracted using the CTAB method [5’-TGTAAAACGACGGCCAGT-3’) to inexpensively label PCR products as described by Schuelke [To validate the polymorphism and transferability of the EST-SSR markers derived from transcriptome deep sequencing, 22 native B method . For ecoSchuelke . PCR conSchuelke . The labThe degree of polymorphism, including the number of amplifying bands per primer pairs with an average and the polymorphism information content (PIC) value were calculated using PowerMarker version 3.25 . A princS1 FigPhalaenopsis species listed in Lanes 1~22 represent 22 (PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


Recently, Press and Dyson have proposed a new class of probabilistic and conditional strategies for the two-player iterated Prisoner’s Dilemma, so-called zero-determinant strategies. A player adopting zero-determinant strategies is able to pin the expected payoff of the opponents or to enforce a linear relationship between his own payoff and the opponents’ payoff, in a unilateral way. This paper considers zero-determinant strategies in the iterated public goods game, a representative multi-player game where in each round each player will choose whether or not to put his tokens into a public pot, and the tokens in this pot are multiplied by a factor larger than one and then evenly divided among all players. The analytical and numerical results exhibit a similar yet different scenario to the case of two-player games: (i) with small number of players or a small multiplication factor, a player is able to unilaterally pin the expected total payoff of all other players; (ii) a player is able to set the ratio between his payoff and the total payoff of all other players, but this ratio is limited by an upper bound if the multiplication factor exceeds a threshold that depends on the number of players. Repeated games have long been exemplary models for the emergence of cooperation in socioeconomic and biological systems145345678910111213141516171920ZD strategies in IPD can be naturally extended to other two-player repeated games2324N-player PGG, each player chooses whether or not contribute a unit of cost into a public pot. The total contribution in the public pot will be multiplied by a factor r (1 < r < N) and then be evenly divided among all N players, regardless whether they have contributed or not. As a simple but rich model, the PGG raises the question why and when a player is willing to contribute against the obvious Nash equilibrium at zero29313234353637383941This paper focuses on a notable representative of multi-player games, the public goods game (PGG)26It is found by surprise that in multi-player repeated games, a single player can pin the total payoff of all others or extort them in a unilateral way. However, different from the observations in IPD, there exist some unreported restrictive conditions related to the group size and multiplication factor, which determine the feasibility to pin the total payoff of all other players and the upper bound of extortionate ratio.N-player repeated game, in which some stage game between N players is infinitely repeated. We prove the theorem (see C) or defection (D), thus there are 2N possible outcomes for each round. For an arbitrary player px is a vector, which consists of conditional probabilities for cooperation with respect to each of these possible outcomes, as:Consider an orem see  that in i-th outcome of the previous round. CCC,CCD,CDC,CDD,DCC,DCD,DDC,DDD}.where C (or D) and the number of cooperators among the opponents in the last round is pC,n . Therefore, the strategy vector for him is represented asIn many well-known multi-player symmetric games  In this determinant, there is one column which can be determined by only player 1’s strategy p1 . Record this special column as p1 properly and makesSince we consider memory-one strategies, the game can be characterized by a Markov chain with a state transition matrix then he can unilaterally enforce a linear relationship among all players’ expected payoffs such thatEx denotes the expected payoff for player x, and α0, p1 resulting in the linear multi-player zero-determinant strategy.Here c > 0 in each stage game31c = 1. Then each chooses either to cooperate by contributing his own endowment c = 1 into a public pool, or to defect by contributing nothing. At the end of each stage game, the total contribution will be multiplied by a factor r (1 < r < N) and divided equally among the N players. An arbitrary player x’s payoff under outcome i is denoted asWe further study the features of multi-player ZD strategies under the iterated public goods game, which is a common paradigm for studying social dilemmas. Consider there are n(i) is the number of cooperators among x’s N − 1 opponents in the outcome i, and hx = 1 if player x chooses to cooperate while hx = 0 otherwise. Hence the payoff vector of player x is where equalizer strategyp1 so thatBy utilizing the multi-player ZD strategy, player 1 can unilaterally set his opponents’ total payoff to a fixed value. Such a unilateral controlling strategy is called the α1 = 0 and αx≠1 = μ. Adopting such a strategy p1, according to which only requires N linear equations, in which there are 2N independent ones corresponding to the 2N independent components. These 2N independent equations have the