IdealPlantArchitecture1 (IPA1), a key regulator of the plant architecture in rice, functions as a direct downstream component of D53 in regulating tiller number and SL-induced gene expression. We showed that D53 interacts with IPA1 in vivo and in vitro and suppresses the transcriptional activation activity of IPA1. We further showed that IPA1 could directly bind to the D53 promoter and plays a critical role in the feedback regulation of SL-induced D53 expression. These findings reveal that IPA1 is likely one of the long-speculated transcription factors that act with D53 to mediate the SL-regulated tiller development in rice.Strigolactones (SLs), a group of carotenoid derived terpenoid lactones, are root-to-shoot phytohormones suppressing shoot branching by inhibiting the outgrowth of axillary buds. DWARF 53 (D53), the key repressor of the SL signaling pathway, is speculated to regulate the downstream transcriptional network of the SL response. However, no downstream transcription factor targeted by D53 has yet been reported. Here we report that  The characterizion of more axillary growth (max) mutants in Arabidopsis thaliana9, dwarf (d) mutants in rice (Oryza SativaL.)16, ramosus (rms) mutants in pea (Pisum sativum)17, and decreased apical dominance (dad) mutants in petunia (Petunia hybrida)20 have indicated that SLs function as root-to-shoot phytohormones that regulate shoot branching22 and also regulate leaf senescence and root development28. Subsequently, the elucidation of the biosynthetic and signaling pathways of SLs has attracted great attention. Carlactone is the conserved endogenous precursor of SLs, which is derived from all-trans-β-carotene through DWARF27 (D27)-mediated isomerization and subsequent cleavage by the carotenoid cleavage dioxygenases 7 and 8 (CCD7 and CCD8)30. In Arabidopsis thaliana, cytochrome P450 MAX1 has been shown to oxidize carlactone to carlactonoic acid (CLA)31, which is then converted into methyl carlactonoate (MeCLA) and further catalyzed to an unidentified strigolactone-like compound by an oxidoreductase-like enzyme, Lateral Branching Oxidoreductase (LBO)32. However, in rice, carlactone is first converted into ent-2′-epi-5-deoxy-strigol by one MAX1 homolog and further into orobanchol by a second MAX1 homolog33. Highly branched phenotypes that can be rescued by SL treatment are displayed by SL biosynthesis deficient mutants that include d27, d10, and d17 in rice; rms1 in pea; and max1, max3, max4, and lbo in Arabidopsis thaliana32.Strigolactones (SLs), a collection of terpenoid-derived compounds produced by plants, were firstly identified as the host-derived germination signals of root parasitic plantsDWARF14 (D14) encodes a member of the α/β-hydrolase fold family protein, which binds and hydrolyses SL to form a covalently linked intermediate molecule (CLIM)37. This reaction triggers a conformational change to form a complex with DWARF3 (D3), which encodes an F-box protein39. D14 could interact with D3 and DWARF53 (D53) in the presence of SLs, leading to the ubiquitination and degradation of the nuclear-localized repressor D5339. D53 contains three ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motifs, which are essential to recruit the transcriptional co-repressor TOPLESS (TPL) proteins and induce their oligomerization to form a repressor-corepressor-nucleosome complex40. In accord, D53 was shown to interact with a TPL-related protein, TPR2, in rice38. However, whether D53 regulates the transcription of genes in the endogenous SL signaling pathway and what are its direct downstream transcription factors remain to be determined. In other angiosperms such as Arabidopsis thaliana and pea, the orthologs of D14, D3 and D53 have similar functions44.Compared with the SL biosynthetic pathways, the knowledge of SL-mediated signaling is still obscure. Through studying SL insensitive mutants in rice, three components have been identified involved in SL perception and signaling. Ideal Plant Architecture1 (IPA1) is a key regulator in determining plant architecture, which encodes a member of the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) family transcription factors, SPL1446. Two microRNAs, miRNA156 and miRNA529, regulate IPA1 expression, and point mutations in the miRNA156 recognition site perturb miRNA156-regulated degradation of IPA1 mRNA, leading to decreased tiller number and increased plant height and panicle branches47. In contrast, the CRISPR/Cas9-generated ipa1 loss-of-function mutants exhibited opposite phenotypes48, reminiscent of the dwarf and high tillering phenotype of SL-deficient or -signaling mutant plants. IPA1 directly binds to the promoter of a negative regulator of tiller bud outgrowth, TEOSINTE BRANCHED1 (OsTB1), to suppress rice tillering47. The ortholog of OsTB1 in Arabidopsis thaliana, BRANCHED1 (BRC1), is a key regulator of branch outgrowth and one of the SL-responsive genes50. Previous studies showed that SL-induced bud growth inhibition occurs at least partially through the regulation of BRC1 transcription in Arabidopsis thaliana and pea53. These results lead to the hypothesis that D53 sequesters transcriptional co-repressor TPL proteins to suppress IPA1, a key transcription factor regulating tillering. In this study, we reported that IPA1 is the direct downstream component targeted by D53 to regulate the SL response and SL-induced gene expression in rice. D53 interacts physically with IPA1 and inhibits its transcriptional activation activity. Furthermore, IPA1 binds directly to the promoter of D53 and plays a critical role in the feedback regulation of SL-induced D53 expression. Together, these findings provide new insights into understanding the function of D53, and identify IPA1 as one of the D53-targeted downstream transcription factors in the SL signaling pathway in rice.IPA1 functions as a transcriptional activator in regulating tiller number and that its expression is regulated at both mRNA and protein levels; mRNA-level regulation was demonstrated by isolating and characterizing the two gain-of-function mutants, ipa1-1D and ipa1-2D55. To understand whether loss-of-function mutations of IPA1 could affect rice plant architecture, especially tillering, we used a targeting induced local lesions in genomes (TILLING) method to generate point mutations in IPA1, which led to amino acid substitutions in the gene product. However, none of the mutant lines exhibited obvious phenotypes, suggesting that these amino acids are not essential for the function of IPA1  and D53 to GAL4 activation domain (AD) to form BD-IPA1 and AD-D53. We found that expression of BD-IPA1 and AD-D53 in co-transformed yeast cells could activate the expression of the ADE reporter gene to allow the transformed yeast cells to grow on the SD medium without adenine, indicating an interaction between IPA1 and D53 . In this system, the LUC (Luciferase) reporter gene was driven by a DNA sequence containing the SQUAMOSA PROMOTER BINDING PROTEIN (SBP) binding element GTAC. When co-infiltrating Agrobacterium hosts expressing IPA1-MYC together with the reporter construct, the expression of LUC was dramatically enhanced . In the presence of the HisTrx-D53 fusion protein, the DNA binding activity of IPA1 was not affected and a super-shifted band could be observed , indicat50. In rice, the D53 transcript is rapidly induced upon GR24 treatment39. By analyzing IPA1 Chromatin Immunoprecipitation (ChIP)-seq data47, we found one IPA1 binding peak on the promoter region of D53 in shoot apexes with the peak summit 286 bp upstream of the D53 transcription start site (TSS). This peak contains a GTAC element which may be responsible for IPA1 binding  plants, the tillering number is dramatically increased , axillary buds, young leaves and nodes66. Further understanding is needed of transcription regulation of OsTB1 in a more specific tissue, especially in the bud whose elongation is regulated by SL. In our previous work, we showed that IPA1 could bind to the promoter of OsTB1 and regulate its expression46. But in Arabidopsis thaliana, loss-of-function mutants of different SPL genes are sensitive to SL treatment, and no evidence has been found of BRC1's regulation by SPL proteins, suggesting that AtSPLs are probably not involved in BRC1 induction by SLs67. It seems that the downstream SL signaling pathway is not fully conserved between monocotyledonous and dicotyledonous plants, and that OsSPL genes and OsTB1 may have diverse functions with AtSPL genes and BRC1 in response to SLs. However, further evidence is required to support this hypothesis. Moreover, in Arabidopsis thaliana, it is reported that SLs modulate polar auxin transport through the endocytosis of PIN-FORMED1 (PIN1)69. PIN1 is localized to the plasma membrane and it is rapidly depleted after SL treatment, independently of protein synthesis69. In rice, it is reported that basipetal polar auxin transport is elevated in d2714, and that IAA level is increased in d328, suggesting that the crosstalk between SL and auxin, two key plant hormones for plant development, is of great importance in both species. However, the mechanism of this crosstalk is still obscure. From IPA1 ChIP-seq data, we found that IPA1 binding sites are located on the promoter of PIN1b47, but whether and how IPA1 and PIN1b are involved in the crosstalk between auxin and SL needs further study.The mechanism of SL perception and signaling mediated by the SL receptor D14, the F-box protein D3 and the repressor D53 are conserved across monocotyledonous and dicotyledonous speciesd mutants have been well studied, but there are other significant phenotypes such as dwarfism and low fertility that need further investigation14. As a pleiotropic regulator, IPA1 regulates many important phenotypes, such as tiller number, plant height and panicle morphology in rice46. More importantly, IPA1 has been widely used in breeding elite rice varieties due to its great potential in improving rice yield55. It will be impoartant to have an in-depth characterization of the regulatory network of IPA1 and D53 to reach a better understanding of rice architecture and provide a powerful tool and new targets for molecular breeding in rice.The high tillering phenotypes of Oryza sativa L.) ssp. japonica Nipponbare, ProIPA1:7mIPA1-GFP transgenic line, ZH11, ipa1-10, ipa1-11, ipa1-3D, ipa1-4D, miR156OE, Ri22, d53 and other mutants were grown either in the green house under 16 h light and 8 h dark at 28 °C or the experimental field of the Institute of Genetics and Developmental Biology. Double mutants of ipa1-1D d53, ipa1-1D d10, and ipa1-1D d27 were generated from crosses between Ri22 (japonica), an ipa1-1D cultivar, and d53, d10, and d27 mutants, respectively.Rice (Oryza sativa L. japonica) using PCR primer pairs targeted to a conserved domain of IPA1. Crude CEL1 endonuclease was extracted from celery71, and used for the cleavage of PCR products at mismatched positions of re-annealed heteroduplexes. Point mutations were detected by capillary electrophoresis using AdvanCEFS96 .TILLING was performed in a population of 6 000 EMS-mutagenized M2 rice plants in the ZH11 background (IPA1 coding sequence (CDS) and D53 CDS were inserted into pGBKT7 and pGADT7 respectively. The Matchmaker® Gold Yeast Two-Hybrid System (Clontech) was used to test the interaction between IPA1 and D53 following the manufacturer's instructions. For the BiFC assay, the D53 CDS and different truncations of the IPA1 CDS were amplified and cloned into pUC-SCYNE(R) and pUC-SCYCE respectively72. The BiFC assay was performed as described previously47. For the Co-IP assay, total proteins were extracted from ProIPA1:7mIPA1-GFP or ProUB:GFP transgenic calli according to the method described previously38. Following the manufacturer's instructions, 30 μl of the agarose-conjugated anti-GFP monoclonal antibody  were added into 500 μl total extracted proteins and incubated at 4 °C for 3 h with gentle rotation. The beads were washed three times with 350 μl extraction buffer, and eluted with 30 μl SDS-PAGE sample buffer. Immunoblotting was performed as described47. For the GST pull-down, purified