The aim of our study was to investigate the role of TrkB pathway in tumor occurrence and development for in order to provide theoretical basis to laryngeal cancer therapy.in vivo experiments were conducted in syngeneic BALB/c mice.Biological characteristics of the cells were studied by migration tests and colony forming assay. Gene and protein expression analysis was performed by RT-PCR or western blot. Significant changes in protein and gene expression, including higher expression level of TrkB, were found in cells and laryngeal cancer specimens. we demonstrated that TrkB activates AKT via c-Src, leading to increased proliferation. Also, TrkB induced EMT via increased expression of EMT related transcription factors such as Twist-1 and Twist-2.Our data indicate TrkB are overexpressed in laryngeal cancer, and TrkB signaling is involved in tumorigenicity of laryngeal cancer. These observations suggest that TrkB is a promising target for future intervention strategies to prevent tumor metastasis, EMT program in laryngeal cancer.What is already known about this subject?• Cancer of the larynx is one of the most common types of head and neck cancer.• The survival rate of advanced laryngeal cancer is only 30 to 40%.• The tropomyosin-related kinase B receptor (TrkB), together with TrkA and TrkC, are neurotrophin receptors regulating the proliferation and differentiation of neuronal cells.What are the new findings?• TrkB are overexpressed in laryngeal cancer.• TrkB signaling is involved in tumorigenicity of laryngeal cancer.• TrkB acts as a key regulator of the PI3K/AKT signal pathway-mediated tumor metastasis.How might these results change the focus of research or clinical practice?• These observations suggest that TrkB is a promising target for future intervention strategies to prevent tumor metastasis, EMT program in laryngeal cancer. Our study provides molecular insight into the tumor metastasis and has important implications in elucidating oncogenic processes. Cancer of the larynx is one of the most common types of head and neck cancer. Laryngeal cancer is also the second most common tumor of the respiratory tract and is the eleventh most common cancer worldwide with high mortality rate. Among them, the laryngeal squamous cell carcinoma accounts for approximately 95% of all malignant tumors of the larynx . LaryngeSeveral risk factors have been implicated in the pathogenesis of laryngeal cancer, including tobacco use, alcohol consumption, air pollution, genetics, nutrition, bacterial infection, life-style and occupational factors –9. Moreovia mitochondrion-driven apoptotic pathway, rendering cancer cells survival, chemoresistance and invasiveness [The phosphoinositide 3-kinase (PI3K) signaling pathway is the most commonly mutated pathway in head and neck squamous cell carcinoma . Activatsiveness . Previousiveness . The ovesiveness . HoweverImmunohistochemical assay was performed to investigate the protein expression of TrkB in 69 carcinomas and 32 tumor-adjacent normal laryngeal tissues.69 samples of laryngeal cancer and 32 samples of tumor-adjacent normal laryngeal tissues were used to detect the expression level of TrkB by immunohistochemistry. It showed that the expression of TrkB was significantly increased in laryngeal cancer than that in tumor-adjacent normal tissue Figures  and B. TFirstly, the western blotting analysis showed that the expression level of TrkB in Hep-2 was higher than that in other laryngeal tumor cell lines, including TU686 cells and M4e cells. Thus, Hep-2 cell line was used in the following studies. Secondly, Transwell assays showed that the down-regulation of TrkB expression could inhibit the migration of Hep-2 cells Figure . SubsequFigure We have demonstrated that c-Src activation by TrkB induces activation of the PI3K/AKT and cyclin D1 expression. To investigate the relationship between Runx3 or Keap1 and TrkB-induced tumorigenesis, we first examined whether the expression of TrkB influences Runx3 and Keap1 expression. We found that Runx3 and Keap1 mRNA expression were significantly increased in Hep-2 TrkB-sh RNA cells Figure . We alsoWe examined whether expression of TrkB was able to promote an EMT in Hep-2 cells. The results showed that the expression of proteins E-cadherin (E-cad) was down-regulated after TrkB overexpression and the expression of proteins of N-cadherin and Vimentin were up-regulated in Hep-2-TrkB cells Figure  and 5B. To investigate the role of TrkB in tumor formation, Hep-2 or Hep-2-shTrkB cells were injected into of BALB/ c mice to establish tumor animal mode. After 30th days, the tumor volume and weight in Hep-2-shTrkB groups were significantly smaller than that in control group, while the inhibitory rate was