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


EGFR/KRAS mutations in NSCLC cell lines with high PD-L1 expression (p < 0.001), we evaluated the relationships between downstream signals and PD-L1 expression, particularly in three KRAS-mutant adenocarcinoma cell lines. The MEK inhibitor U0126 (20 μM) significantly decreased the surface PD-L1 levels by 50–60% compared with dimethyl sulfoxide (p < 0.0001). Phorbol 12-myristate 13-acetate stimulation  increased (p < 0.05) and two ERK2 siRNAs as well as KRAS siRNAs decreased (p < 0.05) PD-L1 expression. The transcriptional activity of the potential AP-1 site (+4785 to +5056 from the transcription start site) in the PD-L1 gene was demonstrated by luciferase assays, which was inhibited by U0126. The chromatin immunoprecipitation assay demonstrated the binding of cJUN to the AP-1 site. Two STAT3 siRNAs decreased PD-L1 expression by 10–32% in two of the three KRAS-mutant lung adenocarcinoma cell lines (p < 0.05), while the PI3K inhibitor LY294002 (40 μM) did not change the expression level. Supervised cluster analysis and gene set enrichment analysis between the PD-L1-high and -low NSCLCs revealed a correlation between PD-L1 expression and genes/pathways related to cell motility/adhesion. These results indicate that MAPK signaling is the dominant downstream signal responsible for ectopic PD-L1 expression, in which STAT3 is also involved to some extent. Furthermore, MAPK signaling may control the expression of PD-L1 and several genes related to enhanced cell motility. Our findings suggest that MAPK, along with STAT3, is important for determining PD-L1 expression, which could be useful for targeted therapies against lung cancers.Ectopic programmed cell death ligand 1 (PD-L1) expression in non-small cell lung cancers (NSCLCs) is related to immune evasion by cancer, and it is a molecular target of immune checkpoint therapies. Although some altered signals in NSCLCs are responsible for ectopic PD-L1 expression, the precise mechanisms remain obscure. Because we found a higher frequency of  Recent advances in immune checkpoint therapies are rapidly changing the clinical applications of cancer therapies . ProgramSTAT3  was amplified from genomic DNA purified from NCI-H1373 cells by PCR with PrimeSTAR GXL DNA Polymerase (Takara) and the following primer set: forward primer, 5′-ACCTGAGCTCGCTAGCAACATGACTCACCTGAGGACAA-3′; and reverse primer, 5′-TATCCTCGAGGCTAGCTCCATCCCAAAGAAAGGGTGTAG-3′. The PCR product was subcloned upstream of the luciferase gene of pGL4.17 basic luciferase plasmid (Promega) with the In-Fusion HD Cloning Kit (Takara) (PDL-P).The candidate promoter region  plasmid (Promega), which encodes Renilla luciferase driven by a thymidine kinase promoter, were co-transfected with FuGENE6 (Promega) at a FuGENE (μL) to DNA (μg) ratio of 6. Two hours later, DMSO or U0126 (20 μM) was added. Twenty-four hours after the transfection, the firefly and Renilla luciferase activities were determined with the Dual-Glo Luciferase Assay System (Promega) and an infinite M200 (TECAN) multiplate reader. After subtracting the background activity, each firefly luciferase activity was normalized using Renilla luciferase activity to determine the transfection efficiency.NCI-H1373 cells were dispensed to Nunclon Delta Surface, a 96-well white-bottomed plate  the day before transfection. Upon transfection, the cell density reached approximately 50%. One hundred nanograms of firefly luciferase plasmid  and 100 ng of pGL4.74 [5′-TCACATTTCAAGCAGGATGACTAAA-3′ and reverse primer, 5′-TGACTCACAGCCACTCTTCCA-3′. The amount of immunoprecipitated AP-1-responsive element was determined using a standard curve constructed from the serial dilution of input DNA, and fold enrichment was calculated using the cJun/isotype control value.ChIP was conducted for cJun in NCI-H1373 cells. Immunoprecipitation of the predicted AP-1-responsive element was conducted using the ChIP-IT Express Enzymatic Kit (Active Motif) and cJun mAb  or isotype control mAb , followed by qRT-PCR with SYBR Green Gene Expression Assays and the following primer set: forward primer, PD-L1-high and -low lung cancer cell lines using CCLE Analysis Tools: Differential Expression and Gene Set Enrichment Analysis (GSEA) .Supervised cluster analysis and GSEA were conducted between the t test was used for comparison of the two groups. Data are expressed as mean ± standard deviation (SD). The correlation between MFI and RQ of PD-L1 