GST, GST-IPA1, HisTrx-D53 and proteins extracted from rice calli were used in the assay, which was performed as described38. HisTrx-D53 fusion protein and D53 protein from rice calli were detected by rabbit polyclonal antibodies anti-D5338 and GST fusion proteins by Ponceau S or a monoclonal mouse antibody anti-GST .The primer pairs used in protein interaction analysis are listed in 47. Primer pairs used in real-time PCR were listed in RNA isolation, reverse transcription and real-time PCR were performed as described previouslyIPA1 or D53 was amplified by the primer pairs listed in Escherichia coli expression vector pGEX-6p-1  or pDEST51. Expression and purification of fusion proteins were conducted as described47.The full-length CDS of ProIPA1:7mIPA1-GFP transgenic seedlings was performed according to the method described previously73. PCR reactions were performed in triplicate for each sample, and expression levels were normalized to the input sample for enrichment detection. The fold enrichment was calculated against the Ubiquitin promoter. No addition of antibodies (NoAbs) was served as a negative control.The ChIP-qPCR using the 47.The probe sequences are listed in ProGTAC:LUC and ProD53:LUC, synthesized GTAC sequence and D53 promoter were cloned into the pCAMBIA1301-LUC vector. To generate the Pro35S:IPA1-MYC and Pro35S:FLAG-D53, CDS of IPA1 and D53 were cloned to the 1300-MYC and pDEST1300-FLAG vectors, respectively. The plasmids used in this study were transformed to Agrobacterium tumefaciens strain EHA105. p19 was used to suppress RNA silencing, pCAMBIA1301 for GUS expression as an internal control. Agroinfiltration and luciferase imaging were performed as described previously74. GUS activities were measured with 4-methylumbelliferyl-β-D-glucuronide (Sigma) according to the manufacturer's instructions.To generate the 75, with plasmids containing GAL4BD, pRTL and 35sLUC as a negative control. The assay was performed as described47.The plasmids containing GAL4BD-IPA1, 35sLUC and pRTL or GAL4BD-IPA1-ΔN, 35sLUC and pRTL or GAL4BD-IPA1-ΔN-ΔSBP, 35sLUC and pRTL were introduced into rice leaf protoplasts as describedrac-GR24 (StrigoLab) or equal volume of acetone, then 0.5 cm shoot base samples were harvested at indicated time points. RNA isolation, cDNA synthesis, qRT-PCR, protein extraction, and immunoblotting were performed as described above.Two-week-old hydroponically cultured rice seedlings were grown in the climatic cabinet at 80% humidity, under 16 h light at 25 °C and 8 h dark at 16 °C. The seedlings were treated with 5 μM IPA1) and LOC_Os11g01330 (D53).Gene sequence used in this study can be found in the Rice Genome Annotation Project under accession numbers: LOC_Os08g39890 (XS, ZL, HY and JL conceived this project and designed all experiments. XS, ZL, HY, GS, JX, XM, YJ, GL, JD, XFY, CML and HL performed experiments. XS, ZL, HY, GX, YW and JL analyzed data. XS, ZL, HY and JL wrote the paper.The authors declare no competing financial interests.


Salivary gland cancers comprise a small subset of human malignancies, and are classified into multiple subtypes that exhibit diverse histology, molecular biology and clinical presentation. Local disease is potentially curable with surgery, which may be combined with adjuvant radiotherapy. However, metastatic or unresectable tumors rarely respond to chemotherapy and carry a poorer prognosis. Recent molecular studies have shown evidence of androgen receptor signaling in several types of salivary gland cancer, mainly salivary duct carcinoma. Successful treatment with anti-androgen therapy in other androgen receptor-positive malignancies such as prostate and breast cancer has inspired researchers to investigate this treatment in salivary gland cancer as well. In this review, we describe the prevalence, biology, and therapeutic implications of androgen receptor signaling in salivary gland cancer. Salivary gland cancers (SGCs) are a group of uncommon, heterogeneous tumors that account for 0.3% of all malignancies and 6% of head and neck cancers in the United States . The majThe androgen receptor (AR) is a nuclear steroid hormone receptor that is physiologically expressed at low levels in many human tissues . Its maiNuclear AR expression based on immunohistochemistry (IHC) is the most widely used marker of active AR signaling, and correlates with the response to ADT in prostate cancer . The preOur group recently identified AR positivity by IHC in 75% of SDCs, and RNA sequencing confirmed extremely low but detectable levels of AR mRNA in AR IHC–negative cases, all of which had typical SDC morphology at the time of pathologic re-evaluation . InteresThe prognostic relevance of AR expression in SDC is difficult to assess, due to the rarity of the disease and the low number of AR-negative cases. Some investigators have identified a trend suggestive of better disease-free survival in AR-positive compared to AR-negative SDC patients ,29, but In other subtypes of SGC, nuclear AR expression is found at lower rates. Adenocarcinoma, not otherwise specified (AC NOS) and acinic cell carcinoma (AcCC) are AR-positive in 26% and 15% of the cases, respectively ,33,34,35A subset of SGCs result from the malignant transformation of a pre-existing pleomorphic adenoma (PA). PA is the most prevalent salivary gland tumor, and is typically benign and non-metastatic. Around 6% of PAs develop into different types of carcinoma, denoted carcinoma ex-PA . WhereasThe full-length AR (AR-FL) gene consists of eight exons, of which exons 4–8 encode the ligand-binding domain. Expression of alternative AR isoforms lacking the ligand-binding domain  is associated with ADT resistance in prostate cancer ,48,49,50An extra copy of chromosome X, which includes the AR gene, is found in almost 40% of SDCs. This may contribute to overexpression of AR, although some of the tumors with an extra chromosome X are negative for AR in IHC . Unlike FOXA1 mutations may potentially be associated with ADT resistance in prostate cancer, although this is being actively investigated [FOXA1 in four of 12 AR-positive SDCs. Conversely, no FOXA1 alterations were found in four AR-negative SDCs [Forkhead box protein A1 (FOXA1) is a transcription factor that mediates the transcription of AR target genes by facilitating the AR/chromatin interaction . FOXA1 mstigated . In a reive SDCs .FASN overexpression can mediate resistance to ADT in prostate cancer, although no clinical data are yet available [FASN were found in four of 12 AR-positive but not in AR-negative tumors [Fatty acid synthase (FASN) is an enzyme that controls fatty acid synthesis and has been shown to promote the growth of prostate cancer as a result of AR signaling. Experimental studies suggest that vailable . In our e tumors .In ACC, which rarely expresses AR, no significant genetic alterations affecting AR signaling have been detected . In otheSeveral ADT drugs have been developed and tested clinically, mainly in patients with prostate cancer. Abiraterone is a CYP17A1 inhibitor which reduces circulating levels of androgen by ultimately blocking the conversion of pregnenolone to DHT. Bicalutamide and flutamide are competitive inhibitors of the AR ligand-binding domain, as is enzalutamide, which was developed more recently and has greater AR affinity compared to the earlier anti-androgens, and may inhibit AR activity via a variety of different mechanisms . TriptorInspired by results from other cancers ,56 and fPatients with AC NOS have been found to respond well to ADT, with partial or complete response in 10 of 11 reported cases, and a median PFS of 20 months. SDC patients appear to have a lower response rate, with partial or complete response in 11 of 26 (42%) reported cases and a median PFS of eight months .Of note, several dramatic responses to ADT in SGC patients were published only as case reports of extraordinary responders. A recent preliminary study including all SDC patients treated with ADT in the Netherlands showed somewhat more modest results, with partial response in four (13%) cases, stable disease in 10 (32%) cases, and progressive disease in 17 (55%) cases, and a median PFS of 3.8 months . On the FOXA1 and FASN. Ongoing and future clinical trials will likely shed light on the clinical benefit and limitations of ADT in AR-positive SGC.AR is expressed in a majority of SDCs and in a minority of other SGCs such as AC NOS, and ADT has emerged as a promising therapy in patients with AR-positive SGC. Several potential mechanisms of resistance to ADT have been described, including the expression of AR splice variants and mutations in


Cannabis spp.) growing operations (MGO) in California have increased substantially since the mid-1990s. One environmental side-effect of MGOs is the extensive use of anticoagulant rodenticides (AR) to prevent damage to marijuana plants caused by wild rodents. In association with a long-term demographic study, we report on an observation of brodifacoum AR exposure in a threatened species, the northern spotted owl , found freshly dead within 669–1347 m of at least seven active MGOs.Marijuana (Liver and blood samples from the dead northern spotted owl were tested for 12 rodenticides. Brodifacoum was the only rodenticide detected in the liver (33.3–36.3 ng/g) and blood (0.48–0.54 ng/ml). Based on necropsy results, it was unclear what role brodifacoum had in the death of this bird. However, fatal AR poisoning has been previously reported in owls with relatively low levels of brodifacoum residues in the liver. One likely mechanism of AR transmission from MGOs to northern spotted owls in California is through ingestion of AR contaminated prey that frequent MGOs. The proliferation of MGOs with their use of ARs in forested landscapes used by northern spotted owls may pose an additional stressor for this threatened species.The online version of this article (10.1186/s13104-018-3206-z) contains supplementary material, which is available to authorized users. Cannabis spp.) growing operations (MGO) in California have increased substantially since the mid-1990s, with a mix of illegal clandestine operations and those growing for medical or, recently, recreational use [The number and extent of marijuana , squirrels, deer, and passerine birds [Secondary poisoning of non-target wildlife  from ARs has become a re-emerging threat in California, especially around outdoor MGOs on or near public lands, which are considered a primary source of AR in wild environments , 9, 14. ne birds . In addine birds . Althougne birds , taking ne birds . For exane birds .Strix occidentalis caurina), a threatened species under the Endangered Species Act [Neotoma fuscipes). Dusky-footed woodrats are also perceived by growers as a threat to marijuana plants because they forage on young plants in the spring and use the plants to build nests [Of particular concern is the threat of secondary AR poisoning in northern spotted owls , Madison, WI for necropsy. Liver and blood samples were taken and sent for AR residue analysis at the USDA-APHIS National Wildlife Research Center, Fort Collins, CO.The liver and blood samples were subsequently tested for 11 anticoagulant rodenticides  and 1 neurotoxicant rodenticide metabolite   around the polygon formed by the owl locations to search for visible AR sources, such as residences and MGOs, within this particular owl territory. We also included locations of illegal MGOs found by law enforcement agencies . All MGOs were known to local law enforcement authorities. We were unable to determine whether the MGOs identified were using ARs. As one study noted [®) to develop maps  with liver residues as low as 10 ng/g [Lynx rufus), which led to a 64% reduction in survival [Brodifacoum was the only rodenticide detected in the liver (33.3–36.3 ng/g) or the blood (0.48–0.54 ng/ml) Table . In this 10 ng/g . Two nor 10 ng/g , 27. For 10 ng/g  and was survival . CombineBrodifacoum is commonly used in household, industrial and agricultural settings and is often found at illegal clandestine MGOs –8 to preUmbellularia californica), into their nests, with evidence that these plants act as larvicides and repellants against fleas [Cannabis contains similar monoterpenes and can also act as a larvicide against mosquito and other insect larvae [One probable mechanism of AR transmission from MGOs to northern spotted owls in this region of California is through ingestion of dusky-footed woodrats, which are a dominant prey of spotted owls in this area . Dusky-fst fleas , 35. Cant larvae –38; anect larvae . Both Cat larvae , 39 and t larvae . In foret larvae , landscat larvae .Based on this, we propose a mechanistic hypothesis on the linkage between MGOs and secondary AR poisoning of northern spotted owls Fig. , where wThe observation of a northern spotted owl with AR residues in proximity to numerous MGOs further suggests the potential linkage between MGOs and AR exposure for this threatened species as another additive stressor. Thus, outdoor MGOs in forested landscapes may provide resources for prey and foraging opportunities for northern spotted owls but with potentially lethal consequences for both.We were unable to definitely identify MGOs as the source for the brodifacoum found in the dead northern spotted owl. However, we were not able to identify any other potential sources of AR within the territory used by this particular owl. Thus, our case study provides evidence to support the hypothesis that MGOs may constitute an additional threat to northern spotted owl populations in northwestern California, a hypothesis that should be examined with further research.


