Sei-1 is an oncogene capable of inducing double minute chromosomes (DMs) formation. DMs are hallmarks of amplification and contribute to oncogenesis. However, the mechanism of Sei-1 inducing DMs formation remains unelucidated.in vivo and gradually decreased following culture in vitro. micro nuclei (MN) was found to be responsible for the reduction. Of the DMs-carrying genes, Met was found to be markedly amplified, overexpressed and highly correlated with DMs formation. Inhibition of Met signaling decreased the number of DMs and reduced the amplification of the DMs-carrying genes. We identified a 3.57Mb DMs representing the majority population, which consists of the 1.21 Mb AMP1 from locus 6qA2 and the 2.36 Mb AMP2 from locus 6qA2-3.DMs formation significantly increased during serial passage Sei-1 overexpression to monitor and characterize DMs in vivo and in vitro. Array comparative genome hybridization (aCGH) and fluorescence in situ hybridization (FISH) were performed to reveal amplification regions and DMs-carrying genes. Metaphase spread was prepared to count the DMs. Western blot and Met inhibition rescue experiments were performed to examine for involvement of altered Met signaling in Sei-1 induced DMs. Genomic walking and PCR were adopted to reveal DMs structure.We employed NIH-3T3 cell line with Met is an important promotor of DMs formation. Sei-1 is a cell cycle promotor and an identified oncogene [Sei-1 is capable of promoting tumorigenesis by enhancing cell proliferation and prohibiting cell apoptosis [Sei-1 in chromosome instability has also been reported [Sei-1's oncogenic ability and potential tumor promoting pathways. Mechanisms that involve chromosome alterations that are inflicted by Sei-1 were not fully elucidated.oncogene . Sei-1 ipoptosis , 6. The reported , 7. ReceMYC and DHFR [Double minute chromosomes (DMs) are round-circle, acentric double-strand extra-chromosome DNA that usually exist in pairs. The formation of DMs is usually regarded as an important sign of genome instability –11. DMs and DHFR –16, are and DHFR –20. DMs and DHFR –24. As tin vitro and in vivo [Although DMs may be naturally lost during cell growth , 26, DMs in vivo –30 while in vivo –33, and  in vivo , 26. Rec in vivo .Sei-1 overexpressing NIH-3T3 cells not only gain a growth advantage but also produce DMs in vivo [Sei-1 overexpression in vitro and in vivo. In this study, we innovatively investigated the mechanism of Sei-1 inducing the formation of DMs in nude mouse. We found the number of DMs induced by Sei-1 was significantly increased during in vivo tissue passage but reduced during in vitro cell passage. Among the DMs-carrying genes, Met was the most prominently amplified and overexpressed. Met signaling pathway was shown to increase the Sei-1-induced DMs population, which indicated a novel function of the well-known oncogene Met to induce the formation of DMs. Additionally, a 3.57 Mb DMs structure was identified to represent the majority population of DMs with two amplicon fragments that were jointed with blunt ends.NIH-3T3 serves as a suitable model for the study of DMs in cancer biology. –38. A pr in vivo . This fiSei-1 and null-vector-transfected NIH-3T3 cell clone Sei-1/NIH-3T3 and Vec  has been well elucidated to directly bind and motivate Met and its downward pathways. An alternative method of Met accumulation that causes self-phosphorylation and activation was also previously reported [Sei-1 overexpressing the Sei-1/NIH-3T3 clone and primary cell pool CPX3, CPX6 with and without mouse recombinant Hgf treatment  were distinctly decreased 24 hours after Met inhibition as a result of the loss of DMs  with nearly identical copy changes were identified in this study: four breakpoints, namely, BP1, BP2 on the edge of AMP1, and BP3, BP4 on the edge of AMP2, which suggests a relatively simple DMs structure. Based on the aCGH results, a set of primers  were desSei-1 is a newly discovered oncogene that is particularly found to be able to induce the formation of DMs. The biological connection between its oncogenic and DMs inducing abilities was barely explored. In this study, we innovatively observed the gain/loss of Sei-1-induced DMs to investigate oncogenic mechanism of Sei-1. Our study discovered Met as a dynamic responder to enhanced Sei-1 oncogenic function. We uniquely found that Met was not only amplified in the form of DMs and overexpressed but also significantly involved in the formation of DMs itself, suggesting a novel function of Met to generate the DMs.in vivo, but progressively DMs lost during in vitro culture. A Darwinian selection pattern was previously implied to explain the increased number of DMs that harbor MYCN in neuroblastoma cells [in vitro passages. They hypothesized that the number of DMs could change dynamically as a response to significant environmental changes [Sei-1-induced DMs probably facilitate the survival of cells under in vivo pressure. In vitro, endogenous microenvironment pressure was retreated, and previous growth advantage conferred by DMs was lost. Therefore, DMs production was restricted, which caused a reduction in DMs populations. Interestingly, we observed the Sei- 1 overexpression level significantly higher in CPX6 compared to Sei-1/NIH-3T3, CPX1, CPX3 both in cells and tumor tissues .Sei-1 CDS were cloned into pcDNA3.1(+) plasmid  and transfected into cells with Lipofectamine 2000 (Invitrogen).NIH-3T3, a mouse embryo originated fibroblastic cell line , was purs.c. xenograft was conducted as previous [Five-week-old Balb/c female nude mice were obtained from SLAC laboratory  and maintained within individual ventilated cages in SPF-degree conditions. The previous , with eaColcemid  was added to the medium of fast growing cells for 2 hours, which were trypsinized, washed with PBS, and subsequently centrifuged. Suspend pellets in pre-warmed 0.075 M KCl and incubate in 37 °C water bath for exact 14 min. Fixate the cells by fresh fixative solution (mixture of 3 methanol: 1 acetic acid) three times. Drop the cell suspension on a clean slide. The slides were dyed with Giemsa and photographed using Olympus BX41 microscope  equipped with JVC TK-C75U color video camera .A micronuclei spread was prepared without colcemid treatment. The protocol, including Giemsa dying and image capturing, was identical to metaphase spread.BAC clones purchased from the BACPAC Resources Center  were labeled with fluorescence dUTPs and hybridized to metaphase/micronuclei spread as described . The sliGenomic DNA samples were extracted from fresh xenograft tissue with the DNeasy Blood & Tissue Kit . Sample quality was guaranteed by Nanodrop . The samples were properly predisposed and applied to a SurePrint G3 Mouse CGH Microarray . The procedures were conducted by Shanghai Biotechnology Co., Ltd. .The DMs were micro-dissected from NIH-3T3 metaphase karyotype that were prepared as previously described . The sam® DNA Mini and Blood Mini Kit (Qiagen) and Trizol . cDNA samples were obtained using Transcriptor First Strand cDNA Synthesis Kit (Roche). For quantitative real-time PCR, the samples were tested using the LightCycler® 480 instrument (Roche) and corresponding SYBR Green I master (Roche). Semi-quantitative PCR was performed as described [Genomic DNA and RNA samples were extracted by QIAampescribed .The Universal Genomic Walker Kit  was employed according to the manufacture instructions. The PCR products were sequenced and aligned with the mouse genome using the BLAT tool online from UCSC. The gene-specific primers (GSP) for BP1 were GSP1, 5′-TTGGTATTTATTCACGACTTTGATAC-3′, GSP2, 5′-GTCATGTACTCTGTTAAGACTGCTTT-3′. The GSP primers for BP2 were GSP1, 5′-AACAGATGAA AAAAAGTTGAGAGGCT-3′, GSP2, 5′- AACAGATGA AAAAAAGTTGAGAGGCT-3′Cells were lysed by RIPA buffer and sonication, and centrifuged at 12000 g 4°C for 30 minutes. Supernatant protein was separated by SDS-polyacrylamide gel, and transferred to PVDF membrane, which was scanned by Odyssey Imaging System  after incubation of corresponding antibodies. Image J Software was used for quantification.Antibodies against Gab1—phospho-Gab1, Met—Phospho-Met, pan-AKT—Phospho-Akt, PI3K, and Phospho-PI3K were purchased from Cell Signaling . Antibodies against Hgf were purchased from Thermo Fisher . Anti-mouse and anti-rabbit antibodies were obtained from Rockland Immunochemicals . Mouse recombinant Hgf and c-Met Kinase Inhibitor III were purchased from Millipore .t-test as well as one-way ANOVA with SNK test were employed as the statistics.Data of the DMs counts and Western blot coming evenly from three parallel studies were presented as the mean ± S.D. All experiments were independently repeated three times. Two-tailed Student's


FOXC2 gene cause a form of lymphedema primarily of the limbs that usually develops at or after puberty. In 90-95% of patients, lymphedema is accompanied by distichiasis. FOXC2 is a member of the forkhead/winged-helix family of transcription factors and plays essential roles in different developmental pathways and physiological processes. We previously described six unrelated families with primary lymphedema-distichiasis in which patients showed different FOXC2 mutations located outside of the forkhead domain. Of those, four were missense mutations, one a frameshift mutation, and the last a stop mutation. To assess their pathogenic potential, we have now examined the subcellular localization and the transactivation activity of the mutated FOXC2 proteins. All six FOXC2 mutant proteins were able to localize into the nucleus; however, the frameshift truncated protein appeared to be sequestered into nuclear aggregates. A reduction in the ability to activate FOXC1/FOXC2 response elements was detected in 50% of mutations, while the remaining ones caused an increase of protein transactivation activity. Our data reveal that either a complete loss or a significant gain of FOXC2 function can cause a perturbation of lymphatic vessel formation leading to lymphedema.Dominant mutations in the  More than 10 Mendelian forms are known, of which three are considered the major forms: Milroy disease (MD) [MIM 153100], lymphedema-distichiasis syndrome (LDS) [MIM 153400] and Meige disease (MGD) [MIM 153200] [Lymphedema is a dysfunction of the lymphatic system, a disorder characterized by abnormal swelling of one or more extremities due to impaired transport of the lymph –3. This ) MIM 15300 and MeFOXC2 gene (MIM 602402), coding for the forkhead transcription factor FOXC2, cause a form of lymphedema with variable age of onset (range: 7-40 years), often associated with distichiasis [FOXC2 plays a key role in regulation of lymphatic endothelial cells differentiation, in formation of smooth muscle cell layers and in morphogenesis of lymphatic valves. FOXC2 and VEGFR-3 act through a common genetic pathway to establish distinct properties of the lymphatic vascular architecture [Dominant mutations in the gene MIM 02402, coichiasis . FOXC2 pitecture .FOXC2 maps to 16q34.3 and it produces a 2.2 kb transcript with a 1.5 kb single exon coding region The FOXC2 protein contains 501 amino acids . The majority of FOXC2 mutations are small insertions or deletions and nonsense mutations causing