expression was evaluated by Pearson’s correlation test. Chi-square test was used for comparing the frequency of the two groups, and a p-value of less than 0.05 was regarded as significant.Unpaired Student’s PD-L1 mRNA levels of a human lung cancer cell line panel as the relative quantity (RQ) to that of BEAS-2B, a human bronchial cell line  and PD-L1-low (PD-L1 RQ < 1) groups (PD-L1-high mRNA (R > 2.7) cell lines expressed a substantial amount of PD-L1 on their surfaces suggests that post-transcriptional regulation is not a dominant mechanism regulating PD-L1 levels.We determined ell line  and disc) groups . MFI of ) groups . The cor) groups . AlthougEGFR-mutant and five KRAS-mutant cell lines, the histological characteristics of which resulted six of them being classified as adenocarcinomas and one as a large cell carcinoma, while the PD-L1-low group contained only two KRAS-mutant adenocarcinoma cell lines  reside outside known functional domains or potential phosphorylation sites, the functional significance of these mutations cannot be specified. The frequency of either EGFR or KRAS mutations in the PD-L1-high group was significantly higher than that in the PD-L1-low group (p = 0.00056). We obtained the same result about mutational status of EGFR or KRAS by COSMIC database. We inferred that the downstream signals of EGFR or KRAS might regulate PD-L1 expression.We obtained information about the mutational status of major driver oncogenes or a tumor suppressor gene in the 20 human lung cancer cell lines from the CCLE. The PD-L1-high group contained two ll lines . BecauseKRAS-mutant lung adenocarcinoma cell lines  were evaluated for PD-L1 expression 24 h after treatment with the MEK inhibitor U0126. Flow cytometry analysis revealed a 50–60% decrease of surface PD-L1 expression with U0126 compared with that with DMSO , miR-200b expression in two (NCI-H358 and NCI-H441), and miR-200c expression in two (NCI-H1373 and NCI-H358) cell lines, suggesting that the MAPK signal suppresses some miRNAs, counteracting the post-transcriptional suppression of PD-L1 by miRNAs in some cell lines. However, the increase in miRNA expression with U0126 was generally weak (9–37%) and inconsistent among the cell lines. So we cannot conclude that miRNA is universally regulated by MAPK signaling.To examine the possible post-transcriptional regulation of PD-L1 under an MAPK signal,  cancers , were qureatment . U0126 tEGFR mutation, in the EBC-1 lung squamous cell carcinoma cell line without an EGFR/KRAS/ALK mutation, and in the NCI-H460 lung large cell carcinoma with a KRAS (Q61H) mutation  than in the U0126 experiments (50–60%)  , which m(50–60%) , residuaor STAT3 .PD-L1 promoter fragment (−2403 to +153 from TSS) contains the binding sites for transcription factors except AP-1. A candidate AP-1 binding site was found at chr9:5455433–5455446 according to GRCh37/hg19 in the first intron of the PD-L1 gene by ChIP-seq data from UCSC Genome Browser  and the PD-L1 promoter plus a candidate AP-1 enhancer (PDL-P+E) vectors were constructed  compared with that with U0126, and PD-L1 expression was not affected in one cell line, indicating that the regulatory mechanism of PD-L1 by STAT3 signaling may not occur as widely as MAPK signaling.Next, we evaluated the contribution of signaling pathways other than MAPK signaling downstream of A levels . The degKRAS, was also evaluated. LY294002, a PI3K inhibitor, did not decrease PD-L1 expression in any of the three KRAS-mutant lung adenocarcinoma cell lines , motility (VAV1 and RAC2), cell proliferation (ANXA3 and IFI16), apoptosis (TNFAIP8 and SH3KBP1), cell signaling , ubiquitination (FBXL13), a ligand for NKG2D (RAET1E), or metabolism (CDA) than PD-L1-low cell lines , the functional significance of these differences is questionable. Next, we compared the expression of RAC2 and CDA mRNAs between DMSO and U0126 treatments in the three KRAS-mutant lung adenocarcinoma cell lines  regulating these molecules seems to be, at least in part, MAPK signaling.GSEA showed that two pathways, actin cytoskeleton regulation by Rho GTPases and integrin-mediated cell adhesion, significantly correlated to the ll lines . Both suEGFR [EML4-ALK [NPM-ALK [EGFR mutation [EML4-ALK fusion [KRAS mutation also contributes to enhanced PD-L1 expression. Ota et al. showed that an MEK inhibitor (U0126) and a PI3K inhibitor (LY294002), but not a STAT3 inhibitor (S3I–201), decrease PD-L1 expression in EGFR-mutant and