However, it remains unclear whether AR signaling also promotes RCC progression by recruiting vascular endothelial cells (ECs), key players in the development of blood vessels. In our study, AR increased EC proliferation and recruitment to the tumor microenvironment and promoted RCC progression. Mechanistically, AR modulated cytokine CXCL5 expression by altering AKT → NF-κB signaling, and interruption of AKT → NF-κB → CXCL5 signaling using either specific inhibitors or siRNA suppressed AR-enhanced EC recruitment and AR-EC-promoted RCC progression. The results obtained using an in vivo mouse model and a human clinical sample survey confirmed the role of AR in promoting RCC progression through enhancement of EC proliferation and/or recruitment via altered AKT → NF-κB → CXCL5 signaling. Targeting this newly identified AR-induced AKT → NF-κB → CXCL5 pathway may facilitate the development of new therapies for slowing RCC progression.Androgen receptor (AR) signaling may promote renal cell carcinoma (RCC) progression  The worldwide incidence of renal cell carcinoma (RCC) has steadily increased over the past two decades124Tumors induce and sustain the growth of new blood vessels through angiogenesis910et al. also demonstrated that AR signaling promotes RCC progression via modulation of HIF-2α/VEGF signalingAndrogen receptor (AR) signaling is crucial during prostate cancer initiation and progressionvia increased endothelial cell proliferation and recruitment by modulating AKT → NF-κB → CXCL5 signaling.Here, we demonstrate that AR signaling promotes RCC progression −) and OS-RC-2  were obtained from ATCC . The 786-O and 769-P cells were maintained in RPMI-1640, whereas the OS-RC-2 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM); both media  were supplemented with 10% fetal bovine serum (FBS) . Cells were grown in a 5% CO2 and 37 °C incubator. Stable clones of OS-RC-2 cells with AR knock-down and AR-over-expressing 769-P and 786-O cells were transfected with siRNA or AR lentivirus as described in our previous studyHuman RCC cell lines 786-O, 769-P  were applied in vitro to activate or inhibit AR signaling at final concentrations of 10 nM and 1 μM, respectively. siRNA was used to knock-down CXCL5, P65 and P110 in RCC samples. Anti-GAPDH (6c5), -β-actin (I-19), -AR (N-20), and - tetramethylrhodamine isothiocyanate (TRITC) IgG antibodies were purchased from Santa Cruz Biotechnology. Anti-CD31 and -P65 antibodies were obtained from Millipore. 5-Bromo-2-deoxyuridine (BrdU) and crystal violet were obtained from Fisher Scientific. Anti-mouse/rabbit secondary antibodies for western blotting were obtained from Invitrogen.DHT (dihydrotestosterone) and Casodex   and PDTC/TNF-α . To determine the role of CXCL5 in EC recruitment, before analysis, we applied a CXCL5 neutralizing antibody for 1 hr at room temperature at a final dilution of 1:300.Switzerland) were used for migration and invasion assays. For in vitro invasion assays, the upper chambers of the transwells were pre-coated with diluted Matrigel . CM (conditioned medium) was obtained by co-culturing HUVECs with RCC cells. Briefly, the two types of cells were cultured in the same dish for 24 hr, and the supernatants were collected and filtered to remove cells.Twenty-four-well (8 μm pores) transwell plates  and serum-free medium were plated in the upper and lower chambers, respectively. After 36 hr of incubation, invaded cells were stained with 0.1% crystal violet and counted. The cell numbers were obtained by averaging the counts from 5 random fields. The migration assay was performed using the same approach as the invasion assay (omitting Matrigel) with an incubation time of 24 hr. The data are presented as triplicate repeats ± SEM.Before performing invasion assays, RCC cells were treated with CM for 48 hr. First, 10The ability of RCC to recruit ECs was monitored using recruitment assays. Briefly, ECs (HUVECs) were plated in the upper chamber (with 8 μm pores), and RCC cells were plated in the lower chamber.BrdU incorporation was used to demonstrate RCC cell/EC proliferation. Briefly, RCC cells/ECs were seeded into 24-well plates and allowed to reach 50–70% confluence. BrdU was added to the medium for 4 hr (3 μg/ml), and the cells were fixed with 4% paraformaldehyde. Then, 0.1% Triton X-100 was used to destroy the cell membrane (15 min), and 2 N HCl (25 min) was used to separate the DNA into single strands. The cells were incubated in 10% bovine serum albumin (BSA) with an anti-BrdU antibody (1:200) overnight at 4 °C, followed by incubation with a TRITC-labeled secondary antibody for 1 hr at RT. The fluorescence intensity of TRITC was monitored using a Super Micro Orifice Plate Spectrophotometer  at 547 nm.USA), and 2 μg of total RNA was subjected to reverse transcription using Revert Aid TM First-Strand cDNA Synthesis Kit  according to the manufacturer’s protocol. Quantitative-PCR was conducted using a Bio-Rad CFX96 system with SYBR green to determine the mRNA expression level of genes of interest. Expression levels were normalized to the expression of GAPDH RNA.Total RNA was isolated using TRIzol reagent ]. A total of 30 μg of protein was separated by 8–10% SDS-PAGE and transferred to nitrocellulose membranes. After blocking, the membranes were incubated with the appropriate dilutions (1:1000) of specific primary antibodies. Next, the blots were incubated with HRP-conjugated secondary antibodies and visualized using Odyssey Detection System .Expression of AR and related proteins was determined by western blotting according to a previous studyUSA) (diluted 1:100 in blocking buffer) for 1 h. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min. The cells were examined using a fluorescent microscope equipped with narrow band-pass excitation filters to individually select for red and blue fluorescence. The cells were observed using Image Pro Plus System® mounted on a fluorescent microscope . All experiments were performed in triplicate.Immunofluorescence staining was performed as described previously6 RCC cells (including 769-P and OS-RC-2) were co-cultured with human umbilical vein endothelial cells (HUVECs) for several weeks. The co-cultured RCC cells were then injected into the kidney capsules of 8 nude mice per group. After 4 weeks, the tumor masses were harvested, weighed, fixed with 4% formalin, and prepared for hematoxylin and eosin (HE) staining. The mouse care and protocols were approved by the Institutional Animal Care and Use Committee of Xi’an Jiaotong University, and all mouse experiments were performed in adherence with the National Institutes of Health Guidelines on the Use of Laboratory Animals.To demonstrate the effect of vascular ECs on RCC tumorigenesis, RCC cells plus or minus HUVECs were implanted into mouse kidney capsules. In brief, 10in vivo, 106 RCC cells (including 769-P and OS-RC-2) were injected into the kidney capsules of nude mice for at least 4 weeks to assess tumorigenesis. HUVECs were tagged by luciferase via a tail vein injection 24 hr before tumor harvest. Further immunohistochemistry (IHC) staining was used to detect the luciferase-tagged HUVECs within the tumor masses.For HUVEC recruitment assays For HE staining, tissue sections were de-waxed and rehydrated using routine methods. The sections were stained with hematoxylin for 5 min and washed in running tap water for 5 min. The sections were then stained with eosin for 30 sec, dehydrated, and mounted using routine methods. Figures show representative fields.Japan). A rabbit polyclonal antibody against AR  and anti-CD31  and anti-luciferase antibodies were used. Slides were further analyzed. Five random fields from each slide were recorded.IHC staining was conducted using Image Pro Plus System Taken together, the results shown in The potential molecular mechanism(s) by which AR signaling enhances EC recruitment to RCC is unknown. To address this, we first utilized a Q-PCR-based super-array analysis to search for key genes that link AR function to EC recruitment. We found that adding AR to 786-O and 769-P cells or that AR knock-down in OS-RC-2 cells significantly increased or decreased expression of several cytokines A. Among Importantly, using interruption approaches with a specific CXCL5 neutralizing antibody, we observed that blocking CXCL5 reduced recruitment of HUVECs to RCC 769-P-AR and OS-RC-2 cells in the presence or absence of DHT . SimilarOverall, the results presented in To study how AR-modulated CXCL5 expression enhances EC recruitment to RCC cells, we focused on NF-κB signaling. NF-κB is a central player controlling inflammation in tumorsNext, to dissect the mechanism by which AR modulates NF-κB → CXCL5 signaling, we focused on the PI3K/AKT pathway, as previous studies have demonstrated that PI3K/AKT signaling is aberrantly activated in many cancers, including RCC232425The results presented in To study the consequences of altering the AR-mediated AKT/NF-κB/CXCL5 pathway to enhance EC recruitment to RCC, we first employed BrdU incorporation assays. CM significantly increased cell proliferation in both AR-positive and -negative RCC cells .Using transwell and wound healing migration assaysin vitro.In summary, the results presented in in vitro cell lines, we first utilized a nude mouse model in which two cell lines (769-P-AR/769-P-Vec cells and OS-RC-2-Si AR/OS-RC-2-Sc AR cells) were orthotopically xenografted into the kidney capsules. Adding AR to the 769-P cells led to increased tumor sizes and masses compared with the control group  alterations and HIF-2α/VEGF are important mediators of this processAR expression is detected in almost 30% of RCC cases, and nearly 91% of RCC cells exhibit positive AR staining via PI3K/AKT signalingPI3K/AKT signaling may play important roles in RCC proliferation and invasion, cancer stem cell maintenance, and angiogenesis within the tumor23243536Angiogenesis requires endothelial cell migration, a process that is directionally regulated by chemotactic stimuli and further involves degradation of the extracellular matrix to enable progression of the migrating cells46in vitro and in vivo evidence that AR signaling positively promotes RCC progression by enhancing recruitment of ECs to RCC, which may occur by modulation of AKT → NF-κB → CXCL5 signaling. Targeting this newly identified interaction may facilitate the development of new therapies to slow RCC progression.In summary, we provide How to cite this article: Guan, Z. et al. Androgen receptor (AR) signaling promotes RCC progression via increased endothelial cell proliferation and recruitment by modulating AKT → NF-κB → CXCL5 signaling. Sci. Rep.6, 