truncated proteins, which have been hypothesized to be responsible for a haplo-insufficiency condition [FOXC2 haplo-insufficient state is associated to hyperplasia and distichiasis in mice [FOXC2−/− mice have been found to exhibit abnormal pericyte recruitment to the lymphatic capillaries and valve agenesis in the collecting vessels, indicating that FOXC2 is essential for the formation of a pericyte-free lymphatic network and lymphatic valve development [FOXC2-null mice [In LD patients, almost 70 different ondition . The FOX in mice . FOXC2−/elopment . Moreoveull mice , 16.FOXC2 haplo-insufficiency also in humans, until some FOXC2 missense mutations, identified in LD patients, were found to cause a gain of protein function [FOXC2 variations are missense mutations of which very few have been functionally investigated. Two mutations located inside the forkhead domain impaired the DNA-binding and transcriptional activation ability of FOXC2 protein [FOXC2 variations. We therefore performed functional characterization of FOXC2 mutations located in different protein domains to provide novel information on structure-function relationships of the FOXC2 transcription factor and to get further insight into the disease-causing mechanism of lymphedema.The pathogenesis of LD was assumed to be associated with function . Approxi protein ; in cont protein . MoreoveGenetic evaluation of LD patients has been previously reported . MoleculFOXC2 cDNA into the pcDNA3.1/NT-GFP-TOPO TA expression vector and then performed site-direct mutagenesis to generate the FOXC2-GFP plasmids with the disease causing mutations.The mutations analysed in this study were all localized outside the forkhead domain, in different regions of FOXC2 protein Figure . The evaFOXC2 mRNA levels  mutation-containing plasmid as a control in order to allow an accurate comparison of intracellular distribution, since the FOXC2(R121H) protein has been previously demonstrated to display a defect in nuclear localization, with partial cytoplasmic signal [FOXC2(M276DfsX186) recombinant plasmid caused the production of nuclear aggregates  plasmid, in our system, induced intranuclear protein aggregation  which leads to the formation of a truncated protein lacking of 235 amino acids in the C-terminal region, and with the addition of 186 nonsense amino acids due to the frameshift  and one nonsense (p.Q420X) mutations. All three AD-2 mutations significantly increased FOXC2 transcriptional ability. Surprisingly, the p.Q420X displayed the highest level of activation, despite the truncated protein lacked more than half of AD-2 sequence and the entire ID-2 region. Consistent with these findings, Van Steensel et al. also reported a gain of function for FOXC2 missense variations localized inside AD-2 domain. We did not detect any evidence of mRNA or protein stability alterations resulting from FOXC2 AD-2 mutations. Thus, it is possible that AD-2 mutations exert their influence by modifying the tridimensional structure of the C-terminal domain of the FOXC2 protein and/or changing the ability of FOXC2 to interact with regulatory proteins. In the central region of Finally, the S370T missense mutation, located in the terminal sequence of central region Figure , resulteTo date, it is not possible to extrapolate general conclusions about the relationship between FOXC2 activity and the protein variation position, because of the very limited number of different types of mutations  that have been functionally tested. However, data obtained so far indicate that missense mutations in FHD and AD-2 domain might represent an exception, causing always a loss and a gain of FOXC2 function, respectively. More data are needed to highlight possible correlation between mutation position and function in other FOXC2 protein domains.FOXC2, that are responsible for regulating the expression of a broad range of genes both during development and in adult tissues [Nowadays, it is possible to estimate the theoretical pathogenic effects of missense mutations utilizing bioinformatics tools. However, the usefulness of these programs in predicting the real contribution of a mutation becomes limited when they provide conflicting data  with the others carrying a gain of function mutations , we noted some differences in the uptake of the radiopharmacon by inguinal lymph glands and its reflux back down into the lower leg. In particular, P1 and P2 patients showed a lymphoscintigram profile/images compatible with a hyperplasia of lymphatic vessels , while P4, P5 and P6 patients reports resembled more a hypoplastic condition . While, Based upon clinical and molecular data, therefore, we can hypothesize that an increased activity of FOXC2 results in disregulated expression of proteins essential for normal development of lymphatic system, causing hypoplasia. In contrast, decreased FOXC2 transcriptional activity appears to be associated with a hyperplastic condition. Our hypothesis on hypo or hyperplasia and activating versus inactivating mutations is consistent with previously reported results , 18 and FOXC2 different mutations and the morphologic changes of lymphatic system, and with distichiasis.The association of FOXC2 mutation function and distichiasis is less clear however. Our patients with activating mutations presented with distichiasis, in contrast, 2 out of 3 patients with inactivating mutation did not. From these clinical findings, it might be tempting to speculate that distichiasis is associated with hypoplasia rather than hyperplasia. However, additional larger clinical studies are required to definitely elucidate correlations between FOXC2 mutations identified in six Italian families with lymphedema-distichiasis, providing evidences of patients with inactivating versus activating mutations of the FOXC2 transcription factor. During development, transcriptional programs of lymphatic endothelial cells are established by networks of transcriptional factors, among which FOXC2 is one of the most important. FOXC2 acts downstream of VEGFR-3 signaling pathway and is essential for the formation of pericyte-free lymphatic network. Moreover FOXC2 regulates the expression of genes essential for all steps of lymphatic valve morphogenesis and maintenance, such as connexins CX43 and CX47 [In conclusion, we have analyzed the molecular consequence of and CX47 , 29, 30.Finally, recent studies show that FOXC2 overexpression is involved in cancer progression, inducing epithelial mensenchymal transition (EMT) , 32. LDSFOXC2 cDNA was amplified by PCR using DNA clone ID 32938 (Thermo Scientific) as template and the following primers: FOXC2-F 5′-ATGCAGGCGCGCTACTCC-3′ and FOXC2-R 5′-TCAGTATTTCGTGCAGTCGTAGGAG-3′. The PCR product was subcloned into pcDNA3.1/NT-GFP-TOPO from Invitrogen to produce NT-GFP-FOXC2, expressing FOXC2 with GFP at the N-terminus. Point mutations were performed using the Phusion Site-Directed Mutagenesis Kit (Thermo Scientific). Mutations in FOXC2 cDNA were introduced using the following primers: A3G forward 5′-ATGCAGGGGCGCTACTCCGTGT-3′ and reverse 5′-ACACGGAGTAGCGCCCCTGCAT-3′; R121H forward 5′- GCAGAACAGCATCCACCACAACCTCTCGC -3′ and reverse 5′- GCGAGAGGTTGTGGTGGATGCTGTTCTGC -3′; S370T forward 5′-GTCGCCCCTGACCGCTCTCAACC-3′ and reverse 5′-GGTTGAGAGCGGTCAGGGGCGAC -3′; Q420X forward 5′- GCCGCGGCGTAGGCGGCCT-3′ and reverse 5′- AGGCCGCCTACGCCGCGGC -3′; L487P forward 5′-ACGCCGCCTCCCTATCGCCAC-3′ and reverse 5′-GTGGCGATAGGGAGGCGGCGT-3′; A492V forward 5′-TCGCCACGCAGTCCCCTACTCCT-3′ and reverse 5′-AGGAGTAGGGGACTGCGTGGCGA-3′.In order to obtain the c.826-827delAT FOXC2 mutation (M276DfsX186) were used the following two couple of primers and two rounds of mutagenesis : forward(1) 5′- CAGCGTGGAGAACATCTGACCCTGCGAACGTC-3′ and reverse(1) 5′- GACGTTCGCAGGGTCAGATGTTCTCCACGCTG-3′ and forward(2) 5′- CAGCGTGGAGAACATCGACCCTGCGAACGTC-3′ and reverse(2) 5′- GACGTTCGCAGGGTCGATGTTCTCCACGCTG-3′.FOXC2 (wild type) and NT-GFP-FOXC2 mutant plasmids were used as template to amplify FOXC2 control and mutants cDNA sequences with FOXC2-F and FOXC2-R primers (reported above). The PCR products were subcloned into pcDNA3.3-TOPO (Life Technologies) to produce control or mutant FOXC2 proteins without any tag. All final expression constructs were sequenced to verify that no additional mutations were introduced.NT-GFP-FOXC2 and GAPDH gene, in order to avoid saturation: regression curves assaying different amounts of cDNAs (corresponding to different mRNA concentrations) and different number of cycles of amplification were performed . After 48 h, cells were carefully washed with PBS, total RNA was isolated with TRIzol (Invitrogen) and 1 μg was converted to cDNA by RT-PCR using random hexamers (0.5 mg), 400 units of MMLV-RT, 1.6 mM total dNTPs, 20 units of Rnasin and 0.4 mM dithiothreitol, in a 50 μl reaction solution containing 10× RT Buffer. Before reverse transcription, RNA was treated with DNase in order to eliminate DNA contamination . RT-PCR erformed . Thirty For transient transfections, HeLa and COS cells were cultured on glass coverslips in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS) and allowed to adhere overnight. The next day, the cells were transiently transfected with recombinant NT-GFP-FOXC2 plasmids using the TurboFect transfection reagent. After 24 h, cells were fixed stained with DAPI, and examined with a Leica MB5000B microscope equipped with 40X and 100X Fluorart oil immersion objectives.HeLa cells were transiently transfected with recombinant NT-GFP-FOXC2 plasmids as described above. Four flasks for each type of recombinant plasmids was transfected. After 48 h, all flasks were evaluated for transfection efficiency. Only samples reaching 80% of transfection efficiency were used to prepare protein cell extracts. Cell extracts were prepared from confluent cultures grown in serum-containing medium. After extensive washing with Dulbecco's phosphate-buffered saline (DPBS), cells were recovered by scraping with a rubber policeman in 300 μl of 0.05% (wt/vol) SDS. The total protein concentration of cell extracts was quantified using a Coomassie (Bradford) Protein Assay Kit (Pierce). Proteins (10 μg/well) were separated by electrophoresis on 8% SDS-polyacrylamide gel (Bio-Rad), transferred to a nitrocellulose membrane (Pierce) and then immunoblotted using a rabbit monoclonal antibody (dil. 1:5000) raised against green fluorescent protein (Invitrogen) and a mouse monoclonal antibody (dil: 1:5000) to GAPDH (Abnova). Specifically bound immunoglobulins were detected using the SuperSignal West Pico Complete Detection Kit (Pierce) containing ImmunoPure Peroxidase Conjugated Goat anti-Mouse and anti-Rabbit IgG (dil 1:20.000). Experiments were repeated three times.HeLa and COS cells were co-transfected with 20 ng FOXC1 luciferase reporter  and 100 The same experimental conditions were used for transient transfection of wild type or FOXC2 mutant plasmids subcloned into pcDNA3.3, a mammalian expression without tag. The dual-luciferase assays were used to obtain sequential quantification of both Photinus pyralis luciferase (reporter vector) and Renilla reniformis luciferase (control vector), as previously described (35). Luminescence detection for all transfected plasmids was performed using the Glomax luminometer (Promega). Reactions were replicated three times, using the Promega Dual Luciferase Assay kit (Promega).The statistical analysis of quantitative data of RT-PCR and of Luciferase assays was made using SPSS v.19 package . The values were compared using Student's t-test. A P-value of ≤0.05 was considered to be statistically significant.The effect of amino acid substitutions on protein function was predicted using ClustalW, SIFT and PolyPhen software. A multiple sequence alignment of mammalian FOXC2 proteins was used as imput for ClustalW. The NCBI reference sequence (FOXC2 protein NCBI accession number: NP_005242.1.) of the human FOXC2 protein was used as the input for SIFT and PolyPhen, with default query options.