EML4-ALK-positive lung cancer cells, as determined by qRT-PCR and flow cytometry [KRAS-mutant adenocarcinoma cell line. Contrary to the previous report mentioned above, our analysis showed the partial contribution of STAT3, but not PI3K signaling, to PD-L1 expression. The different mutational status may lead to different signaling pathways for PD-L1 expression.The mechanisms of ectopic PD-L1 expression in human cancers have been studied in lung cancers –13, melaEGFR ,11, EML4[NPM-ALK , with ac[NPM-ALK –8. It ha[NPM-ALK ,17–19, S[NPM-ALK , or PI3K[NPM-ALK  are relemutation ,11 and EPD-L1-high cell lines included three squamous cell carcinomas without any known oncogenic driver mutations and one large cell carcinoma with a KRAS (Q61H) mutation  without AP-1 sites  in BEAS-2B does not induce PD-L1 expression [KRAS is insufficient for PD-L1 expression. Skoulidis et al. recently reported three subsets  of KRAS-mutant lung adenocarcinoma cell lines according to the co-occurring genetic events as determined by an integrative analysis of genomic, transcriptomic, and proteomic data [KRAS-mutant lung adenocarcinoma cell lines may reflect their heterogeneity.Two n levels . Akbay epression , suggestmic data . These sRAC2 and CDA as genes with significantly higher expression in PD-L1-high lung cancer cell lines. The expression of these genes as well as PD-L1 was dependent on MAPK signaling, suggesting that MAPK signaling constitutes a common pathway for an immune checkpoint molecule, and other functional molecules regulating cell motility, proliferation, or pyrimidine metabolism. GSEA identified that the pathways of the actin cytoskeleton regulation by Rho GTPases and integrin-mediated cell adhesion are significantly related to PD-L1-high lung cancer cell lines. These signatures may be related to the enhanced cellular motility or invasive ability and are reminiscent of the findings in a previous report by Chen et al. describing the correlation between PD-L1 expression and epithelial—mesenchymal transition (EMT) [KRAS-mutant lung adenocarcinoma cell lines, which was also dependent on MAPK signaling (unpublished data). Although the functional significance of these molecules requires further investigation, we speculate that MAPK signaling regulates gene expression related to EMT and immune evasion. Furthermore, we found CCL17 (TARC) expression was higher, while CXCL3 expression was lower in PD-L1-high cell lines than in PD-L1-low cell lines, respectively (We identified on (EMT) . We alsoon (EMT) –30, in Kectively . CCL17 iKRAS-mutant lung adenocarcinoma cell lines. We also demonstrated the MAPK-dependent transcriptional activity of AP-1, as well as the binding of cJUN to AP-1, in the PD-L1 gene. MAPK signaling also regulates PD-L1 in squamous and large-cell lung carcinoma cell lines. PD-L1 is correlated with the expression of other functional genes (RAC2 and CDA) and to pathways related to the cytoskeleton and cell adhesion, suggesting possible implications for EMT.In summary, this study showed that PD-L1 expression is regulated by MAPK and partially by STAT3 signaling in S1 FigPD-L1-high cell lines (RQ > 2.7 in PD-L1-low cell lines (RQ < 1). Empty and shaded histograms indicate isotype control and PD-L1, respectively. BEAS-2B, a normal human bronchial cell line, is shown as a reference.A, Ten (PDF)Click here for additional data file.S2 FigKRAS-mutant lung adenocarcinoma cell lines showed inconsistent increases in the expression of miR-200a, miR-200b, and miR-200c with U0126 . Each miRNA level was normalized by miR-16  and DMSO-treated cells (reference). Data are the mean of three independent experiments.qRT-PCR (ΔΔCt method) of (PDF)Click here for additional data file.S3 FigEGFR mutation ; (B) EBC-1, a squamous cell carcinoma cell line; and (C) NCI-H460, a large cell carcinoma cell line. Data are the mean of three independent experiments.(A) NCI-H1975, an adenocarcinoma cell line harboring (PDF)Click here for additional data file.S4 FigA, Surface protein (relative MFI) (upper) and mRNA levels (RQ) (lower) of PD-L1. Data are the mean of three independent experiments. B, Immunoblot shows KRAS knockdown and decreased ERK phosphorylation. One representative result of two or three independent experiments.(PDF)Click here for additional data file.S5 FigCCL17 expression is higher, while CXCL3 expression is lower in PD-L1-high than in PD-L1-low lung cancer cell lines.(PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.