37085; doi: 10.1038/srep37085 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Many mechanisms or pathways are involved in secondary post-traumatic brain injury, such as the ubiquitin-proteasome pathway (UPP), axonal degeneration and neuronal cell apoptosis. UCH-L1 is a protein that is expressed in high levels in neurons and may have important roles in the UPP, autophagy and axonal integrity. The current study aims to evaluate the role of UCH-L1 in post-traumatic brain injury (TBI) and its potential therapeutic effects. A novel protein was constructed that fused the protein transduction domain (PTD) of trans-activating transduction (TAT) protein with UCH-L1 (TAT-UCH-L1) in order to promote neuronal transduction. The TAT-UCH-L1 protein was readily detected in brain by immunoblotting and immunohistochemistry after i.p. administration in mice. TBI was induced in mice using the controlled cortical impact (CCI) model. TAT-UCH-L1 treatment significantly attenuated K48-linkage polyubiquitin (polyUb)-protein accumulation in hippocampus after CCI compared to vehicle controls, but had no effects on K65-linkage polyUb-protein. TAT-UCH-L1 treatment also attenuated expression of Beclin-1 and LC3BII after CCI. TAT-UCH-L1-treated mice had significantly increased spared tissue volumes and increased survival of CA3 neurons 21 d after CCI compared to control vehicle-treated mice. Axonal injury, detected by APP immunohistochemistry, was reduced in thalamus 24 h and 21 d after CCI in TAT-UCH-L1-treated mice. These results suggest that TAT-UCH-L1 treatment improves function of the UPP and decreases activation of autophagy after CCI. Furthermore, TAT-UCH-L1 treatment also attenuates axonal injury and increases hippocampal neuronal survival after CCI. Taken together these results suggest that UCH-L1 may play an important role in the pathogenesis of cell death and axonal injury after TBI. Traumatic brain injury (TBI) is often associated with diffuse axonal pathology that leads to significant motor and cognitive deficits , 2. Althin vivo. TBI was induced in mice using the controlled cortical impact (CCI) model. The effect of TAT-UCH-L1 or vehicle upon Ub-protein accumulation, hippocampal neuron cell survival and axonal injury after TBI was determined. These studies address whether UCH-L1 expression is an important determinant of cell survival and axonal injury after TBI and whether TAT-UCH-L1 has potential as a novel therapeutic strategy in TBI treatment.The UPP is composed of numerous components which include ubiquitin, the 26S proteasome, ubiquitin activationg enzyme (E1), ubiquitin conjugating enzyme (E2), ubiquitin ligating enzyme (E3), and deubiquitinating enzymes (DUBs) , ubiquitThis study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the University of Pittsburgh Institutional Animal Care and Use Committee (Protocol Number: IS00001941). All surgery was performed under isoflurane anesthesia, and all efforts were made to minimize suffering. Animals were housed in a temperature and humidity controlled environment with 12 h light cycles and free access to food and water.Mouse monoclonal anti-poly-ubiquitinated conjugates antibody (clone FK1) were obtained from Enzo Life Sciences , anti-ubiquitin (linkage-specific K63) antibody was from Abcam  and anti-ubiquitin (K48-specific) was from Millipore . Anti-NeuN antibody was from Millipore; Anti-HA, anti-LC3B and anti-Beclin-1 rabbit antibodies were from Cell signaling technology . Monoclonal anti-GAPDH and anti-HA antibodies were from Covance ; Cy3-conjugated monoclonal mouse anti-biotin and Alexafluor 488-conjugated secondary antibodies were from Jackson Immunoresearch Lab . Anti-β-actin antibody and all other chemicals were from Sigma-Aldrich.The DNA sequence encoding full-length mouse UCH-L1 was amplified by PCR, and cloned into a modified pET30a vector containing the N-terminus PTD of TAT protein and HA sequences  . ConstruPlasmids were introduced into Escherichia coli Rosetta (DE3) strains  producing 6X His-tagged TAT-UCH-L1. Protein production was induced by shaking the E. coli Rosetta cells in medium containing 0.8mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) at 35°C for 4h before harvest by centrifugation. Cell pellets were then lysed and purified with a QIAexpress Ni-NTA Fast start kit . Purified protein was dialyzed against 1XPBS buffer and protein concentration was determined by BCA protein assay (Pierce). Protein purity was assessed by SDS-PAGE and Coomassie blue staining.o). Injury settings used were designed to generate underlying cortical and hippocampal tissue loss and were consistent with previous studies. Body temperature was maintained at 36.5–38.0°C using a warming pad. Following TBI, the incision was closed and anesthesia was discontinued. Bupivacaine was applied topically to the wound for pain relief. Mice remained on the warming pad until recovered from anesthesia then were returned to their home cage. Sham surgery was identical to that described above without trauma. After randomization, mice were administered 60 mg/kg TAT-UCH-L1 or PBS as vehicle control via intraperitoneal injection 1h prior to CCI or sham surgery. The TAT-UCH-L1 dose was determined based on preliminary experiments detecting the TAT HA tag in brain at various timepoints and concentrations (data not shown). Mice were monitored daily post injury. Animals exhibiting signs of infection  or pain  or weight loss greater than 20% of controls were removed from the study and euthanized by carbon dioxide inhalation.Animal studies were conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and with approval of the University of Pittsburgh Institutional Animal Care and Use Committee. Male mice  obtained from The Jackson Laboratory , were housed in a humidity and temperature controlled environment with free access to food and water and 12 h light cycles. The controlled cortical impact (CCI) model of traumatic brain injury used in this study was performed as described in detail previously, 21. BriPericontusional brain cortex and hippocampus were dissected from mice at 1h, 4h or 24h after TAT-UCH-L1 treatment and injury and rapidly frozen on dry ice until homogenization with T-PER tissue protein extraction reagent (Pierce) supplemented with protease and phosphatase inhibitors. Protein concentrations were measured by BCA assay. Western blotting was performed as previously described . For LC32O/O2 (2:1) and transcardially perfused with 20 mL of heparinized saline (4 u/ml) followed by 20 mL of 10% buffered formalin 21 days post TBI. Whole brains were embedded in paraffin and 7 μm thick serial sections were collected every 1 mm. Sections were stained with hematoxylin and eosin and hippocampi were examined using 20X brightfield magnification. All morphologically normal neurons (those with nuclear and cytoplasmic staining) in ipsilateral and contralateral CA1, CA3 and CA4 regions at Bregma -2.2 mm (center of the contusion) were counted by a blinded observer [Mice were anesthetized with 4% isoflurane in Nobserver . Data arSpared tissue volume was calculated using the method of Swanson et al by measuring surviving brain tissue in ipsilateral and contralateral hemispheric areas at each slice using ImageJ 1.50i software. Spared volume was determined by multiplying slice area by slice interval thickness then adding together all slices, 25. Spa2 fields from 20X fluoromicrographs located in thalamus (-2.2 mm from Bregma). N = 5 animals per group. Brain sections incubated without application of primary antibody served as controls.Amyloid precursor protein (APP) immunofluorescent staining was performed as previously described , 26. MicData are expressed as means +/- SE. Cell counting and spared tissue volumes were analyzed using Student’s T test; Densitometric comparison of immunoblots and APP-positive neurites were analyzed using ANOVA with Dunnett’s or Bonferroni post hoc analysis, respectively, using IBM SPSS statistical software. Results were considered to be significant when p < 0.05.in vivo, a TAT-UCH-L1 wild type (WT) fusion protein was generated. In addition to the PTD sequence and UCH-L1, TAT-UCH-L1 also contains a 6X His tag for purification and an HA tag for detection  1h before TBI or sham surgery. TAT-UCH-L1- or vehicle- treated mice were sacrificed 21 days post CCI or sham surgery and the brains perfused. Neurons exhibiting normal morphology (yellow arrows) with visible nucleoli were counted in ipsilateral and contralateral CA1, CA3 and CA4 regions of hippocampus . Cell suFor spared tissue volume analysis, serial sections were cut and percent contralateral spared tissue volume was measured. As shown in To detect the effect of TAT-UCH-L1 on TBI-induced APP accumulation, mice were treated with 60 mg/kg TAT-UCH-L1 or vehicle 1 h prior to TBI and sacrificed by perfusion fixation 24 h after trauma. Brain sections taken at the center of the contusion in trauma- and sham- operated mice were immunostained. APP-positive neurites were counted in ipsilateral and contralateral thalamus . APP accTo detect the effect of TAT-UCH-L1 treatment on TBI-induced Ub-protein accumulation and autophagy activation, a separate cohort of animals underwent trauma or sham surgery and was sacrificed at 24 h. Poly-Ub proteins and the autophagy markers, Beclin-1 and LC3B, were detected in hippocampus with immunoblotting. As shown in Hippocampal protein levels of both beclin-1 and LC3BII were increased in vehicle-treated mice 24 h after TBI as compared to sham mice. Compared with vehicle-treated TBI mice, TAT-UCH-L1 treatment significantly decreased both beclin-1  and LC3BII levels , suggesting that TAT-UCH-L1 treatment attenuates autophagy activation induced by TBI .in vivo. 2) Treatment with TAT-UCH-L1 decreases concentrations of K48-linked poly-Ub proteins after TBI and attenuates increases in Beclin-1 and LC3BII. 3) TAT-UCH-L1 treatment decreases accumulation of APP in damaged axons and increases cell survival in CA3 after TBI.The major findings of the study are: 1) Modification of UCH-L1 by fusion of the protein with the PTD of the TAT protein produces a systemically active protein that transduces brain neurons in vivo. Several such constructs have been used in animal models of neurological diseases including: Fusion of the PTD of TAT with the anti-apoptotic protein Bcl-XL produces a brain permeable protein that decreases activation of caspase 3, reduces infarction volume and improves motor outcomes after temporary focal ischemia in mice[The PTD domain of TAT confers neuronal permeability to the HIV virus. This sequence has been exploited by construction of several TAT proteins that cross the blood brain barrier and readily transduce neurons a in mice; A TAT 1a in mice; A TAT fa in mice. Thus, TUCH-L1 is expressed almost exclusively in neurons. The protein is highly prevalent, constituting as much as 1% of brain protein, hence the protein may have a role in many neuron-specific functions. UCH-L1 Clearance of misfolded and damaged proteins from neurons is also mediated by autophagy in addition to the UPP. UCH-L1 In addition to UCH-L1’s role in the neuronal UPP, it may have other functions. Many of the components of the UPP found in other cell types are present in the neuron; thus UCH-L1 may not only be required for UPP function. UCH-L1 may also be important in other neuron-specific transport functions. The neuron has unique requirements for transport of proteins and organelles from the soma down the axon to maintain the integrity of the axon. UCH-L1 interacts with cytoskeletal proteins such as tubulin and neurofilaments , 36. UCHThere is also evidence that UCH-L1 is important in synaptic function. UCH-L1 expression is regulated by synaptic activity and alters synaptic protein distribution, thus UCH-L1 may play an important role in synaptic function and remodeling . InhibitThe current results support a role for UCH-L1 in maintaining and repairing damaged axons after TBI. APP is actively transported down axons and also has a role in axonal transport itself . APP accin vitro and improve memory function in a mouse model of Alzheimer’s disease [The current study is the first to show that TAT-UCH-L1 treatment may reduce histological damage and axonal integrity after TBI. There are several limitations to this study: These studies use only pretreatment paradigms and do not assess whether the treatment has an effect on cognitive or motor function. The studies provide limited data regarding the mechanisms by which TAT-UCH-L1 prevents axonal damage and cell death after TBI. These studies do not address long term effects on axonal pathology, neuronal cell death and function outcome; therefore, additional studies are needed. Despite these limitations, the current work demonstrates the promise of TAT-UCH-L1 for the treatment of axonal injury after TBI. In order to optimize TAT-UCH-L1 efficacy, pharmacokinetic studies are needed to design an optimal dosing paradigm. In addition, further modification of the protein construct such as mutation of C152A or truncation of the N-terminus may enhance potency and half-life of the TAT protein , 43, 44. disease . Thus, T disease . Thus, mS1 Fig(DOCX)Click here for additional data file.S2 Fig100nM of recombinant TAT-UCH-L1 WT, TAT -UCH-L1 C90S fusion proteins or recombinant UCH-L1 WT or C90S proteins were incubated with 500 nM Ubiquitin-AMC substrate and hydrolase activity was measured by detecting fluorescence intensity (arbitrary fluorescence units) generated by the cleavage of Ubiquitin-AMC. n = 4 per group. Data is expressed as means +/- SE.(DOC)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