Here, we investigated whether nerve and muscle cell activities regulate presynaptic conventional PKC (cPKCα and βI) via BDNF/TrkB signaling to modulate synaptic strength at the NMJ. To differentiate the effects of presynaptic activity from that of muscle contraction, we stimulated the phrenic nerve of rat diaphragms  with or without contraction (abolished by μ-conotoxin GIIIB). Then, we performed ELISA, Western blotting, qRT-PCR, immunofluorescence and electrophysiological techniques. We found that nerve-induced muscle contraction: (1) increases the levels of mature BDNF protein without affecting pro-BDNF protein or BDNF mRNA levels; (2) downregulates TrkB.T1 without affecting TrkB.FL or p75 neurotrophin receptor (p75) levels; (3) increases presynaptic cPKCα and cPKCβI protein level through TrkB signaling; and (4) enhances phosphorylation of cPKCα and cPKCβI. Furthermore, we demonstrate that cPKCβI, which is exclusively located in the motor nerve terminals, increases activity-induced acetylcholine release. Together, these results show that nerve-induced muscle contraction is a key regulator of BDNF/TrkB signaling pathway, retrogradely activating presynaptic cPKC isoforms (in particular cPKCβI) to modulate synaptic function. These results indicate that a decrease in neuromuscular activity, as occurs in several neuromuscular disorders, could affect the BDNF/TrkB/PKC pathway that links pre- and postsynaptic activity to maintain neuromuscular function.The neurotrophin brain-derived neurotrophic factor (BDNF) acts via tropomyosin-related kinase B receptor (TrkB) to regulate synapse maintenance and function in the neuromuscular system. The potentiation of acetylcholine (ACh) release by BDNF requires TrkB phosphorylation and Protein Kinase C (PKC) activation. BDNF is secreted in an activity-dependent manner but it is not known if pre- and/or postsynaptic activities enhance BDNF expression  Nerves and skeletal muscles interact via two modes of communication: electrical activity and neurotrophic regulation  release at the neuromuscular junction (NMJ) and the TrkB receptor is normally coupled to this process  activation  were used to evaluate the following techniques.Diaphragm and levator auris longus (LAL) muscles were obtained from Sprague-Dawley rats . The animals were cared for in accordance with the guidelines of the European Community Council Directive of 24 November 1986 (86/609/EEC) for the humane treatment of laboratory animals. All the procedures realized were reviewed and approved by the Animal Research Committee of the Universitat Rovira i Virgili . At least five independent animals (Primary antibodies used for Western blot were rabbit anti-BDNF (Cat# sc-20981), rabbit anti-PKCα (Cat# sc-208), rabbit anti-PKCβI (Cat# sc-209), rabbit anti-TrkB (Cat# sc-8316) and goat anti-glyceraldehyde 3-phosphate dehydrogenase  polyclonal antibodies, purchased from Santa Cruz Biotechnology. Rabbit anti-pPKCα , goat anti-p75 (Cat# AB1554), rabbit anti-neurotrophin-4  and rabbit anti-pTrkB  antibodies were purchased from Merck Millipore. Rabbit anti-pPKCβI  polyclonal antibody was purchased from Abcam. The secondary antibodies used were donkey anti-rabbit conjugated to horseradish peroxidase (HRP) from Jackson Immunoresearch Labs (Cat# 711-035-152) and rabbit anti-goat conjugated to HRP from Molecular probes (Cat# R21459). Immunohistochemistry was performed with antibodies that are commonly used as markers to differentially detect the components of the NMJ : mouse anti-syntaxin (Cat# S0664) and mouse anti-neurofilament-200 (Cat# N2912) monoclonal antibodies were purchased from Sigma. Mouse anti-S100 monoclonal antibody (Cat# AM10036FC-N) was from Acris Antibodies. Rabbit anti-PKCβI polyclonal antibody was purchased from Santa Cruz Biotechnology (Cat# sc-209). The secondary antibodies used were donkey anti-rabbit or anti-mouse conjugated to Alexa Fluor 488 and Alexa Fluor 647 from Molecular Probes . Postsynaptic AChRs were detected with α-bungarotoxin (α-BTX) conjugated to tetramethylrhodamine (TRITC) from Molecular Probes . As a control, primary antibodies were omitted from some muscles during the immunohistochemical and Western blot procedures. These control muscles never exhibited positive staining or revealed bands of the appropriate molecular weight with the respective procedures. In double-staining protocols, omitting either one of the two primary antibodies completely abolished the corresponding staining and there was no cross-reaction with the other primary antibody. Pretreatment of a primary antibody with the appropriate blocking peptide (between three- and eight-fold by weight) in skeletal muscle tissue prevented immunolabelling.In presynaptic stimulation treatments and electrophysiological experiments, muscle contraction was blocked using μ-conotoxin GIIIB . This toxin selectively inhibits sarcolemmal voltage-dependent sodium channels (VDSCs) without affecting synaptic ACh release  was used for TrkB inhibition assays. The working solution was 10 μg/ml. For BDNF exogenous incubations we used h-BDNF  10 mM.Phosphatase inhibition experiments were performed using a phosphatase inhibitor cocktail from Sigma-Aldrich  in a 100-fold dilution.To block cPKCβI activity we used the specific translocation inhibitor peptide βIV5–3 at 10 μM (kindly provided by Dr. Mochly-Rosen from Stanford University). It is derived from the V5 domain of cPKCβI and binds to the anchoring protein RACK (receptors for activated C-kinase), disrupting the interaction between cPKCβI and its specific βI-RACK. This inhibits its translocation to the membrane and its activation.2 2, MgSO4 1, NaH2PO4 1, NaHCO3 12 and glucose 12.1 mM) continuously bubbled with 95% O2/5% CO2 at room temperature. The phrenic nerve was stimulated ex vivo at 1 Hz by an A-M Systems 2100 isolated pulse generator . The frequency of 1 Hz allows the maintenance of different tonic functions  without promoting synaptic plasticity . To study separately the effect of synaptic transmission from the effect of the muscle cell contraction, we performed experiments in which contractions were prevented using μ-CgTx-GIIIB or not. Visible contractions of the diaphragm muscle served to verify successful nerve stimulation. Three main experiments were performed to discern the effects of synaptic activity from that of muscle activity  in ice-cold lysis buffer  50, EDTA 1, NaF 50, PMSF 1, sodium orthovanadate 1; NP-40 1%, Triton X-100 0.1% and protease inhibitor cocktail 1% . Protein lysates were obtained collecting supernatants after removing insoluble materials by centrifugation at 1000 Protein concentrations were determined by DC protein assay . The BDNF Emax ImmunoAssay System (Promega Cat# G7610) was used to measure the amount of total BDNF (pro- and mature) in each sample following standard protocols. Plates were coated with a specific anti-BDNF monoclonal antibody and then washed once using tris-buffered saline, 0.1% Tween 20 (TSBT). Plates were blocked using 200 μL Promega 1× Block and Sample buffer. After washing, diaphragm samples and BDNF protein standards (0–500 pg BDNF protein) were added in triplicate to the plates and incubated for 2 h at room temperature. Plates were washed five times and anti-human BDNF polyclonal antibody was added to each well. Plates were washed again five times using TBST wash buffer. The sandwich was completed by adding anti-IgY HRP conjugate. Finally, plates were developed using stabilized chromogen (tetramethylbenzidine) and the reaction was stopped using 1 N HCl. Absorbance was read at 450 nm using a Sunrise Tecan A-5082 microplate reader and data was analyzed with Magellan software (Tecan Group Ltd.). Afterwards, the amount of detected BDNF was normalized to a standard curve and to the total protein content determined with the colorimetric assay (Bio-Rad).Diaphragm muscles with the phrenic nerve were dissected, frozen in liquid nitrogen, and stored at −80°C before use. Homogenization, lysate obtainment and determination of protein concentration were performed with the same protocols and solutions described previously for BDNF ELISA assay. The linear and quantitative dynamic range for each target protein and the appropriate dilutions of samples were determined for accurate and normalized quantitation of densitometric analysis.g at 4°C for 15 min, and the resulting supernatant was further centrifuged at 130,000 g at 4°C for 1 h. The supernatant corresponded to the cytosolic fraction and the pellet, to the membrane fraction. The latter was resuspended in lysis buffer  50, EDTA 1, NaF 50, PMSF 1, sodium orthovanadate 1; NP-40 1%, Triton X-100 0.1% and protease inhibitor cocktail 1% ). Fractionation was assessed by blotting of GAPDH, a specific cytosolic protein. GADPH immunoreactivity was never observed in the membrane fraction. Protein concentrations were determined as previously described.To isolate the membrane and cytosolic fractions, diaphragm muscles were dissected and homogenized in ice-cold lysis buffer without detergents  50, EDTA 1, NaF 50, PMSF 1 and sodium orthovanadate 1 and protease inhibitor cocktail (1/100). The homogenized samples were cleared with a centrifugation at 1000 TM-P; Amersham, GE Healthcare). The membranes were blocked in TBST containing 5% (W/V) nonfat dry milk or 5% bovine serum albumin (BSA) and probed with the primary antibody overnight at 4°C. The membranes were incubated with the secondary antibody and visualized by chemiluminescence with the ECL kit .Protein samples of 15 μg or 30 μg were separated by 8% or 15% SDS-polyacrylamide electrophoresis and electrotransferred to polyvinylidene difluoride (PVDF) membranes .The blots were visualized with the ChemiDoc XRS+Imaging System . The densitometry of the resultant bands was analyzed with the ImageJ software (ImageJ). The integrated optical density of the bands was normalized with respect to: (1) the background values; and to (2) the total protein transferred on PVDF membranes, measured by total protein analysis , as described above for protein analysis. After 30 min of treatment  was used to extract RNA from muscle samples. Tissue was mechanically homogenized with a PRO200 homogenizer (Pro Scientific). Following extraction, RNA was quantified on a spectrophotometer (Beckman DU 530) by measuring 260 nm absorbance values. Extracted RNA was then reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) with the following