markedly higher than control group Figure . We nextLaryngeal cancer is one of the most common malignant tumors of the head and neck, accounting for about 1% to 5% of the systemic tumors in our body, and the pathological type of squamous cell carcinoma, accounts for 93% to 99% of all laryngeal cancel higher incidence of laryngeal cancer is in northern China, and the age of onset is 50 to 60 years old and male with a high incidence than women . In receIn this study, we show that TrkB is frequently overexpressed in highly metastatic laryngeal cancer cell lines and clinical laryngeal cancer samples. Furthermore, TrkB expression was significantly up-regulated in laryngeal cancer samples in comparison with normal laryngeal tissue. Activation of the PI3K/AKT pathway through TrkB-mediated c-Src activation induced tumorigenic and metastatic potential of laryngeal cancer.Tumor metastasis is an important reason causing tumor morbidity and mortality. Several studies have shown that TrkB play an essential role in invasion and metastasis of various tumor cells , 21. In Studies have shown that PI3K/AKT signaling pathway involved in development of human malignant tumors, and the pathway is related with tumor proliferation, apoptosis, metastasis, invasion and other cellular processes , 23. PI3c-Src played essential roles in multiple aspects of tumor progression, including proliferation, disruption of cell/cell contacts, migration, invasiveness, resistance to apoptosis, and angiogenesis . MoreoveIn conclusion, TrkB are overexpressed in laryngeal cancer, and TrkB signaling is involved in tumorigenesis and metastasis of laryngeal cancer. It indicates that TrkB pathway may be a promising target for future intervention strategies to prevent tumor metastasis, EMT program in laryngeal cancer. The future study should focus on the targeting regulation of TrkB pathway via non-coding RNA which may provide a novel therapeutic plan for laryngeal cancer.2. The inhibitors of protein kinase (K252a and SU6656) and PI3K inhibitor (LY294002) was purchased from Calbiochem .Human laryngeal cancer cell lines  were obtained from the Cell Bank of the Chinese Academy of Sciences  and were maintained as previously described –35. BrieFresh-frozen human laryngeal cancer samples and tumor-adjacent normal laryngeal tissues were obtained from the Affiliated Hospital of Southwest Medical University. The subject was approved by the institutional review board and the ethics committee of the Affiliated Hospital of Southwest Medical University (K2013166) and all patients have signed informed consents.2O2 and 10% goat serum and they were subsequently incubated overnight with a primary antibody for TrkB  at 4°C. The samples were incubated with HISTOFINE simple stain MAX-PO (R)  and finally visualized by 3,3’diaminobenzidine (DAB) with hematoxylin-counterstain.The paraffin sections of laryngeal cancer were deparaffinized and rehydrated routinely. The sections were immersed with 3% HShort-hairpin RNA (shRNA) constructs targeting TrkB (shTrkB) and AKT1 (shAKT1) and pLKO were obtained from Sigma-Aldrich  and introduced into cells via retroviral infection or with lipofectamine2000 reagent according to the manufacturer’s protocol. shRNA that did not match any known human cDNA was used as a control. For TrkB overexpression, pcDNA3.1 plasmids containing the full length cDNA sequences for human TrkB was stably transfected into Hep2 cells.Western blotting was performed as previous reported , 37. BriTrkB: forward: 5-GGAAAAGCAAAAACCCTGTCTAGA-3;reverse: 5-TGTAGCATCACTTCCTGCCATT-3β-actin: forward: 5-TTGCCCCGAGGCTCTCTT-3;reverse: 5-AGTTGAAGGTGGTCTCGTGGAT-3.RT-PCR was performed as previous reported . BrieflyAfter a reverse transcription reactions at 55°C for 30 minutes, there was an initial 10-minute denaturation at 95°C followed by the 40 cycles run consists of a 15-second denaturation step at 94°C and an annealing step at 60°C for 30 seconds, and extension step at 68°C for 1 minute. The density of bands was determined by using Un-Scan-It gel Software  and statistical analysis was performed by utilizing either conventional Student’s t test. The PCR results were averaged with three independent experiments, which were used in the statistical analysis.5 cells in PBS per mouse) of the nude mice (n=10 in each). Immunohistochemistry of TrkB, cyclinD1, AKT, E-cadherin were also performed in the xenograft tumor tissue. The primary antibodies for TrkB , cyclinD1 , AKT , E-cadherin  were purchased from Santa Cruz Biotechnology . The procedures were similar with the part of immunohistochemistry. Briefly, the paraffin sections of xenograft laryngeal cancer were deparaffinized, rehydrated and immersed with 3% H2O2 and 10% goat