The purpose of this study was to clarify psychosocial factors/problems, social participation, quality of life (QOL), and rehabilitation needs in chronic-phase traumatic brain injury (TBI) patients with cognitive disorder discharged from the level-1 trauma center (L1-TC), and to inspect the effects of rehabilitation intervention to these subjects.A mixed-method research  was conducted at an outpatient rehabilitation department.Inclusion criteria of subjects were transfer to the L1-TC due to TBI; acute-stage rehabilitation treatment received in the L1-TC from November 2006 to October 2011; age of ≥18 and <70 years at the time of injury; a score of 0–3 on the Modified Rankin Scale at discharge and that of 4–5 due to physical or severe aggressive behavioral comorbid disorders. Study details were sent, via mail, to 84 suitable candidates, of whom 36 replied. Thirty-one subjects , who had consented to study participation, were participated. Cognitive function, social participation, QOL, psychosocial factors/problems, rehabilitation needs, and chronic-phase rehabilitation outcomes were evaluated using the Wechsler Adult Intelligence Scale, Third Edition, the Wechsler Memory Scale-Revised, the Zung Self-Rating Depression Scale, the Sydney Psychosocial Reintegration Scale, Version 2, and the Short Form 36, Version 2, qualitative analysis of semistructured interviews, etc.P < 0.05). Linear regression analysis showed that the social participation status was greatly affected by the later cognitive disorders and psychosocial factors/problems not by the severity of TBI. No changes were observed in these scores following chronic-phase rehabilitation intervention.Participants were classified into achieved-social-participation , difficult-social-participation , and no-cognitive-dysfunction groups . Relative to the achieved-social-participation group, the difficult-social-participation group showed greater injury and cognitive dysfunction and lower Sydney Psychosocial Reintegration Scale and Short Form 36 role/social component summary scores (64.9/49.1 vs 44.3/30.4, respectively, Chronic-phase TBI with cognitive disorder led to rehabilitation needs, and improvement of subjects’ psychosocial problems and QOL was difficult. There was no estimate for the number of people with nonfatal TBI who did not attend emergency departments. In 2010, the Centers for Disease Control and Prevention estimated that 2,500,000 individuals experienced TBI in the USA. Of these individuals, 2,213,826 (87%) were treated and released by emergency departments, 283,630 (11%) were hospitalized and discharged, and 52,844 (approximately 2%) died. The annual age-adjusted rate of total TBI-related emergency department visits, hospitalizations, and deaths has increased by 45%, from 566.7 to 823.7 per 100,000 persons from 2007 to 2010, in the USA.According to the Centers for Disease Control and Prevention, 1,700,000 individuals sustained traumatic brain injury (TBI) annually in the USA between 2002 and 2006. estimated that 3,170,000 people  in the USA were living with a long-term disability resulting from TBI at the beginning of 2005. In 2008, Watanabe et al estimated that in Tokyo , 379 individuals sustained TBI involving cognitive dysfunction annually.Zaloshnja et alCognitive dysfunction occurs during TBI, but the functional disorders at the core of this dysfunction include executive dysfunction and memory, attention, cognitive-behavioral, and psychobehavioral disorders, which combine to create a plethora of sociobehavioral disorders. Furthermore, psychological and sociobehavioral disorders are affected by psychosocial factors such as preinjury developmental and lifestyle history, psychosocial problems related to postinjury social life, and the quality and amount of rehabilitation. If psychosocial problems are magnified, activity limitation, participation restriction, and quality of life (QOL) degradation increase Fig. . TherefoHowever, there is no clear definition for psychosocial factors/problems. Generally, psychosocial factors/problems include issues involving interpersonal relationships, family members, school- and occupation-related problems, availability of social support, economic problems, and indemnity. following injury. In most cases, movement disorders or self-care issues are almost resolved at the time of transfer or discharge from the L1-TC, but cognitive screening tests performed by occupational therapists show that some type of cognitive impairment could be present.At the university hospital in Yokohama City, Japan, TBI patients are transported to the level-1 trauma center (L1-TC). Once patients have been stabilized, they are discharged home or transferred to a general, rehabilitation, or long-term care hospital. In the L1-TC, interventions involving acute-phase rehabilitation are initiated during the early “confused-agitated” or “confused, inappropriate, and nonagitated” stageCognitive-behavioral, psychobehavioral, and sociobehavioral disorders do not commonly manifest during acute rehabilitation. During the chronic phase, psychosocial problems resulting from cognitive dysfunction manifest subsequent to discharge. However, individuals with TBI are often reluctant to visit rehabilitation departments and agree only after being urged to do so by important family members or significant others.However, during the chronic stage, neurosurgery departments are the main providers of medical services. In addition, the rehabilitation provided in follow-up studies involving TBI patients who have been discharged from the hospital's emergency department or L1-TC is insufficient, and issues surrounding their social participation and QOL are not considered.Rehabilitation outcomes typically involve either employment or domestic life. If the outcome is that the patient is unfit for occupational and domestic life, the use of social participation resources is inadequate, which could increase the risk of psychosocial problems such as social withdrawal at home. In addition, even when patients are employed, rehabilitation support should be offered at workplace. The lack or cessation of rehabilitation support could make continuing occupational activity difficult for patients.  was designed. Semistructured interviews were conducted to determine the psychosocial factors/problems, social participation, QOL, and extent of subjects’ rehabilitation needs. Rehabilitation interventions were then implemented according to subjects’ rehabilitation needs, and intervention outcomes were evaluated using changes in social participation and QOL.The purpose of this study was to elucidate the psychosocial factors/problems of chronic-phase TBI patients who had been discharged from the L1-TC at the university hospital. A cross-sectional study involving mixed methods2The study was conducted between June 2012 and March 2014.2.1Figure In the 1st step, the database at the university hospital rehabilitation department was used to identify 206 consecutive TBI patients who had been referred for rehabilitation intervention between November 2006 and October 2011.Patients’ medical information pertaining to the L1-TC was collected from the hospitalization medical records department at the L1-TC.The 2nd step involved application of the following inclusion criteria: mild to severe TBI, according to the Glasgow coma scale (GCS), and transfer to the L1-TC (192 cases); acute-phase rehabilitation treatment received in the L1-TC (192 cases); age of ≥18 years and <70 years at the time of TBI (136 cases); and a score of 0–3 on the Modified Rankin Scale (m-RS) at discharge (81 cases) and that of 4–5 at discharge due to physical or severe aggressive behavioral comorbid disorders (16 cases).The exclusion criteria were: a score of 4–5 on the m-RS at discharge due to the state that do not fit into the above-mentioned inclusion criteria of m-RS 4–5 state (33 cases); cognitive or apparent psychiatric impairment that existed prior to TBI (6 cases); died (6 cases); residence in other prefectures (1 case); and absence of cognitive dysfunction (6 cases).Consequently, 52 patients were excluded according to the exclusion criteria from 136 patients and 84 suitable candidates were selected.In the 3rd step, study details were sent, via mail, to 84 suitable candidates, of whom 36 replied. Three additional candidates were referred directly from the neurosurgery department (1 subject had been omitted from the database). Of 39 potential subjects, 31 consented in a document to study participation.2.2 the modified grounded theory approach developed by Kinoshita is the most popular grounded theory approach in Japan. Modified grounded theory approach amendments intended to clarify the coding method were made following application of the basic properties of original grounded theory .The qualitative data were collected mainly via semistructured interviews. Of available qualitative approaches,Two rehabilitation doctors, who were certified specialists of the Japanese Association of Rehabilitation Medicine, conducted the initial intake and semistructured interviews with 31 subjects at home or in an interview room at the rehabilitation department. The interviewer requested family members or significant others to attend the interviews whenever possible.During the semistructured interviews, subjects could answer the following questions freely: “What are the conditions of your current lifestyle, occupation, friends, and family;” “What are the residual TBI effects that you currently experience;” “Which 3 incidents in your life have been influenced most by TBI, in consecutive order;” and “What are your hopes for rehabilitation?” Interviews were transcribed verbatim using shorthand. The main interviewer documented the content of conversations on the day of the interview, and another interviewer confirmed whether this content was accurate. concept formation and category generation were performed.Descriptive content regarding subjects’ psychosocial factors/problems, social participation restriction, QOL, rehabilitation needs, and occupational or educational status was extracted using the interview transcripts. Following application of the modified grounded theory approach coding method,2.3 Behavioral Assessment of the Dysexecutive Syndrome (BADS), and Rivermead Behavioral Memory Test (RBMT) were used to assess cognitive dysfunction, regarding which information was also obtained from recent data held at rehabilitation hospitals implementing follow-up.Assessment of cognitive dysfunction: The Wechsler Adult Intelligence Scale, Third Edition (WAIS-III), Wechsler Memory Scale-Revised (WMS-R), Cognitive-Behavior