thermocycle: 25°C for 10 min, 37°C for 2 h and 85°C for 5 min. Each qRT-PCR sample included 2.5 ng of cDNA, primers, and Power SYBR Green PCR Master Mix (Applied Biosystems). Thermocycle for the quantitative step on the ABI PRISM 7000 Sequence Detection System was as follows: 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 15 s and 60°C for 1 min. A dissociation curve was determined for each well to confirm that only one product was amplified. Each sample was run in triplicate. Samples without reverse transcriptase during the cDNA conversion were also assessed to ensure that there was no DNA contamination. The reference gene was 18S (400 nM primers: GGAGCCTGCGGCTTAATTTG and CCACCCACGGAATCGAGAAA). In each experiment, we confirmed that levels of the reference gene were equivalent between treatment groups. Transcripts of four BDNF exons were quantified: IV , VI , VIII  and IX . Optimal concentrations and amplification efficiencies were calculated for each primer set.p < 0.05 (*).Relative Expression Software Tool (REST) was used to assess statistical significance and fold change of genes . The muscles were permeabilized with 0.5% Triton X-100 in PBS, and nonspecific binding was blocked with 4% BSA. Then, muscles were incubated overnight at 4°C in mixtures of three primary antibodies raised in different species  and then rinsed. The muscles were then incubated for 4 h at room temperature in a mixture of appropriate secondary antibodies. Acetylcholine receptors (AChRs) were detected with α-BTX conjugated with TRITC. At least three muscles were used as negative controls as described above. No crossover was detected between antibodies. For improved localization of the cPKCβI isoform at the NMJ, the muscles were processed to obtain semithin cross-sections from whole-mount multiple-immunofluorescent stained muscles. This method provided a simple and sensitive procedure for analyzing the cellular distribution of molecules at the NMJ  and evoked EPPs (EPPs) were recorded intracellularly with conventional glass microelectrodes filled with 3 M KCl (resistance: 20–40 MW). Recording electrodes were connected to an amplifier , and a distant Ag-AgCl electrode connected to the bath solution via an agar bridge (agar 3.5% in 137 mM NaCl) was used as a reference. The signals were digitized , stored and computer-analyzed. The software Axoscope 9.0  was used for data acquisition and analysis. To prevent muscle contraction during EPP recordings, we used μ-CgTx-GIIIB . After a muscle fiber had been impaled, the phrenic nerve was continuously stimulated  using two platinum electrodes that were coupled to a pulse generator (CIBERTEC CS-20) linked to a stimulus isolation unit. Thus, in stimulated muscles, we recorded and measured control EPPs and then, we incubated the muscle in βIV5–3 inhibitor peptide for 1 h. The last 50 EPPs were recorded. We selected fibers with membrane potentials of no less than −70 mV and used only those results from preparations which did not deviate by more than 5 mV during the recording. The mean amplitude (mV) per fiber was calculated and corrected for non-linear summation . Differences were considered significant at p < 0.05 (*).Diaphragm muscles were removed surgically and incubated in a Sylgard-Petri dish containing normal Ringer solution (in mM): NaCl 135, KCl 5, CaClin vivo experimental system in which we can distinguish the effects of synaptic activity from that of muscle contraction (see Table Stimulation condition in the figures). The effects of muscle contraction were determined by comparing muscles which were both stimulated via their nerves, but in one case muscle contraction was blocked (referred to as the Contraction condition in the figures). Finally, presynaptic Stimulation with Contraction treatment comprises the effects of synaptic activity and muscle contraction, showing complete neuromuscular activity.To determine the relationship between neuromuscular activity and neurotrophic control, we developed an per se, but this has not been directly shown. Therefore, our first objective was to determine whether synaptic activity and/or muscle contraction regulate BDNF protein levels in the skeletal muscle. Thus, we compare BDNF protein levels in muscles stimulated via the nerve (1 Hz) for which contraction was blocked or not. Each hemidiaphragm was compared to a corresponding control from the same animal  increases BDNF expression in the skeletal muscle, CNS and plasma  increase in mBDNF levels with respect to basal conditions whereas muscle contraction further increased mBDNF levels by 45.2% ± 7.5 (p < 0.05). Although the source of this increased BDNF is not clear, our results show that activity in either the synapse or the muscle increase mature BDNF without affecting pro-BDNF protein levels in skeletal muscle.Although results of the ELISA allowed us to quantify accurately the total BDNF protein levels in muscle for each experiment, this technique cannot tell us how the mBDNF and pro-BDNF are modulated by activity. Because pro-BDNF and mBDNF produce different, and even opposite, effects Lu, , we usedbdnf mRNA transcription in skeletal muscle under our activity stimulation treatments. These results suggest that the maintenance of pro-BDNF could be due to an increase in BDNF mRNA translation from mRNA pools in the cells.That pro-BDNF levels were maintained in the face of increases in mBDNF levels suggests that synthesis of this isoform is tightly regulated, otherwise, pro-BDNF levels would decrease after the upregulation of its cleavage into mature BDNF. Thus, we examined gene expression via qRT-PCR to determine whether BDNF mRNA levels were enhanced after the different stimulation conditions. Surprisingly, no difference was found in the expression of any of the four BDNF mRNA transcripts  examined after any condition Figure . Therefop > 0.05; muscle contraction: −7.23 ± 5.84, p > 0.05; presynaptic stimulation with contraction: −8.61 ± 5.34, p > 0.05), suggesting that activity-dependent enhancement in TrkB-signaling is likely mediated by BDNF.Because both BDNF and NT4 bind TrkB receptors, we also tested whether synaptic activity with or without muscle contraction regulates NT4 protein level in the skeletal muscle. Results showed that NT4 protein levels did not change under these conditions . Moreover, none of the stimulation conditions affected TrkB.FL or p75 levels. Thus, the basal FL:T1 ratio was ~1:11 and contraction treatment elevated it to ~1:6. Furthermore, preincubation with exogenous BDNF with contraction yielded similar results . Therefore, BDNF, which is enhanced by contraction, could contribute to the contraction-dependent decrease of TrkB.T1. However, presynaptic stimulation did not alter the level of any BDNF receptor indicating that muscle contraction per se is necessary for synaptic activity to reduce the amount of TrkB.T1 protein.Having established that muscle contraction driven by presynaptic stimulation enhances mBDNF protein levels, we next sought to analyze BDNF receptors that are expressed the most in the skeletal muscle . In order to study TrkB receptors, we used an anti-TrkB antibody raised against a peptide sequence corresponding to the amino acids 160–340 of TrkB, the extracellular domain shared by TrkB.FL (145–150 kDa) and TrkB.T1 . We used an antibody which specifically recognizes the tyr816 phosphorylation of TrkB.FL, known to trigger the PLCγ signaling pathway that activates PKC. Unexpectedly, we did not detect any change in TrkB.FL phosphorylation in muscle after 30 min of nerve stimulation, regardless of whether the muscle contracted or not Figure .p < 0.05; 30 s: −41.63 ± 6.49, p < 0.05). These results indicate that while synaptic activity without muscle contraction positively regulates TrkB.FL phosphorylation, muscle contraction has the opposite effect, negatively regulating phosphorylation of TrkB.FL on the tyr816 very rapidly, suggesting a quick sequence of activation-phosphorylation-dephosphorylation for TrkB.FL. Because several phosphatases are involved in regulating TrkB signaling . Presynaptic stimulation for 1 min without muscle contraction significantly increased TrkB.FL phosphorylation . However, 47/TrkB did not change pTrkB.FL levels after 1 min of presynaptic stimulation with muscle contraction (−1.98% ± 2.92 p > 0.05). Possibly, phosphorylation of TrkB.FL in this condition is too low for 47/TrkB to decrease it further.To confirm that phosphorylation of TrkB.FL is ligand-specific, we analyzed the effect of 47/TrkB, an anti-TrkB antibody that extracellularly competes with the binding of endogenous TrkB to any ligand. We found that this inhibitor under presynaptic stimulation, with or without muscle contraction significantly decreases pTrkB.FL after 30 min , synaptic activity and muscle contraction regulate phosphorylation of TrkB.FL and, at longer times (30 min), the regulation involves altering the level of TrkB.T1 without effects on TrkB.FL phosphorylation. Within 10 min, presynaptic stimulation induces phosphorylation of TrkB.FL whereas muscle contraction decreases it by the action of phosphatases. These results suggest that muscle contraction performs a regulatory control on the TrkB.FL signaling. After 30 min of stimulation, neither synaptic activity nor muscle contraction has any effect over TrkB.FL phosphorylation. However, this prolonged postsynaptic activity regulates BDNF/TrkB signaling via a reduction of TrkB.T1. Next, we focused on the effects of decreased TrkB.T1 after 30 min of stimulation on the PKCs.2+. Moreover, Besalduch et al.  and cPKCβI . Furthermore, we previously demonstrated that MARCKS phosphorylation (PKC’s substrate) is increased after presynaptic stimulation . TrkB tyr816 phosphorylation directly activates PLCγ1  under presynaptic stimulation  and cPKCβI  and pPKCβI protein levels  and PKCβI . This might indicate that the activity-dependent ratio FL/T1 could decrease pPKCs due to an activity-dependent pPKC translocation, action and subsequent degradation.We next determined the involvement of BDNF/TrkB pathway on pPKC levels. Preincubation with exogenous BDNF under presynaptic stimulation increases the ratio of pPKCα  to modulate synaptic function.In the neuromuscular system, evidence supports BDNF/TrkB signaling as a regulator of neuromuscular function. However, it remained unknown if nerve-induced muscle contraction Diaphragm muscle has been described as a useful model of flat skeletal muscle to study synaptic function , allowing a build-up of mBDNF and leaving net pro-BDNF levels unchanged.Previous results indicate that 2+ signaling mechanisms  and TrkB receptors among others, cooperate by stimulating PLCγ pathway. In this regard, it should also take into account the possible involvement of M1, A1 in PKC activation in the NMJ.BDNF binding to the TrkB.FL activates the intrinsic tyrosine kinase domain, leading to autophosphorylation in the activation loop  through TrkB. This suggests distinct roles of presynaptic vs. postsynaptic induced-BDNF. Moreover, exogenous BDNF enhances the opposite effects of presynaptic and postsynaptic activities on cPKC protein levels through a different ratio of TrkB.FL/T1. Muscle contraction increases TrkB.FL/T1 ratio and the reduction of the T1 dominant negative fashion over FL signaling upregulates presynaptic cPKCs. Therefore, the apparent distinct roles of the pre- and postsynaptic BDNF are consequence of the muscle contraction-induced decrease of TrkB.T1. This TrkB.T1 downregulation could be related with the recycling events of the receptor. TrkB isoforms seem to be differentially recycled after BDNF-induced endocytosis with TrkB.FL receptor degraded (targeted to the lysosomes) more quickly than TrkB.T1 (Huang et al., PKC isoforms need to be phosphorylated to be active Newton, . It is wTogether, the data support a regulatory mechanism which has been represented in Figure Several neuromuscular disorders show progressive loss of the connection between nerve and muscle. This leads to the pathological non-communication of the two tissues, eventually resulting in muscle weakness. Our results suggest that a decrease in neuromuscular activity, as it occurs in most neuromuscular disorders, could affect the BDNF/TrkB retrograde pathway linking pre- and postsynaptic components to correctly maintain neuromuscular function.EH: data collection, quantitative analysis, literature search, data interpretation and statistics; VC: data collection, literature search, data interpretation and design graphic abstract; TO: electrophysiological analysis; LN, AS, MT and MMS: data collection; KH and CLJ: PCR experiments and data interpretation; JT, MAL and NG: conception and design, literature search, data interpretation and manuscript preparation.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.