form:where According to pC,N−1 and pD,0. The opponents’ total payoff thus depends on the number of players N, the multiplication factor r and the parameter γ. Player 1 can thus adjust the opponents’ total payoff by adopting strategies that results in different values of γ. Note that the same equalizer effect can be realized by different equalizer strategies with the same γ. pC,N−1 or a small pD,0 brings a small γ, and consequently increases the total payoff of the opponents. The range of possible total payoff of the opponents is also strongly affected by r and N: (i) when N − 1) to r(N − 1), or equivalently, he can set the average payoff of co-players from 1 to r; (ii) when r; and (iii) when where N − 2 strategy components and the coefficients μ and ξ can be represented by pC,N−1 and pD,0. In pC,N−1 ≤ 1 and 0 ≤ pD,0 ≤ 1 are satisfied, the nontrivial equalizer strategies exist. Generally, the feasible regions of equalizer strategies are the intersections of two half-planes determined by pC,N−1 and pD,0, which can be obtained by linear programming. In r and N, as well as the allowed upper bound of r versus different N. It is shown that as the increase of the number of player N, the allowed upper bound of r decreases with the number of players N, namely the feasible regions of equalizer strategies get narrow. Thus it is difficult for player 1 to pin his opponents’ payoff when more players participate in the game.Moreover, according to χ-fold of the sum of opponents’ surplus. This is the so-called χ-extortion strategy. Formally, the extortion strategy is defined as:Besides setting the opponents’ total payoff, a ZD player can also extort all his opponents and guarantee that his own surplus over the free-rider’s payoff is χ is the extortionate ratio and Φ is a free parameter. This vector equation gives us 2N linear equationswhere where χ > 0. By analyzing the probability constraints and the sign constraints  is the number of cooperators among x’s opponents in state i. x’s action in state j. Conventionally, if player x's action in state j is C, then Here M′ = M − I, where I is the unit diagonal matrix. After some elementary column operations on this matrix, the joint probabilities will be finely separated, leaving one column solely controlled under player x's strategy but not dependent on other players anymore , which links the stationary vector and the determinant of transition matrix. Here we briefly summarize their proof. By applying Cramer’s rule to the matrix M′, we have Adj(M′)M′ = det(M′)I = 0. Meanwhile from vT · M′ = 0. Comparing the above two equations implies that every row of Adj(M′) is proportional to v. Thus for an N-vector u, M′ji. This is exactly the definition of determinant of the matrix which by replacing the i-th column of M′ with u. Assume i the last column, we have:The stationary vector N × 2N matrix and u is the last column of M′. This theorem is of much significance since it allows us to calculate one player’s long-term expected payoff by using the Laplace expansion on the last column of M′. Let u1 denote the payoff vector for the player 1, player 1’s long-term expected payoff E1 is given by M′ by u1, we can calculate player 1’s long-term expected payoff as:where u1. Thus making a linear combination of all the players’ expected payoffs yields the following equation:where 1 is an all-one vector introduced for normalization. Player 1’s expected payoff depends linearly on its own payoff vector α0 = and αxM′ there exists a column p1 in terms of M′ is zero, the strategy p1 is a multi-player ZD strategy of player 1.where How to cite this article: Pan, L. et al. Zero-Determinant Strategies in Iterated Public Goods Game. Sci. Rep.5, 13096; doi: 10.1038/srep13096 (2015).


The main objective of a phase II selection trial is to identify the most promising treatment amongst multiple competing experimental regimens when it truly exists, with moderate sample sizes.Utilising response-adaptive randomization (AR) in such designs has ethical advantages as it steers patients away from the inferior treatment arms. A common approach uses Bayesian AR, whereby the randomization probability to an arm is based on the posterior probability that arm has the highest response rate. We also consider a frequentist alternative called sequential elimination , which is a special form of AR. Using a simulation study based on an Acute Myeloid Leukaemia trial with four experimental arms, we compared the two approaches as well as a single stage pick-the-winner selection design . A Bayesian futility monitoring rule based on comparison to the historical response rate of standard treatment in such patients is also incorporated.Under scenarios where all arms are futile, both AR designs perform better in terms of treatment selection and in-trial allocation than a single stage design. However, under scenarios where there is a winner, all approaches are comparable in terms of selection properties. Nevertheless, both AR approaches are more superior in allocating more patients to the best arm and are hence more ethical. The improved performance is more evident if there is a clear winner. The challenges of practical implementation of such approaches will be discussed, and one notable advantage of the Frequentist approach is that it is considerably more straightforward.