Scale for TBI, is a self-assessment questionnaire consisting of 31 items used in cognitive dysfunction screening. Seven factors, including amnesia (9 items) and executive function (4 items), are extracted through factor analysis. The standard deviation for control subjects is used as the measurement unit, with scores for each factor expressed as z values and healthy individuals’ mean scores for each factor designated as the zero point. The higher a subject's z score, the more severe the impairment. In this study, the amnesia factor (9 items) was included in the analysis.The Cognitive-Behavior Scale for TBI used the cut-off point according to the level of memory disturbance; 22–24 in normal, 17–21 in poor memory, 10–16 in moderately impaired, and 0–9 in severely impaired.The total score of RBMT are 24 point. The original English version of RBMT (SDS) was used to assess depressive state. The SDS is a 20-item self-assessment questionnaire that measures depression. Item scores range from 1 to 4 . In the Japanese edition of the SDS, scores of 40–49 and ≥50 indicate depressive state and depression, respectively.Assessment of psychological disorders: The Zung Self-Rating Depression Scale,14 was used to assess social participation restriction. The SPRS-2 consists of 12 items concerning work and leisure activities, interpersonal relationships, and independent living skills. Higher scores indicate favorable social participation. If total scores (0–48) are converted to logit values (0–100 logit) using Rasch analysis, no ceiling or floor effects are observed. The reliability and validity of the scale have been demonstrated statistically. Form A (physician-led assessment questionnaire) measures change since injury, and form B (self-assessment questionnaire) measures current status. Because there is no Japanese version of the scale, the SPRS-2 (forms A and B) was translated into Japanese for use in the study.Assessment of social participation restriction: The Sydney Psychosocial Reintegration Scale, Version 2 (SPRS-2),16 was used to assess QOL. The SF-36 is a 36-item health-related QOL measure that was developed in the USA; the reliability, validity, and national standard value (norm-based scoring) of the scale have been demonstrated. The SF-36v2 contains 8 domains, for which scores range from 0 to 100; higher scores indicate better QOL. Physical, mental, and role/social component summary scores are calculated based on the scores for the 8 domains. Norm-based scoring  is published according to sex and age. Furthermore, the SF-36v2 was used to assess the effect of subjects’ cognitive dysfunction on significant others.Assessment of QOL: The Japanese edition of the 2nd version of the 36-Item Short-Form Health Survey, or Short-Form 36 (SF-36v2),2.4Content related to subjects’ social participation and rehabilitation needs was extracted from the semistructured interview transcripts and classified into concepts or categories concerning social participation and rehabilitation needs.Consent for prospective chronic-phase rehabilitation intervention was obtained from the subjects, and the services were implemented based on subjects’ specific rehabilitation needs.2.5Approximately 12 to 18 months subsequent to the initial interviews, an additional self-assessment questionnaire measuring participation restriction and rehabilitation needs and including the SPRS-2 (Form B) and SF-36v2 was sent to subjects via mail. The self-assessment questionnaire items were intended as follow-up for those used in the interviews. Scores for the SPRS-2, SF-36v2, and variables, such as social participation, included in the additional assessment were compared to those of the initial interviews.Rehabilitation intervention outcomes were assessed according to differences between the scores for variables in the initial interview and additional assessment. Relative to that recorded at initial interview, QOL at additional assessment was categorized as improvement, no change, decline, or breakdown of the life.2.6t test was 17 .The sample size was calculated to examine changes in SF-36v2 scores. With regard to norm-based scoring for the Japanese population, the mean SF-36v2 score was 50 (standard deviation: 10). Therefore, the required sample size for the independent t test, analysis of variance, and a chi-square (χ2) test were performed in the statistical analysis. A stepwise linear regression analysis was conducted. Dependent variables were social participation status, Sf-36v2, and SPRS-2 score at the initial interview. Independent variables were 23 items/domains such as education years, TBI severity (GCS score), cognitive function (WAIS-III), psychological disorders (SDS), psychosocial factors, QOL (SF-36v2), etc. In order to confirm the validity of the participant selection, Cronbach-alfa was calculated. A P value of <0.05 was considered statistically significant.IBM SPSS Statistics version 21 was used for statistical analysis. A The clinical ethical review board at the university with which the authors were affiliated approved the study in June 2012. This study was conducted in accordance with the ethical standards established in the Declaration of Helsinki (1964) and later amendments.33.1Thirty-one subjects participated in the interviews. Cronbach-alfa test for 8 subdomains of SF-36v2 and 12 items of SPRS-2 were 0.832 and 0.963, respectively. Subject characteristics are presented in Table The content of the initial intake and semistructured interviews was clear and logical for 8 subjects, who did not show memory disturbance or attention disorder in everyday life or display participation restriction in their work, school, or community lives. These 8 subjects did not display cognitive dysfunction and were assigned to the group without cognitive dysfunction (no-cognitive-dysfunction group). They were subsequently excluded from the analysis and the chronic-phase rehabilitation intervention.In the 11 subjects who showed cognitive dysfunction, social participation levels ranged from good  to inadequate . These subjects were assigned to the group that achieved social participation despite cognitive disorder .For 12 subjects, social participation was difficult  or impossible . These subjects were assigned to the group that experienced difficult social participation .P = 0.137), with injury most common between youth and middle age. The educational background did not show significant difference between 3 groups statistically (P = 0.724). Most subjects from all 3 groups had suffered trauma in a traffic accident; all 3 groups included individuals classified as having severe TBI (GCS: 3–8) upon transfer to the L1-TC.Participants’ median age at injury was 33.4 years . The duration of the L1-TC hospitalization was long (median: 40 days) for all groups; this was particularly true for the difficult-social-participation group. Forty-five percent of achieved-social-participation group and 50% of the difficult-social-participation group subjects were subsequently transferred to a convalescence rehabilitation hospital or special rehabilitation hospital for TBI. Outpatient rehabilitation period of the difficult-social-participation group (n = 8) was long. Despite experiencing cognitive dysfunction, 9 (81.8%) and 6 (50.0%) subjects from the achieved- and difficult-social-participation groups, respectively, did not receive follow-up treatment from the rehabilitation department , which was also true for all no-cognitive-dysfunction group subjects .Diffuse axonal injury was observed in 58% of the difficult-social-participation group, which also included 9 subjects with severe TBI; this number exceeded that of similar subjects in the achieved-social-participation group (P = 0.883).The median period from TBI to 1st interview was 50.7 months .Approximately 20% of subjects in both social-participation groups showed a history of developmental issues such as parental divorce and poor academic performance. In addition, 33.3% of the difficult-social-participation group subjects exhibited psychobehavioral disorders such as sleeplessness, depression, self-injurious behavior (with wrist scars indicating self-cutting), and panic. Approximately 27% and 42% of achieved- and difficult-social-participation group subjects, respectively, reported a history of family issues such as divorce. Moreover, 19% and 25% of achieved- and difficult-social-participation group subjects, respectively, reported work-related problems, with no significant between-group difference  and 8 (66.7%) achieved- and difficult-social-participation group subjects, respectively, exhibited rehabilitation needs involving social participation such as occupational support. In both groups, all subjects required intervention via rehabilitation.3.3Relative to the achieved-social participation group, the difficult-social-participation group obtained significantly lower WAIS-III (performance intelligence quotient), WMS-R , and BADS scores and higher Cognitive-Behavior Scale for TBI amnesia scores Table .t = 1.398, P = 0.178).SDS scores for the achieved- and difficult-social-participation groups were somewhat high but did not differ significantly .SPRS-2 scores (form A) were significantly lower in the difficult-social-participation group relative to those of achieved-social-participation group . Further, SF-36v2 scores related to significant others did not differ significantly between the 2 groups.Furthermore, regarding QOL, the difficult-social-participation group obtained significantly lower SF-36v2 role/social component summary scores relative to those of the achieved-social-participation group , outpatient rehabilitation for cognitive dysfunction (4 subjects), and others. In the difficult-social-participation group, interventions included cognitive dysfunction evaluation (6 subjects), outpatient rehabilitation for cognitive dysfunction (6 subjects), referral to the rehabilitation institution for social participation promotion (3 subjects), and others. However, because the Japanese healthcare insurance system has not adopted holistic day treatment rehabilitation, the program was not offered.Of the subjects who were referred to the rehabilitation institution, 2 underwent consultation. Of these, 1 withdrew prior to initiation of an employment support program. Another subject, who had experienced repeated dismissal subsequent to TBI injury, secured employment independently via the Internet and withdrew from the employment support program prior to completion. These subjects no longer underwent follow-up at the rehabilitation institution.Regarding the outcomes of the 12 to 18-month prospective rehabilitation intervention, in the difficult-social-participation group, social participation improved slightly and the number of unemployed subjects decreased from 9 . Three subjects from the difficult-social-participation group were considered to have experienced a breakdown of the living.At additional assessment, 11.1% and 36.4% of achieved- and difficult-social-participation group subjects, respectively, showed improved QOL, but significant difference between both groups was not shown .Parents were divorced during a high school student. He was a part-time worker after graduation from senior high school. He was injured for brain contusion  and diffuse axonal injury. GCS at the time of the L1-TC import was E1V2M2. Length of stay in the L1-TC was 43 days. He was transferred to TBI rehabilitation hospital, and discharged to mother's house after 153 days from injury.Rehabilitation goal was “social welfare work,” but he employed as a handicapped worker to a company for handicapped . Follow-up by rehabilitation department was not done.His self-awareness for moderate cognitive dysfunction  was insufficient. His psychobehavioral disorders were making excuses and lie for his failure, easiness of anger , poor managing money or life, etc.Rehabilitation needs of mother and the social worker was to have rehabilitation again, and that of him was vague hope to living alone. He was pressed mother and a social worker for visiting the rehabilitation medicine 54 months after the injury. .He answered in