Shared use of recreational facilities is a promising strategy for increasing access to places for physical activity. Little is known about shared use in faith-based settings. This study examined shared use practices and barriers in faith communities in North Carolina.Faith communities in North Carolina (n = 234) completed an online survey (October–December 2013) designed to provide information about the extent and nature of shared use of recreational facilities. We used binary logistic regression to examine differences between congregations that shared use and those that did not share use.Most of the faith communities (82.9%) that completed the survey indicated that they share their facilities with outside individuals and organizations. Formal agreements were more common when faith communities shared indoor spaces such as gymnasiums and classroom meeting spaces than when they shared outdoor spaces such as playgrounds or athletic fields. Faith communities in the wealthiest counties were more likely to share their spaces than were faith communities in poorer counties. Faith communities in counties with the best health rankings were more likely to share facilities than faith communities in counties that had lower health rankings. The most frequently cited reasons faith communities did not share their facilities were that they did not know how to initiate the process of sharing their facilities or that no outside groups had ever asked.Most faith communities shared their facilities for physical activity. Research is needed on the relationship between shared use and physical activity levels, including the effect of formalizing shared-use policies. Increasing access to safe, affordable recreational facilities is one way to increase physical activity. The strategy is particularly important for increasing physical activity in low-income or racial/ethnic minority communities, where issues related to safe access to recreational facilities are well-documented ,2. ShareFaith communities are effective partners for promoting healthful eating and physical activity among their members . Public Given the importance of ecological frameworks in the design and implementation of physical activity interventions, community and environmental supports for physical activity are increasingly promoted as a strategy to increase individual physical activity levels among community members ,11. SharResearch on health promotion in faith communities is increasing . Formal The objectives of this study were to 1) create a baseline assessment of shared use of physical activity facilities among North Carolina faith communities, 2) determine barriers to shared use, and 3) determine priorities for future programs to support shared use of physical activity facilities in faith communities.We conducted a survey of faith-based organization in North Carolina in fall 2013. Three organizations collaborated to implement this assessment: North Carolina State University, Partners in Health and Wholeness, and the North Carolina Division of Public Health (DPH). North Carolina State University and DPH have a history of working together. In 2007, they collaborated to create the practice-tested faith-based intervention Faithful Families Eating Smart and Moving More (Faithful Families) , and permitted faith communities with limited access to the Internet to submit paper copies of the survey. Using program records, partners distributed the survey to faith communities that had participated in Faithful Families or the PHW program (262 faith communities). CTG coordinators throughout the state also distributed the survey to faith community contacts in their counties. Any faith community could participate, regardless of tradition or religious background. Emails were addressed generically but were tailored by local staff members  to be delivered to their local contacts in the faith community. The survey was completed by clergy, deacons, health committee members, and faith community members. Using survey distribution approaches developed from the tailored-design method , we sentBecause the survey was distributed across several networks, we do not know how many faith communities received the survey. However, according to the Association of Religion Data Archives, North Carolina has more than 15,000 faith communities . Two hunThe questionnaire comprised 25 questions related to shared use of facilities, including questions about the county where the faith community was located, congregation size, types of facilities that were shared, type of policy or agreement governing the use , and perceived barriers to shared use.Using census data and the county information reported by survey respondents, we measured the percentage of black residents living in the county where each participating faith community was located. We classified the percentage of black residents as low (≤10%), moderate (11%–30%), and high (≥31%) . We obtaP = .05.We used descriptive statistics to describe faith community characteristics and type of shared use. We used binary logistic regression to examine differences between congregations that shared use and those that did not share use. The regressions focused on faith community size, county economic tier, county health ranking, percentage of county population living in rural areas, and percentage of black residents in the county as key explanatory variables. Initial unadjusted models were estimated without controlling for other variables, followed by models estimated that controlled for other variables. Statistical significance was established at Of the 234 faith communities that responded to the survey, 78 (34.4%) were small (<120 members), 75 (33.0%) were medium sized (120–299 members), and 74 (32.6%) were large (≥300 members) . Forty-fOf the 100 counties in North Carolina, 53 were represented in the survey . The disMost respondents (82.9%) indicated that their faith communities had facilities that were shared with outside groups or individuals . Of the P = .03) than faith communities in the poorest counties (Tier 1) to share facilities (P = .01) to share facilities than faith communities in counties ranked lowest in health outcomes. In addition, faith communities in counties with a moderate percentage of rural residents were less likely  than faith communities with a high percentage of rural residents or a low percentage of rural residents to share facilities.In unadjusted models, faith communities in the wealthiest (Tier 3) counties were significantly more likely . The second most common reason given was not being asked by outside groups or individuals to share .Our study yielded findings that can help shape future projects and practices related to shared use of physical activity facilities in faith communities. First, these data illustrated that faith communities are opening up their spaces for shared use. As the first study of shared use among faith communities, this study can encourage public health practitioners to expand or enhance partnerships with faith communities to encourage shared use. To aid in this work, and as a direct result of this research and a request by DPH, ChangeLab Solutions developed a guide to implementing shared-use practices in faith communities .Second, our study showed that faith communities are sharing various types of spaces: classrooms, gymnasiums, playgrounds, and athletic fields. As a part of this study, we asked faith communities that shared facilities whether they were willing to share their information in a public database, which is now available online. Additional research is needed to understand how these spaces are used and whether use differs between faith communities that have formal agreements and faith communities that have informal agreements or no agreements.Third, our study found that several factors were associated with differences in sharing facilities. Faith communities in counties with the greatest wealth and highest health rankings were more likely to share their facilities than faith communities in poorer counties and counties with lower health rankings. Additionally, faith communities with a moderate percentage of rural residents were less likely to share facilities than faith communities in counties with a high percentage or low percentage of rural residents. These findings correspond to findings from a study by Edwards et al , which nFourth, our study found that faith communities did not cite liability concerns or maintenance costs as the primary reasons for not sharing their facilities. These finding are similar to those of another study in North Carolina that examined shared-use practices in schools . In our Our study identified faith communities as potentially untapped resources for shared use and increasing physical activity. From a social-ecological perspective, place-based physical activity interventions should consider the social, physical, and organizational environment to maximize usage and promotion of shared use of facilities for physical activity . A folloThis study has several limitations. First, it was not designed to serve as a comprehensive assessment of the facilities of faith communities in North Carolina. Because the study was based on a convenience sample, the results may not be representative of all faith-based organizations in the state. Because we sent the survey to a wide network of practitioners and partners, we could not determine a baseline number of faith communities to whom the survey was distributed, and therefore cannot calculate a response rate. This research focused on how faith communities shared