serum. Subsequently, the sections were incubated overnight with primary antibodies, followed by incubation with HISTOFINE simple stain MAX-PO (R)  and visualization by DAB with hematoxylin-counterstain.Five-week-old male athymic nude mice (BALB/c nu/nu) were purchased from Chengdu Dashuo Biological Technology Co. Ltd and acclimated for two weeks. All animal procedures were approved by the Animal Care and Use Committee at animal experimental center of Southwest Medical University. The animal experiment was also approved by the institutional review board and the ethics committee of the Affiliated Hospital of Southwest Medical University (K2013167), Hep-2 cells transfected with TrkB-targeting shRNA or non-targeting control shRNA were subcutaneously implanted into flank regions  and were shown as the mean ± standard deviation (SD). Student’s t-test was used for the comparison of mean values between two groups. A value of P< 0.05 was considered significant.


Programmed death-1 receptor (PD-1) and its ligand (PD-L1) play an integral role in regulating the immune response against cancer. This study investigated the prognostic significance of PD-L1 expression on tumor cells and tumor-infiltrating immune cells (TILs) in the tumor microenvironment in Chinese patients with esophageal squamous cell carcinoma (ESCC). Archival formalin-fixed, paraffin-embedded ESCC samples from treatment-naïve patients with ESCC after surgery or by diagnostic endoscopic biopsy were collected between 2004 and 2014. Expression of PD-L1 in ESCC tumor specimens was assessed by immunohistochemistry (IHC), and the degree of TIL infiltration was evaluated by examining hematoxylin and eosin-stained (H&E) specimens. PD-L1+ as defined as ≥1% of tumor cell membranes showing ≥1+ intensity. In 428 patients, specimens from 341 (79.7%) were PD-L1+. In the definitive treatment group (patients who received curative esophagectomy or definitive [chemo-]radiation therapy), PD-L1 positivity was associated with a significantly shorter DFS and OS. In the palliative chemotherapy group exhibited, neither PFS nor OS correlated significantly with PD-L1 expression. PD-L1 expression was positively associated with TIL density. In 17 paired tumor tissues collected before and after treatment, an increase in PD-L1 expression was associated with disease progression, whereas a decrease in PD-L1 expression was associated with response to chemotherapy or disease control. So, PD-L1 expression was associated with a significantly worse prognosis in patients with ESCC. These observations suggest that PD-L1 may play a critical role in ESCC cancer progression and provide a rationale for developing PD-L1 inhibitors for treatment of a subset of ESCC patients. Esophageal cancer is one of the most aggressive and lethal malignancies among gastrointestinal cancers, ranking sixth in cancer-related death. It has two main subtypes: squamous-cell carcinoma (ESCC), which is mostly found in Asia, Africa, and South America, and adenocarcinoma (EAC), which is mostly found in North America and Europe . In ChinProgrammed cell death 1 (PD-1), a member of the CD28 family of co-stimulatory receptors, is a T cell immune-checkpoint involved in providing inhibitory signals in T-cell activation when engaged by its ligands: PD1 ligand 1 (PD-L1) and PD1 ligand 2 (PD-L2) . It is wTo explore the prognostic significance of PD-L1 expression and TILs for ESCC, we investigated the extent of PD-L1 expression and TILs in formalin-fixed samples from 428 Chinese ESCC patients, and analyzed the association between PD-L1 expression, TIL density and outcome.A total of 428 ESCC specimens, one from each patient, were obtained from patients admitted to the Affiliated 307 Hospital Cancer Center, Beijing, P. R. China during 2004 to 2014. Samples were collected either by surgery  or by endoscopic biopsy  from treatment-naïve patients. The median follow-up was 34.4 months (range 0.3-147.1 months) from the date of sample collection. The last follow-up date was December 31, 2015. At the time of last follow-up, 22.43% (n=96) of patients with ESCC were still alive.Treatment modalities depended on the stage and localization of ESCC. Patients with localized tumors underwent curative esophagectomy , while those with unresectable disease or who were medically unfit for surgery received definitive (chemo-)radiation . Patients with advanced metastatic disease received palliative chemotherapy , palliative R1/R2 resection , best supportive care , or chose not to receive any treatment .Tumor PD-L1 expression was assessed via IHC, using rabbit monoclonal antibody clone 73-10 proprietary of Merck KGaA. Table PD-L1 expression was found primarily on the membranes of the tumor cells Figure , and