the initial interview; “I want my mother not to say ‘You could do it before’.” “Colleagues say to me ‘Work is slow, and to do that work than the work earlier’.” “I can intend to quit work. I am working but do not think it is ‘good.”Case 2: Male .Parents were divorced at a primary schoolboy. He was a national public servant (a round of inspection and supervisory duties) after graduation from senior high school. He was injured for brain contusion  and multiple bone fractures such as the fracture of orbit. GCS at the time of the L1-TC import was E1V2M4. Length of stay in the L1-TC was 60 days. He was transferred to a general hospital of the hometown. He discharged the hospital to mother's house in 72 days from the injury, and then continued outpatient cognitive rehabilitation for 4 months. He returned to an office work in 13 months from the injury, but there were no rehabilitation follows afterward. He completely returned to his former duties in 43 months.After injury in 48 months, he consulted the rehabilitation medicine for the purpose of aftereffect diagnosis. The rehabilitation needs to the cognitive disorder was vague.He was aware of his memory disturbance and difficulty of handling multiple tasks. Memory disturbance was remarkable, but the recent memory was kept. The psychobehavioral disorder such as easiness of anger or impulsiveness was not found. .He could input a promise and a schedule into i-phone promptly. He was going to hide cognitive disorders from a boss and fellow workers. Therefore, he was not able to talk about his cognitive disorders in the workplace.He answered in the initial interview; “In patrolling work, observations to a wide range cannot be reached every corner.” “I forget the place where I put a memo for the patrol report.” “I will panic when I receive various instructions continuously.” “My boss strictly warned me what I forgot and failed.”4 The results showed that 62% and 22% of subjects with moderate  and severe TBI , respectively, were initially discharged home. Most subjects (94%), including those who were discharged via rehabilitation hospitals, general hospitals, and nursing homes, had returned home 1 year subsequent to injury . One year subsequent to injury, 1 in 4 patients exhibited cognitive disorders, behavioral disorders, and physical disabilities, with only 32% able to resume their preinjury occupations. Almost half of TBI patients needed outpatient care for cognitive-behavioral disorders. Of those initially discharged home without follow-up, 10% subsequently required outpatient rehabilitation. The researchers concluded that long-term aftercare for chronic-phase TBI was required to improve social participation.The pathways of TBI patients discharged from an L1-TC in The Netherlands were studied retrospectively by de Koning et al.The qualitative findings of the present study showed that, in the chronic stage of TBI, cognitive-behavioral disorder fluctuated according to psychosocial issues. TBI patients with cognitive dysfunction sometimes discontinued the rehabilitation department's serial observation inappropriately after returning home and engaging in social participation involving activities such as working. Subjects or their significant others subsequently become aware of cognitive-behavioral disorders and the need for chronic-phase rehabilitation. Results of the stepwise linear regression analysis showed that the social participation status in the chronic phase TBI was greatly affected by the later cognitive disorders  and psychosocial factors/problems not by the TBI severity (GCS).Cognitive-behavioral and psychobehavioral disorders observed in chronic-phase TBI were affected by psychosocial factors/problems and tended to manifest gradually. When rehabilitation and support for these disorders were insufficient, social participation was inhibited and QOL decreased. In contrast, with self-awareness of cognitive disorder, social reintegration could be achieved with social support, adequate support from significant others, and chronic-phase rehabilitation. systematically reviewed the prognostic value of self-reported traits/problems/strengths and environmental barriers/facilitators related to TBI patients’ participation outcomes. Of the self-reported variables, the number of postconcussive symptoms, fatigue, and physical competence were predictive of employment and need for supervision, whereas self-efficacy was unlikely to predict employment. Subjective well-being, pain, and social interaction, but not coping style, were possibly predictive of employment. The researchers concluded that self-report variables were likely to contribute to participation outcome prediction.Because of TBI patients’ impaired memory and awareness, the reliability of the content of their responses to self-assessment questionnaires should be considered. Sherer et alThe present study involved self-assessment via the Cognitive-Behavior Scale for TBI, SPRS-2 (Form B), and SF-36v2, but the objectives of the study did not include identification of outcome predictors or observation of disparities between subjects’ self-assessment and evaluation performed by their significant others. To minimize the influence of subjects’ under- and overestimation, information provided by significant others was reviewed, semistructured interview transcripts were considered, and concepts and categories related to psychosocial factors/problems, social participation, QOL, and rehabilitation needs were extracted. Accordingly, the concepts and categories identified by the self-assessment questionnaires and semistructured interviews presumably exerted little influence on outcomes, despite errors in subjects’ self-assessment. assessed psychosocial TBI outcomes using neurophysical, mental, and social scales.Generally, psychosocial factors include issues involving family members, interpersonal relationships, school and occupational issues, availability of social support, economic problems, and compensation. However, the definition and range of psychosocial factors remain unclear. Bond reviewed current approaches to the community integration assessment. The Community Integration Questionnaire, SPRS-2, Reintegration to Normal Living Index, and Community Integration Measure were used to assess community integration. In these measures, the 3 core elements of community integration included relationships with others, independence, and meaningful activities. Only the SPRS-2 assessed both postinjury changes (form A) and current status (form B), which were well correlated.Social participation and community integration, which are affected by psychosocial issues, are rehabilitation goals for TBI patients. Salter et al systematically reviewed neurobehavioral rehabilitation programs for adults with TBI and made evidence-based recommendations for program adoption. The researchers concluded that the greatest overall improvement in psychosocial functioning was achieved via comprehensive-holistic rehabilitation programs, which could be considered a treatment standard for TBI patients with behavioral and psychosocial disorders. Cicerone et al systematically reviewed cognitive rehabilitation in individuals with TBI and stroke. They made evidence-based recommendations for practice standards and provided evidence to support interventions to improve attention, memory, social communication skills, and executive function and the implementation of comprehensive-holistic neuropsychological rehabilitation following TBI.Cattelani et al,24 developed and implemented the comprehensive-holistic day-treatment rehabilitation program for chronic TBI. In a review, Malec and Basford found that the program involved neuropsychological orientation, integrated treatment, group intervention, dedicated resources, a neuropsychologist as a team member, participation of significant others, dedicated vocational or independent living, and evaluation of multifaceted results. However, the program is seldom offered in Japan, because it has not been adopted by the healthcare insurance system. Therefore, in its modified form, the program should be offered as standard cognitive rehabilitation for individual issues in cognitive disorders. Therefore, if subjects’ exhibit little or no awareness of deviation in their social behavior , they experience difficulty in solving complicated psychosocial problems and maintaining social participation involving activities such as work, increasing their risk of breakdown of the living.Ben-Yishay et alThe effect of chronic-phase rehabilitation intervention was insufficient in this study; this may have occurred because a comprehensive-holistic day treatment rehabilitation program was not offered.4.1Cultural and social differences between Japan and Australia should have been considered when translating the original English SPRS-2 into Japanese. However, because this would have required additional research, and the sample size was small, these differences were not explored.In addition, the actual number of subjects did not match the sample size. Because this was a mixed-methods study involving qualitative data collection, it was difficult to perform a clinical study involving numerous subjects. Nevertheless, the number of subjects should be increased to reduce type I and II errors in future.It was difficult to obtain accurate information in a single semistructured interview with TBI patients with cognitive dysfunction, because they engaged in circumlocution and deviated from the point. In particular, statements of participants relating to rehabilitation needs were often some ambiguity. Therefore, for confirming their rehabilitation needs, it was necessary to check the verbatim record or to conduct re-interview in some cases. This necessitated the repetition of interviews, which increased the possibility of bias such as that involving interviewers’ opinions.In qualitative studies involving conversational data, it is important to use study methods that minimize bias in the analyst. Sufficient discussion regarding mismatched concepts/categories and computer software was necessary.5Even if initial TBI severity was severe, some subjects experienced no residual cognitive dysfunction or social participation restriction. Relative to the achieved- and difficult-social-participation group, the difficult-social-participation group included a significantly higher number of subjects with severe TBI (GCS: 3–8) and cognitive dysfunction in the chronic phase. Furthermore, there was a decrease in social participation and QOL measured via the SPRS-2 and SF-36v2. The social participation status in the chronic phase TBI was greatly affected by the later cognitive disorders  and psychosocial factors/problems not by the TBI severity (GCS). There were no significant differences in QOL in subjects’ significant others in either group; therefore, they did not appear to be strongly affected by subjects’ cognitive dysfunction. Despite experiencing cognitive dysfunction, the achieved-social-participation group tended to discontinue their follow-up participation at the rehabilitation department. In addition, psychological disorders, such as anxiety, were observed in 70% of subjects, regardless of the extent of their social participation. Further, irrespective of the severity of social participation restriction, most subjects exhibited rehabilitation needs, and rehabilitation intervention was required in the chronic-phase of TBI. During this phase, the provision of supportive outpatient rehabilitation programs and continued follow-up at the rehabilitation department were important in social participation maintenance and enhancement, despite the fact that cognitive function did not improve in practice.The authors thank to Hidetaka Wakabayashi, MD  for statistical advice. The authors also thank Grant-in-Aid for Scientific Research (C) from the Japanese Ministry of Education, Culture, Sports, Science, and Technology: “A qualitative study of rehabilitation needs and participation restriction of chronic-phase subjects with cognitive disorder due to traumatic brain injury” (2012–2014), Project Number: 24500599 for the support.