their facilities, not whether they used the facilities of another organization. Second, the organizations that completed the assessment might represent those most interested in the topic of shared use or in promoting physical activity. As a result, a higher number of faith-based organizations that allow community access to their facilities might be represented in our data. Our data suggest the need for additional, larger studies that examine shared use policies and practices in faith communities. Third, we did not include denomination or religious affiliation in the survey, which has led us to adapt the survey instrument to include that information for future use. Fourth, the logistic regression had limitations. Because of the small sample size, some variables had small numbers of outcome events, which led to wide confidence intervals for the odds ratios. Therefore, associations between variables should be interpreted with caution; a larger sample of faith communities is needed for more precise estimates.Our findings suggest that faith communities are apt partners for increasing shared use in the community setting. Faith communities have facilities that can be used for various physical activities: indoor classrooms can be used for fitness classes; gymnasiums can be used for free play, games, or fitness classes; outdoor spaces can be used for organized games or free play; and large open spaces (including parking lots) can be opened up for biking, walking, and other activities. Faith community partnerships that promote shared use could be particularly important for communities that have persistent health disparities, including low-income and racial/ethnic minority communities, where access to spaces for physical activity may be limited.


The ability to assess another person’s direction of attention is paramount in social communication, many studies have reported a similar pattern between gaze and arrow cues in attention orienting. Neuroimaging research has also demonstrated no qualitative differences in attention to gaze and arrow cues. However, these studies were implemented under simple experiment conditions. Researchers have highlighted the importance of contextual processing  in attentional orienting, showing that attentional orienting by social gaze or arrow cues could be modulated through contextual processing. Here, we examine the neural activity of attentional orienting by gaze and arrow cues in response to contextual processing using functional magnetic resonance imaging. The results demonstrated that the influence of neural activity through contextual processing to attentional orienting occurred under invalid conditions (when the cue and target were incongruent versus congruent) in the ventral frontoparietal network, although we did not identify any differences in the neural substrates of attentional orienting in contextual processing between gaze and arrow cues. These results support behavioural data of attentional orienting modulated by contextual processing based on the neurocognitive architecture. Similar to eye gaze, non-social stimuli also play important roles in influencing attention, such as an arrow on a road sign. However, compared with eye gazes, non-social directional stimuli are not helpful when making conclusions regarding someone’s cognitive state, such as speculating about what a person wishes to do.The ability to assess another person’s direction of attention is paramount in social communication. For example, we are able to identify a person’s focus based on their eye gaze, thus enabling an understanding of other people’s inner state 2. For example, Friesen and Kingstone3 presented non-predictive gaze cues at the centre of a screen prior to the presentation of a peripheral target (right or left). Before the onset of the target, a centrally presented directional cue  appears on screen. Under valid conditions, the cue will accurately indicate the subsequent target location, whereas under invalid conditions, the cue will indicate the opposite location. A rapid response to a validly cued target indicates an allocation of attention  to the target location prior to target onset. In contrast, a delayed response to an invalidly cued target occurs when the onset of the target at the opposite location, indicating a reorienting of attention to the target. Previous studies9 have commonly demonstrated that arrow cues automatically trigger attentional shifts in the same manner as gaze cues. These studies have demonstrated that both gaze and arrow cues trigger attentional shifts when they are counterpredictive of a target location7, facilitate response time when discriminating the target following the cue6, have comparable sensitivity to object-based selection8 and the stimulus onset asynchrony between the cue and target9.Over the past two decades, cognitive psychologists have focused on comparing the role of directional gaze and arrow cues in attentional orienting. These studies have typically investigated attentional orienting based on gaze and arrow cues using a modified version of Posner’s cueing paradigm11). The dorsal frontoparietal network, with regions centred around the intraparietal sulcus (IPS), superior parietal lobule (SPL)/Brodmann’s area (BA)5, 7, and frontal eye field (FEF)/BA8, may be responsible for orienting of attention to a validly cued target in the cueing paradigm, but also for reorienting attention to an invalidly cued target11. The ventral frontoparietal network, with regions centred on the temporoparietal junction (TPJ)/BA39, 40, 22 and ventral frontal cortex (VFC)/BA44, 45, 47  and inferior frontal gyrus (IFG)), may only be responsible for reorienting attention. Most previous studies18 have demonstrated that the differences in cortical activity associated with social gaze and arrow cues are quantitative rather than qualitative, although some studies21 have reported evidence suggesting different mechanisms for these cues. For example, Tipper et al.17 reported attentional orienting to both eye gaze and arrow cues engaged extensive dorsal and ventral frontoparietal networks, but the magnitude of activation differed between these networks. However, these studies only examined the differences between gaze and arrow cues under simple conditions (e.g. a dot or letter as the target). Given that Birmingham and Kingstone22 suggested that the apparent difference in attentional orienting between gaze and arrow cues might be distinguished only when the cues were embedded in a rich environment, it is thus important to examine the differences between gaze and arrow cues under more complex conditions.Recent neuroimaging studies regarding attentional orienting have attempted to investigate differences in cortical activity between gaze and arrow cues. These studies have focused on two attentional networks  in attentional orienting when using arrows or eye gaze as cues. Previous studies28 reviewed previous evidence, indicating that the TPJ  was engaged in terms of “contextual updating” in attention. For example, Weidner et al.29 demonstrated that cortical activity in TPJ increased when the contextual processing of the relationship between the cue and target was incongruent as opposed to congruent  was incongruently rather than congruently cued). Moreover, Noppeney et al.30 observed that the activity in STS increased when a sound or speech target was incongruent  as opposed to congruent  with prior visual information. This finding indicated that context also modulated the activity of STS. Consistently, when a strong relationship was established between the target word and a word sound that had been previously presented, the results showed the enhanced activation for thematically related categories  and response suppression for taxonomically related categories  in the left STS31. In the present study, we focused on these brain regions to examine the influence of neural systems in relation to gaze and arrow cues through contextual processing focused on the relationship between the cue and target in attentional orienting.At a neural level, researchers have shown activity in the TPJ and superior temporal sulcus (STS)/BA21, 22 associated with contextual processing in attention. Geng and Vossel27, two sounds  were manipulated as targets to determine the contextual relationship between cue and target; that is, social gaze and social voice and arrow and tone as congruent meaning conditions, and social gaze and tone and arrow and social voice as incongruent meaning conditions. The aims of this study are as follows: (1) We first wanted to examine whether the influence of neural activity in TPJ and STS differed between gaze and arrow cues in response to contextual processing of the relationship between cue and target. (2) Furthermore, given that previous studies11 have characterised the functional mechanisms of the orienting and reorienting of attention  in dorsal and ventral frontoparietal networks, respectively, we considered it important to specifically investigate these functional mechanisms and how they were modulated through the contextual processing of the relationship between cue and target. Specifically, if different neural activity was observed for gaze and arrow in response to the contextual processing of cue-target, we would subsequently examine whether the neural activity for attentional orienting differed with contextual processing between gaze and arrow cues at the dorsal and ventral frontoparietal networks, respectively. In contrast, if no difference between gaze and arrow was evident, we would then examine only the influence of neural activity for attentional orienting by contextual processing in both gaze and arrow cues in these two attentional networks.In the present study, we examined the neural activity of attentional orienting with social gaze and arrows as cues using Posner’s cueing paradigm. Based on a previous study32, and had normal or corrected-to-normal visual and auditory acuity.This research was approved by the local ethics committee of Capital Medical University, Beijing, China. No foreseeable risk to the participants was present, and personal identifying information was not collected. Participants provided written informed consent and background information. All procedures complied with the ethical standards of the 1964 Declaration of Helsinki regarding the treatment of human participants in research. In total, 22 volunteers  participated. All participants were right-handed, as assessed by the Edinburgh Handedness Inventory27. Previous studies have demonstrated that female faces are less resemblance to angry expressions than male faces, and male faces are perceived as less likeable34 and more powerful35. To avoid any differential influence of expression , a Japanese female face with neutral expressions was used for this task ask Fig. . The imaver. 