Flow cytometry was used to examine the expression of PD-L1 on ALDH+ MMICs. To determine the role of PD-L1 in MMICs self-renewal, we cultured melanoma cells with anti-PD-L1 and measured tumorsphere formation and apoptosis. In addition, the effects of anti-PD-L1 on tumorigenicity and residual ALDH+ MMICs in tumors were evaluated in vivo. We demonstrated that melanoma cell-intrinsic PD-L1 was expressed in ALDH+ MMICs. Blocking PD-L1 in melanoma cell lines impaired tumorsphere formation and induced the apoptosis of sphere cells. In addition, blocking PD-L1 inhibited tumor growth in vivo. We observed residual ALDH+ MMICs within the tumor. The results showed that blocking PD-L1 also significantly decreased the residual ALDH+ MMICs in the tumors. In conclusion, these results suggest a new mechanism underlying melanoma progression and PD-L1-targeted therapy, which is distinct from the immunomodulatory actions of PD-L1.Recent studies have indicated that therapeutic antibodies targeting PD-L1 show remarkable efficacy in clinical trials in multiple tumors and that a melanoma cell-intrinsic PD-1: PD-L1 axis promotes tumor growth. However, few studies have shown tumor-intrinsic PD-L1 effects in malignant melanoma initiating cells (MMICs). Here, we aim to determine the possible regulatory effects of PD-L1 on MMICs. The ALDEFLUOR kit was used to identify ALDH Metastatic melanoma is an extraordinarily challenging cancer, with a 16% 5-year survival rate, and it responds poorly to most standard chemotherapies , 3. For Recently, it has been established that programmed death-1 (PD-1) is a prominent checkpoint receptor that, upon binding its ligands PD-L1 or PD-L2, dampens T effector functions by inhibiting signaling downstream of the T cell receptor . PD-L2 i+ tumor-initiating cells [In contrast to the immunosuppressive effect of PD-L1, it is also known to contribute to the promotion of tumor cell growth and downregulation of quiescent cells , 9. Furtng cells . PD-L1 ang cells . These rμg/mL streptomycin (Gibco). Cells were cultured at 37°C in 95% humidity and 5% CO2 atmosphere. All cell lines were routinely screened for mycoplasma contamination.B16-F0 and B16-F1 melanoma cell lines were maintained in RPMI 1640 (Gibco) containing 10% fetal bovine serum , 100 U/ml penicillin (Gibco), and 100 ∗106/ml cells were suspended in Aldefluor Assay Buffer (AAB) and incubated with 5 μL ALDH substrate (BAAA) for 45 min. 5 μL diethylaminobenzaldehyde (DEAB) was added to a separate sample containing BAAA for an ALDH-inhibited control. Then, samples were washed and resuspended in AAB. Fluorescence-activated cell gates were established using the inhibited control, DEAB, with the fluorescein isothiocyanate (FITC) channel with excitation and emission wavelengths of approximately 495 nm and 521 nm, respectively. To evaluate the expression of PD-L1 in ALDH+ cells, PD-L1 antibody  and immunoglobulin G (IgG) isotype-matched control (GeneTex) containing BAAA were added to the cells separately. All samples were incubated for 30 minutes at 4°C. Following incubation, the material was centrifuged, and pellets were resuspended with 500 μl assay buffer prior to data acquisition. Flow cytometry analysis was performed on a BD Biosciences FACSCanto, and data analysis was conducted using CellQuest Pro (B&D Biosciences).The ALDEFLUOR kit  was used to identify the stem/progenitor cells that expressed high levels of the aldehyde dehydrogenase (ALDH) . Brieflyμg/mL heparin (Sigma-Aldrich), 4 mg/mL bovine serum albumin (Sigma-Aldrich), 20 pg/mL insulin (Sigma-Aldrich), N2 supplement (Invitrogen), supplemented with 10 ng/mL bFGF , and 20 ng/mL EGF (Peprotech), as previously described [μg anti-PD-L1  or control rat immunoglobulin G (IgG). Tumorspheres were observed under microscope 14 days later. Individual spheres with diameters larger than 100 μm from each replicate well were visualized and counted with an inverted microscope.The B16-F0 and B16-F1 melanoma cells were plated as single cells in ultralow attachment six-well plates  and cultured in RPMI 1640 containing 6 mg/mL glucose (Sigma-Aldrich), 1 mg/mL NaHCO3 (Sigma-Aldrich), 5 mM HEPES (Sigma-Aldrich), 4 escribed . On the Cells were double-stained with FITC-annexin V and PI according to the manufacturer's instructions (Annexin-V FITC/propidium iodide (PI) Apoptosis Detection Kit; BD Pharmingen). Analysis was performed by flow cytometry. Early apoptotic cells were stained with Annexin-V