The spatial context has strong effects on visual processing. Psychophysics and modeling studies have provided evidence that the surround context can systematically modulate the perception of center stimuli. For motion direction, these center-surround interactions are considered to come from spatio-directional interactions between direction of motion tuned neurons, which are attributed to the middle temporal (MT) area. Here, we investigated through psychophysics experiments on human subjects changes with spatial separation in center-surround inhibition and motion direction interactions. Center-surround motion repulsion effects were measured under near-and far-surround conditions. Using a simple physiological model of the repulsion effect we extracted theoretical population parameters of surround inhibition strength and tuning widths with spatial distance. All 11 subjects showed clear motion repulsion effects under the near-surround condition, while only 10 subjects showed clear motion repulsion effects under the far-surround condition. The model predicted human performance well. Surround inhibition under the near-surround condition was significantly stronger than that under the far-surround condition, and the tuning widths were smaller under the near-surround condition. These results demonstrate that spatial separation can both modulate the surround inhibition strength and surround to center tuning width. Contextual interactions in visual processing are ubiquitous across a large panel of feature processing aspects and contribute to binding or segregating spatial elements in the visual field  participated in the study. They had normal or corrected-to-normal vision. All subjects were naïve to the purpose of the experiment (excluding the two authors) and provided written consent for participating in the study. The experiment was approved by the Ethics Committee of the University of Science and Technology of China and conformed to the tenets of the Declaration of Helsinki.2 mean luminance. A circular window cut into a black cardboard centered on the screen, was used to limit the screen area to avoid local cues of vertical/horizontal positions  in a center-surround configuration with two surround conditions, which were called near- and far-surrounds. The center target motion had a 2° diameter, and the width of the surround annulus was 2° as well. For the far-surround condition, there was a blank annulus between the center target and surround context with a width of 2°; therefore, the total visual angle was 10°. However, the near-surround condition did not have a blank annulus, and the total visual angle was 6°. For both near- and far-surround conditions, the RDP had a density of 10 dots/square degree; each dot had a speed of 8°/s and a 0.1° diameter. The target motion stimulus in the center of the display had all dots moving in the same direction, which was maintained in a vertical upward direction with a staircase procedure. The surround direction of motion was defined with respect to the direction of the center target and had a variation range from −180° to 160° in steps of 20°; all of the surround dots had the same motion direction. In this experiment, a minus sign indicates counterclockwise from 0°, which was defined as a vertical upward motion.Subjects performed the experiment after their head was stabilized with a chin-rest at a distance of 1.5 m from the screen. They were instructed to fixate on a fixation point (~0.067° size) displayed in the center of the screen throughout the entire measurement period, and the stimulus was centered at the fixation point. Each subject started the trial by pressing the spacebar on the keyboard, and 200 ms after the trial started, the whole center-surround stimulus was presented for 150 ms. Subjects were asked to report whether the center target direction was tilted clockwise/counterclockwise from the vertical upward direction in both near- and far-surround conditions. They answered by pressing one of two predefined keys on the keyboard. No feedback was provided to the subjects. Subjects experienced near- and far-surround conditions in random order. For the purpose of test-retest reliability, each subject performed the experiment twice.*2*25).A weighted up-down adaptive procedure . This function was adjusted to the data using Bayesian fitting , σ – gamma probability distribution with parameters Gamma , and midpoint had uniform prior. After fitting, all of the midpoints of a given block of measures were adjusted to a mean of zero by subtracting their average.where The model of motion contextual interactions developed in previous research ; n is the number of neurons (n = 1440 for this study); θc and θs are the motion directions for the center and surround, respectively; Amax is the maximum firing rate of the center neuron tuned to its preferred input value θi under the circumstances of no surround being present; Ainh is the surround-to-center inhibition strength of the maximum response rate of the center neurons; σc is the motion tuning width of the center neuronal population; and σs is the motion direction tuning width of the surround-to-center inhibitory effect due to the presence of a surround with direction θs.where s being equal to σc for the near-surround condition, and in the far-surround condition, σc was fixed at the value from the near-surround condition and σs was set as free. This allowed us to extract changes of the surround-to-center tuning width. The midpoint fitted from the psychometric function across different surround directions of motion was the parameter to be predicted by the model. The method of model fitting to the midpoint was as follows. For a given population characteristic , the predicted model directions of motion θpred were computed for all surround stimuli directions θs (θc fixed at 0 in the model). They were used in the sum-of -square equation:Because of entanglement of the two tuning width parameters, and thus overfitting, their relation to each other was fixed in the near-surround condition. In the main text, the first results correspond to the condition of σs and s = 1.5σc, and (2) σs = σc/1.5.where μr, mean 0.93, range [0.85:0.98], all p < 0.0001; see r = 0.39, p = 0.11), while the remaining 10 subjects all had highly significant correlations . The 11th subject, who did not show reliable reproducibility, was also among the subjects with the smallest repulsion effect for all conditions, and one far-surround data set did not exhibit a repulsion effect (see F-test below). Thus, the far-surround condition included only 10 subjects' data set.First, the test-retest reliability between the first and second experimental run was analyzed for each subject. The raw repulsion effect was compared between the first and second run of each condition using Pearson's Product Moment correlation coefficient . In contrast, variation in center tuning width σc affects both the peak position and amplitude of repulsion .To understand the putative neuronal changes in the center-surround effects with surround distance, we used a computationally simplified theoretical model of center-surround interactions in motion processing . First, we found that the model was able to predict all of the direct repulsion effects in the near-surround condition of the 11 subjects well  and the tuning width was 24.6 ± 0.87° (n = 11)  and mean surround-to-center tuning width was 30.7 ± 2.6° (n = 10).Under the far-surround condition, the model allowed the surround amplitude of inhibition and surround-to-center tuning width to be extracted, or their relative changes, by using the individual fit results of the near-surround condition. Under this far-surround condition, we found that the model predicted the direct repulsion effects of 10 of the 11 subjects well  = 18.1, p < 0.0001, Figure t(9) = 3.59, p < 0.05, Figure Comparing the results of the near- to far-surround conditions resulted in significant decreases in the amplitude of inhibition  and σs in the near-surround was significantly smaller than that in the far-surround . For σs = σc/1.5, the near-surround condition had a larger Ainh  and smaller σs  than the far-surround condition.The above results are based on the near-surround condition surround-to-center tuning modulation being equal to center tuning width. But the exact relation between these two values might be different, especially since physiological results showed slightly larger surround tuning widths than the center Born, . TherefoIn this study, we found that near- and far-surround motion discrimination tasks led to different motion repulsion results. The motion repulsion effects caused by the near-surround condition were stronger than those in the far-surround condition, as reported previously (Kim and Wilson, Various behavioral measures support the concept of suppressive surround-to-center motion interactions. In one type of research, when using simple grating stimuli, it was found that human perceptual performance was decreased by high contrast and large sized motion stimuli (Tadin et al., In these behavioral and modeling studies, the way that the surround modulates the center as a function of their separation was assumed to be a simple decaying amplitude effect. Our results further showed that, in addition, the direction width of the surround modulation increased with spatial distance, similar to that reported in the orientation domain (Shushruth et al., Researchers have reported that contextual modulation in the motion domain can be either antagonistic or integrative, which strongly depends upon the visual stimulus design and parameters (Nawrot and Sekuler, Interestingly, the behavioral results in the orientation domain were supported by equivalently carried neurophysiological measures (Shushruth et al., Based on the similarity of center-surround effects in the motion direction and orientation features (Tzvetanov, Conception and design of the experiments: TT, HW, and ZW. Acquisition of simulation and behavioral data: HW and TT. Analysis of data: HW and TT. Critical revisions of the paper and approval of the final version: HW, ZW, YZ, and TT.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.