1.3.13; Audacity store.com). The duration of the stimulus presentation was 150 ms.Furthermore, two types of auditory stimuli were presented as targets. One type was sampled from a woman: an/i/voice sound (F0 frequency of 300 Hz), which is similar to the /iy/sound in English. The other type was a pure tone of similar frequency to the F0 voice (300 Hz), which was produced using the Audacity software package  was used to generate auditory and visual stimuli on a Windows computer. In addition, the participants generated their responses using a keypad .These stimuli were generated on a computer and presented to the participants via a custom-built, magnet-compatible audio-visual system during magnetic resonance (MR) scanning. To attenuate the acoustic noise that accompanies fMRI  scanning, shooting earmuffs were used. Participants viewed visual stimuli on a back-projection screen. The auditory stimuli were identical to those presented in a previous study39, the participants were asked to answer quickly and precisely whether or not they heard the auditory target on the left or right side of the headphones by pressing the corresponding key on the switch keypad using their dominant index or middle fingers, respectively. Response times (RT) were measured in each trial. A standard procedure for the Posner’s cueing paradigm removed cue stimuli before a target stimulus appeared on the display. However, when using a facial gaze as a cue, many studies e.g. refs The sequence of stimulus presentation is shown in Fig. 42. Consistent with a previous study43, alternating blocks of experimental trials of cue condition and blocks of baseline were presented. Within the condition blocks, congruence trials were presented in a pseudorandom event-related distribution.The fMRI analysis relied on a within-subject three factorial design, with the cue condition (gaze or arrow), context condition  or incongruent ), with validity condition  as the repeated factors. Sixty trials were performed under each condition. Our experimental design was based on a mixed block/event-related paradigm, facilitating a more complete utilisation of the BOLD signal and enabling a deeper interpretation of how the regions of the brain function on multiple timescales3. The slices covered most of the brain, including the entire temporal cortex, but excluding the most inferior parts of the cerebellum. We also acquired high-resolution isotropic T1-weighted images .The images were acquired using a 3.0-T Trio Tim Scanner-vision whole-body MRI system  to measure activation using a head coil. The functional images comprised 33 consecutive slices parallel to the anterior-posterior commissure plane, covering the entire brain. A T2*-weighted gradient-echo planar imaging (EPI) sequence was used with the following parameters: TR = 2000 ms, TE = 30 ms, flip angle = 90°, field of view = 220 × 220 mm, matrix size = 64 × 64, and voxel size = 3.4 × 3.4 × 3.5 mmThe data were analysed using the SPSS software package (ver. 21.0). Incorrect responses  and RT of less than 100 ms or more than 1000 ms were excluded from the RT analysis , and trials in which a response occurred prior to the target onset were also excluded. The mean RT under conditions was calculated for each participant. The mean RT was analysed using a three-way analysis of variance (ANOVA) with cue , context , and validity  as within-participant factors. To examine whether an interaction was significant, if present, a follow-up simple main effect (i.e. assessing the effect of each independent variable at each level of the other independent variable) analysis was conducted to interpret the result.http://www.fil.ion.ucl.ac.uk/spm/software/spm12) implemented in MATLAB 2013b (Math Works). The functional images from each run were realigned using the first scan as a reference to correct for head movements. The movement parameters generated during spatial realignment indicated that all subjects moved less than 2 mm during the course of the trial. The T1 anatomical image was preprocessed using an intensity inhomogeneity correction. Then, T1 anatomical images were coregistered to the first scan of the functional images. Next, the coregistered T1 anatomical image was normalised to the Montreal Neurological Institute space using a unified segmentation-spatial normalisation approach44. The parameters from this normalisation process were subsequently applied to each of the functional images. Finally, these spatially normalised functional images were resampled to a voxel size of 2 × 2 × 2 and were spatially smoothed in three dimensions using an 8-mm full-width-at-half-maximum Gaussian kernel.Data preprocessing and statistical analyses were performed using the Statistical Parametric Mapping software package , which yielded regressors in a general linear model (GLM) for each condition. We used a high-pass filter comprising a discrete cosine basis function with a cut-off period of 128 to eliminate the artefactual low-frequency trend. To correct the global fluctuation related to motion artefacts, global scaling was conducted. Serial autocorrelation, assuming an AR (1) (first-order autoregressive) model, was estimated from the pooled active voxels with a restricted maximum likelihood procedure and used to whiten the data and design matrix47.We used random-effects analyses48, the statistical maps exhibited a spatial extent threshold at p < 0.05, family-wise error (FWE)-corrected for multiple comparisons, and an intensity threshold at p < 0.001, uncorrected for multiple comparisons at the whole-brain level was used to protect against false-positive activations. The peak voxels of clusters exhibiting reliable effects are reported in MNI coordinates. We had an a priori hypothesis regarding the activity of contextual processing in TPJ and STS, and the influence of contextual processing in dorsal and ventral frontoparietal networks. Based on anatomical masks using the WFU Pickatlas tool, a small-volume correction was also employed to the a priori regions of interest, attributed to the anatomical structures in left/right hemisphere of the STS with BA21, 22, the IPS and SPL with BA5, 7, the FEF with BA8, and the IFG with BA44, 45, 47, separately. Consistent with the whole-brain level, we used small-volume correction at a voxel spatial extent threshold at p < 0.05, FWE corrected, and an intensity threshold at p < 0.001, uncorrected for multiple comparisons. Finally, to quantify neural responses with the influence of attentional orienting under context conditions, we used the MarsBaR software package49 to extract percentage changes in BOLD signals for congruent and incongruent contexts under valid and invalid conditions, averaged across voxels with given regions of interest (ROI) using spheres with a radius of 8 mm. Then, the means of the percent signal change (PSC) between the conditions were compared using repeated-measures ANOVA. All statistics were calculated using the SPSS software package (ver. 21).The contrast images from the first-level analyses from all subjects were subsequently used for the second-level group statistics. First, for each participant, the data were best fitted at every voxel using a combination of effects of interest. These data were delta functions representing the onsets of the eight conditions, given by the crossing of our 2 × 2 × 2 factorial design: cue  × context  × validity , convolved with the SPM12 haemodynamic response function. Second, based on the behavioural results, a 2 × 2 × 2  factorial ANOVA was used to investigate the relationship between behavioural results and brain activation. Based on a methods analysisp > 0.05), thus indicating that the participants suffered no speed-accuracy trade-off  = 12.412, p = 0.002, ηp2 = 0.371), with faster responses under the eye gaze (339.7 ms) versus arrow (351.3 ms) condition. In addition, we also observed a significant main effect of context  = 8.213, p = 0.009, ηp2 = 0.281), with faster responses under congruent (341.3 ms) versus incongruent (349.7 ms) conditions, and validity  = 25.247, p < 0.001, ηp2 = 0.546), with faster responses under valid (334.8 ms) versus invalid (356.2 ms) conditions.The mean RT under each condition are listed in Table F  = 4.907, p = 0.038, ηp2 = 0.189), but no significant interaction was detected for cue × context  = 0.166, p = 0.688, ηp2 = 0.008), cue × validity  = 0.048, p = 0.828, ηp2 = 0.002), or cue × context × validity  = 0.108, p = 0.746, ηp2 = 0.005).A significant interaction of context × validity was observed  but not under valid conditions (p = 0.182) with a faster response for congruent (349.9 ms) versus incongruent (362.5 ms) under invalid conditions, indicating an RT benefit for targets that match the context  of the cue under invalid conditions but not under valid conditions. This result suggests that the disengagement of attention from cued locations is facilitated through contextual processing. These findings demonstrated that attentional orienting is modulated through contextual processing only under invalid conditions, enabling the investigation of the neural substrates underlying the behavioural response of the contextual processing between the cue and target in attentional orienting induced by gaze and arrow cues.The F  = 9.591, p = 0.006, ηp2 = 0.324) but not between gender and other factors  ≤ 2.54, p > 0.1). However, the post hoc test did not reveal a difference between genders under valid or invalid conditions (both p > 0.1), although a faster response was observed for valid compared with invalid conditions in both male  and female  participants. Thus, the results of the present study suggest that attentional orienting through contextual processing was not influenced by the gender of the participants.Because previous studies have reported that gaze-triggered orienting is different between genders  × 2 context conditions  × 2 validity conditions  repeated-measures ANOVA.21, thus indicating that gaze versus arrow cues increased activation in various occipital and temporal areas, whereas the reverse contrast evoked activation in occipital regions.In a whole-brain analysis, the gaze trials evoked significantly greater activity than arrow trials in a many clusters of voxels. One of these clusters included the fusiform gyrus (BA 19), extending from the extra-striate visual areas into the occipital and temporal cortices . Additionally, anatomical region-based small-volume corrections revealed significant activation in the temporal lobe, including STS BA21/22). Fig. , Table 21/22. FigA 2  × 2  ANOVA was performed to investigate the influence of activation by the contextual relationship of cue-target in attentional orienting networks. The results of the whole-brain analysis revealed a significant interaction in left hemisphere TPJ BA 40). Additionally, anatomical region-based small-volume corrections revealed significant activation in the left hemisphere IFG (BA 47) regions  but not under valid conditions  for gaze or arrow cues. Compared with a previous study23 in which attentional orienting based on a contextual effect for gaze and arrow was investigated in the context of colour , the present study examined attentional orienting through contextual processing using facial gaze and voice, which seems to more closely resemble a real-world environment. Furthermore, although another study25 examined attentional orienting through gaze cues influenced by emotional context for 80 different images  as targets, the present study only manipulated two sounds as targets, potentially