wasThree major patterns of PD-L1 expression were found in ESCC specimens: (i) a diffuse PD-L1 expression in the presence of TILs; (ii) regional expression of PD-L1 colocalized with TILs; and (iii) PD-L1 expression at the invasive front Figure . PD-L1 eth IUCC/AJCC staging system, 157 (36.7%) and 191 (44.6%) had stage 0-II and III disease, respectively. And the majority  of tumor specimens showed a moderate to high differentiated tumor. The clinicopathological features of patients are summarized in Table Among the 428 patients, the majority 335 (78.3%) were male and 247 (57.7%) were older than 60 years. According to the 7Statistical analyses using Spearman Rank Correlation indicated that PD-L1+ specimens were associated with aggressive clinicopathological features, which included deeper tumor invasion (p=0.037) and nodal metastasis (p=0.013). No significant association was found between PD-L1 expression and gender (p=0.545), age (p=0.943), level of differentiation (p=0.955), primary tumor location (p=0.231) or TNM stage (P=0.581).The correlation of PD-L1 expression with disease prognosis, as measured by disease free survival (DFS/PFS) and overall survival (OS), was analyzed separately in the definitive treatment cohort and palliative treatment cohorts.The definitive treatment cohort comprised 250 patients, including patients who received curative esophagectomy or definitive (chemo-)radiation therapy, and excluding those who had hospital mortality (n=16) and occurrence of other primary cancer (n=7). The DFS rates at 1, 3, and 5 years, respectively, were 79.5%, 53.9%, 32.3% for PD-L1– patients and 57.8%, 31.1%, 21.3% for PD-L1+ patients. Similarly, the OS rates at 1, 3, and 5 years, respectively, were 92.5%, 55.8%, 36.5% for PD-L1– patients and 84.5%, 36.3%, 29.8% for PD-L1+ patients. DFS and OS was significantly related to PD-L1 expression , PD-L1+ patients exhibited worse DFS and OS than PD-L1– patients Figure  & 4B. StPatients in the palliative treatment cohort exhibited no significant association between PD-L1 expression and PFS  or OS  .The correlation between TIL density and patient prognosis was examined. Similar to PD-L1 expression, there was no significant difference in TIL density between surgical and biopsy specimens (p=0.243), suggesting that TIL density was not affected by size of the specimens. In definitive treatment cohort, no significant correlation was observed between DFS/OS and TIL density  Figure  & 5B. HoExpression of PD-L1 and numbers of CD8+ and Foxp3+ cells was examined in 17 paired tumor specimens. This included 3 patients who did not receive any treatment between the times when paired tumor samples were harvested (mean duration 1.16 ± 0.28 months), 9 paired specimens collected before and after 2 cycles of chemotherapy (mean duration 1.23 ± 0.24 months), and 5 paired specimens collected before and after 4 or more cycles of chemotherapy (mean duration 4.97 ± 2.22 months).There was an increase in PD-L1 expression as disease progressed (accompanied by an increase in the depth of tumor invasion) in patients who did not receive any treatment , with a mean duration of 1.16 months between obtaining harvesting of the first and second tumor specimens. Representative changes in PD-L1 expression are shown in Figure No statistically significant change was found in CD8+ lymphocyte numbers levels between samples obtained before and after chemotherapy (p=0.151), including patients who received treated with 2 or 4 or more cycles chemotherapy between obtaining harvesting tumor specimens. Of the 14 patients with paired specimens who received chemotherapy, 10 showed an increase of CD8+ lymphocytes while 4 showed a decrease Figure . Of the Based on PD-L1 status and TIL density, ESCC specimens were classified into 4 groups. In this classification, PD-L1+ was defined as specimens with ≥1% expression on tumor cell membranes with ≥1+ intensity and a TIL score of 3 (high) was considered as TILs+. Type I was PD-L1+ and TILs+ (PD-L1+ with TILs driving adaptive immune resistance), type II was PD-L1– and TILs– (PD-L1–with no TILs indicating immune ignorance), type III was PD-L1+ and TILs– (PD-L1+ with no TILs indicating intrinsic induction), and type IV was PD-L1– and TILs+ (PD-L1– with TILs indicating the role of other suppressor(s) in promoting immune tolerance). The proportions of four types were 13.6% (58/428), 17.1% (73/428), 66.1% (283/428), and 3.3% (14/428), respectively . As a validation for our hypothesis that PD-L1 expression is associated with an aggressive tumor progression, we found that PD-L1 expression increased as disease progressed. In contrast, PD-L1 expression decreased between baseline and second tumor sampling obtained after 2 cycles of