alone, whereas necrotic and late apoptotic cells were stained with both Annexin-V and PI.5 B16-F0 or 5 × 105 B16-F1 melanoma cells. Then, 100 μg anti-PD-L1 or control rat IgG was administered intraperitoneally 3, 6, and 9 days following melanoma cell inoculation. All animals were randomly assigned to two groups of 5 mice each. Tumor size was monitored every two days. All surgical procedures and care given to the animals were in accordance with institutional guidelines.Adult SPF male C57BL/6 mice were implanted subcutaneously on the right flank with either 5 × 10t-test was used to determine significant differences. P values < 0.05 were considered statistically significant.All data were reported as the mean ± standard error. Statistical analysis was performed using GraphPad Prism 5.0 Software (San Diego). A two-tailed paired +/ALDH+ subpopulations from these two cell lines. As shown in + cells were identified in melanoma cell lines by flow cytometry with the ALDEFLUOR kit. Cells were then incubated for 30 min with mouse monoclonal antibodies specific for PD-L1. The analysis of the percentage of PD-L1+ALDH+ cells was gated by ALDH+ cells. We found that approximately 10% to 18% of the cultured murine B16-F0 cells and B16-F1 cells were ALDH+. Approximately, 5% of the ALDH+ cells were PD-L1+/ALDH+. These data suggest that PD-L1 may be involved in regulating MMICs.Previous studies have described the isolation of MMICs from mice using ALDEFLUOR/ALDH as a marker , 15. To To determine whether PD-L1 can mediate MMIC self-renewal, we cultured melanoma cell lines with anti-PD-L1. The results showed that anti-PD-L1 significantly inhibited tumorsphere formation in B16-F0 and B16-F1 melanoma cells compared to the control groups . Cancer Tumorsphere formation has been reported as a measure of the presence of MMICs in enriched cell populations. We further explored the effects of anti-PD-L1 on apoptosis in melanoma tumorspheres. The data illustrated that anti-PD-L1 induced significant apoptosis in melanoma tumorspheres . Anti-PDμg anti-PD-L1 or control rat IgG 3, 6, and 9 days following melanoma cell injection. Other studies have reported that anti-PD-L1 significantly suppressed tumor growth compared with PBS-injected animals in two animal models. Anti-PD-L1 promoted tumor rejection in 50% of B16-F0 melanoma challenged mice (P = 0.031) and 50% of B16-F1 melanoma challenged mice  and 1.4-fold residual ALDH+ MMICs in the B16-F0 animal model (P = 0.045). These results suggest that one mechanism for the anti-tumor effects of anti-PD-L1 is related to its ability to suppress the tumorigenicity capacity of MMICs.Mice were challenged with melanoma cells (B16-F0 and B16-F1) and treated with 100 + MMICs and induced tumorsphere formation. PD-L1 further inhibited the apoptosis of MMIC-enriched cells. Blockage of PD-L1 directly inhibited tumorigenesis in vivo and significantly decreased the residual percentage of MMICs. These results may indicate that melanoma cell-intrinsic PD-L1 promotes self-renewal and the tumorigenic capacity of MMICs.Previous studies have shown that PD-L1 expression is a common phenomenon in immunotherapy-naive melanomas –19. FurtTraditionally, PD-1 ligands have been expressed in tumor cells, leading to T-cell exhaustion and tumor cell evading the immune response, which was thought to require its receptor interaction . Accordi+ MMICs. Glioma stem cells expressed lower levels of the PD-L1 than those of differentiated glioma cells, which contributed to the higher sensitivity of glioma stem cells to the cytotoxicity of the IL-2-activated NK cells [+ tumor-initiating cells and inhibits IFN-γ secretion by tumor-infiltrating lymphocytes (TILs) incubated with CD44+ cells [ in vivo, which is in agreement with these published studies.MMICs are known to have high ALDH. Previous studies have successfully used ALDH as a marker to isolate MMICs from mice , 15. OurNK cells . In head4+ cells . These p4+ cells –29. We sIt has been established that the expression of PD-L1 is an indicator of poor prognosis for patients' survival in many cancers, such as pulmonary adenocarcinoma, gastric cancer, colorectal cancer, and esophageal cancer –33. In cTaken together, our results suggest that melanoma-PD-L1 can enhance tumorigenesis by maintaining the stem cell properties in MMICs. Future studies are needed to elucidate the underlying cellular and molecular mechanisms, which will be helpful to maximize its clinical benefits.