easing the establishment of a pairing between the cue and target. That is, the pairing of gaze and voice was easily established, and arrow and tone represented the other pair. Thus, the present study observed attentional orienting through contextual processing when using gaze and arrow as cues. Based on these findings, the results of the present study extended those of previous studies25, indicating that attentional orienting through centrally presented cues, irrespective of cue characteristics , could also be modulated by contextual relationship processing between the cue and target.We examined attentional orienting by gaze and arrow cues under a localising task in which participants were asked to indicate whether the target (voice and tone) was heard on the left or right side of the headphones. The combination of cues and targets varied across trials, reflecting contextual relationship processing, where the contextual meaning of the cue-target was congruent  or incongruent . Although the behavioural results showed no difference between gaze and arrow cues, a RT benefit was observed for targets matching the context of the cue under the invalid condition. This finding suggested that a disengagement of attention from cued locations was facilitated through contextual processing. Previous studies31 have revealed that neural substrates in the regions of the TPJ and STS were associated with contextual processing. Consistently, to highlight the neural underpinnings of RT modulated by contextual processing, the results of the present study also demonstrated that the left STS and bilateral TPJ were specifically involved in contextual processing under both gaze and arrow conditions. The results also indicated that these regions were weakly activated when the relationship of cue-target was congruent (the expected target matching the context of the cue) compared with incongruent (the expected target non-matching the context of the cue). We suggest that these regions may be the locations of an inhibitory mechanism, which enhanced neural activity to suppress the processing of incongruent predictions for targets from cue stimuli. Given that the activity of these regions did not differ between social gaze and arrow cues, we further suggest that a comparable neural system was elicited by contextual processing for gaze and arrow cues and further influenced the attentional process. This idea is consistent with previous studies18 that demonstrated the differences in attention to social and non-social cues were quantitative rather than qualitative.Importantly, consistent with the behavioural results, the main analyses of fMRI data revealed that neural substrates were not different in response to the contextual relationship processing of cue-target for gaze and arrow cues. Previous studies54. These studies reported an amplitude enhanced at P1  in attentional orienting with gaze as the cue under valid versus invalid conditions, whereas a greater amplitude at P3  was observed under invalid versus valid conditions. However, in previous studies56, the N300  - N400  wave reflected an updating of context information. For example, Demiral et al.55 observed a stronger N300-N400 effect elicited through a semantic context when the contextual scene was presented before the target in a spatial attention task. Given that the pattern of attentional orienting by contextual processing differed between valid and invalid conditions, we suggest that this finding may be influenced through associations with the activities of temporal staging between the influence of attentional orienting  and the contextual processing component. Compared with the early stage (70–100 ms) under valid conditions, the time window of processing overlapped with that of contextual information and attentional orienting under invalid conditions at a later stage (300–500 ms), in which these processes could be integrated to suppress a violation of expectancies (the expected target matching the context of the cue) when the contextual relationship of cue-target was incongruent. In addition, we speculated that varying SOA conditions may influence expectations, and the patterns of attentional orienting by contextual processing for valid and invalid conditions could be modulated in the present experiment. That is, if the target was presented at a short SOA prior to the expectation of the subject, then attentional orienting by contextual processing may not be influenced under valid or invalid conditions, whereas if the target was presented at a long SOA after the expectation of the subject, then attentional orienting by contextual processing might be influenced under invalid conditions.Interaction analyses in behavioural results indicated that a different pattern of attentional orienting by contextual processing was elicited for valid and invalid conditions. That is, a RT benefit was observed for targets matching the context  of the cue under invalid conditions, but not under valid conditions. We propose that the different patterns between valid and invalid conditions may be influenced through the overlap of the time window between contextual processing and attentional orienting. Electrophysiological studies have demonstrated that the activity of temporal staging differed between valid and invalid conditions57. Compared with incongruent conditions, we suggest that the lower activity in the ventral frontoparietal network when the contextual processing of the relationship between cue and target is congruent under invalid conditions may reflect a disengagement of attention from cued locations at a lower cost, which is readily elicited. This idea may explain the behavioural data obtained under invalid conditions, indicating that a lower cost is associated with processing when the contextual processing of the relationship between the cue and target was congruent versus incongruent; thus, participants could disengage attention from the cued location to rapidly capture a target.Furthermore, the results of the interaction analyses in fMRI also demonstrated the influence of contextual processing on neural activity for attentional orienting under invalid but not valid conditions. Such neural activities were observed in the left hemisphere TPJ and IFG, an area in the ventral frontoparietal network that may be responsible for invalidity orienting  in individuals with autism spectrum disorder (ASD)First, we tested attentional orienting by contextual processing using gaze and arrow as cues with two types of targets  under only visual-auditory cross-modal conditions. Given the complexity of real life, future studies should examine attentional orienting by contextual processing using two types of targets under visual-visual unimodal or visual-tactile cross-modal conditions in which attentional orienting by contextual processing may also play an important role.F  = 5.09, p = 0.04, ηp2 = 0.195), two 3-way repeated-measures ANOVA (block × cue × context) was performed under valid and invalid conditions separately. Although no significant interaction was detected for block conditions under invalid conditions, , we observed that the main effect of context  = 11.74, p = 0.003, ηp2 = 0.36) was significant with faster response to congruent than incongruent conditions (349.0 vs. 361.5 ms), thus indicating that contextual processing between the cue and target was immediately established at both gaze and arrow pairings. However, under valid conditions, although we observed no significant main effect of context  = 1.41, p = 0.25, ηp2 = 0.063), a significant interaction of block × cue × context was observed  = 12.67, p = 0.002, ηp2 = 0.38). The post hoc test revealed a significant difference between context conditions (p = 0.045) with a faster response to congruent than incongruent conditions (327.4 vs. 346.9 ms) when using arrows as cues in the last half of the block, thereby indicating that attentional orienting by contextual processing could be influenced by increasing the number of trials throughout the experiment under valid conditions when the tone target was matched with arrow cue. In the present study, attentional orienting was modulated through contextual processing under invalid conditions. Thus, we suggest that this effect may be elicited through immediately established pairing between arrow and tone, rather than increasing the number of trials in the experiment. However, future studies should investigate the mechanism of how contextual processing is influenced by increasing the number of trials through the experiment.Second, the present study involved two types of contextually related cues and targets. In contrast with faces and voices, which are immediately paired in a congruent context, the pairing of the tone and arrow might be influenced through the increasing number of trials throughout the experiment. To evaluate this possibility, all experimental blocks were divided into two parts , and a four-factor repeated-measures ANOVA  was used to analyse the RTs. Given that a significant 4-way interaction was observed  as a baseline condition to examine differences in the neural mechanisms between valid and invalid attentional orienting conditions. However, compared with a non-directional arrow as a neutral cue, Engell et al.19 suggested that a direct gaze, as a neutral cue, was perceived as directional rather than non-directional, which was problematic in terms of comparing social versus non-social cueing in an fMRI study. Future research should investigate the need for a baseline condition in which the neural gaze and arrow cues involve no spatial information and have the same effect on neural activity, such as closed eyes and non-directional arrows.Finally, in the present study, we directly contrasted the context conditions by gaze and arrow cues in invalid and valid trials to reveal differences in attentional orienting. Previous studiesIn this study, we observed that the response time in attentional orienting by gaze and arrow cues was modulated through contextual processing between the cue and target when contextually congruent and incongruent under invalid conditions in behavioural studies. Additionally, on the neural level, activity in the left TPJ and STS was observed with attentional orienting by gaze and arrow cues in response to contextual processing of the relationship between the cue and target. However, we did not observe any difference in the neural substrates between social gaze and arrows by contextual processing in attentional orienting. This finding adds further evidence in support of the notion that the differences in attention to social and non-social cues are quantitative rather than qualitative. Importantly, both behavioural and fMRI results indicated that the influence of contextual processing on neural activity for attentional orienting occurred under invalid conditions. Such an increase was observed in the ventral frontoparietal network when the cue and target were incongruent rather than congruent. This finding may provide an explanation for the behavioural data regarding attentional orienting by contextual processing based on the neurocognitive architecture.Supplementary_material