chemotherapy in those with satisfactory treatment responses. Interestingly, PD-L1 expression increased after 4 or more cycles of chemotherapy in patients with worse treatment responses. Recently, a study investigated changes of PD-L1 after neoadjuvant therapy in ESCC patients. Similarly, they found a decrease in PD-L1 expression after 2 cycles of neoadjuvant chemotherapy (NAC) and an increase after neoadjuvant concurrent chemoradiotherapy . HoweverWe also investigated the immunological impact of chemotherapy on CD8+ and Foxp3+ T cells in the ESCC tumor microenvironment. Among 14 patients who received chemotherapy, 10 exhibited an increase in CD8+ T cells. Increased levels of CD8+ T cells were associated with a better outcome, although the difference was not significant due to small number of available samples. This finding was also seen in several other studies –47, inclIn conclusion, PD-L1 is expressed on the cells of most (>70%) ESCC tumors, and is significantly associated with tumor aggressiveness and an enhanced risk for postoperative recurrence (shorter DFS and OS). As such, PD-L1 may represent a target for treatment of ESCC and is a potentially predictive biomarker for anti-PD-1/PD-L1 antibodies. The results presented in this study support the clinical development of PD-1/PD-L1 blockade as a treatment for ESCC. Further research investigating the effect of PD-L1/PD-1 blockade in ESCC is warranted.Archival formalin-fixed, paraffin-embedded ESCC samples were collected from patients diagnosed with primary ESCC after surgery or by endoscopic biopsy at the Affiliated 307 Hospital Cancer Center, Beijing, P. R. China between 2004 and 2014. Serial sections of 4 μm thickness were obtained from each specimen. Histopathologic data was obtained from pathology reports, and patient survival data was obtained from the patient's attending physician. The retrieval of tissue and clinical data were performed according to the regulations of the local institutional review boards and data safety laws with specific regard to ethical standards and patient confidentiality (Ethics No. ky-2016-2-12).Conventional clinicopathologic variables, including age, gender, level of differentiation, primary tumor location, tumor invasion depth (T), nodal metastasis (N), distant metastasis (M), TNM stage, and treatment received were collected from patients' attending physicians. Tumor stage was determined according to the 7th American Joint Committee on Cancer/International Union Against Cancer tumor-node-metastasis (TNM) classification system (2009).A subset of patients underwent right or left thoracotomy for curative resection by total or subtotal thoracic esophagectomy, in addition to regional lymphadenectomy; none underwent transhiatal esophagectomy. Regional lymph nodes included not only mediastinal but also perigastric nodes, thus regional lymphadenectomy represented at least a two-field lymphadenectomy. Esophageal reconstruction was performed using the stomach, colon, or jejunum. Postoperative treatment was recommended for patients with pathologically confirmed metastasis to the lymph nodes or deep tumor invasion (≥T3), and patients without severe postoperative complications. Postoperative treatment included adjuvant chemotherapy, radiotherapy or chemoradiotherapy. The first cycle of adjuvant chemotherapy was administered 4-5 weeks after surgery, and consisted of cisplatin and 5-FU or cisplatin and paclitaxel at standard doses. Adjuvant radiation therapy was administrated 4-5 weeks after surgery, and included external beam radiation with four fields of 6 MV photons to a total dose of 50.4-60 Gy using 1.8-2 Gy daily fractions in 5 weeks. Positioning of the fields and dosimetry were studied using a CT scan and 3D treatment plan. For adjuvant chemoradiotherapy, the radiation therapy program was the same as that described above, and chemotherapy regimens consisted of cisplatin or paclitaxel or 5-FU administered concurrently with radiation.Patients who were unfit for surgery due to the presence of comorbidities or other risk factors received definitive (chemo-)radiation at ≥50.4 Gy. For tumors of the cervical esophagus, high dose (60-66 Gy) definitive (chemo)-radiation therapy was prescribed. Patients with metastatic or unresectable esophageal cancer received palliative chemotherapy. Chemotherapy regimens consisted of cisplatin, taxanes (paclitaxel or docetaxel), fluorouracil  or others. All chemotherapeutic drugs were prescribed at standard doses. The clinical criteria for response were defined by RECIST 1.1.TM FLEX Target Retrieval Solutions (Dako), and then cooled to room temperature in TBST wash buffer for 5 min, then incubated with the primary antibodies. Antibody detection was visualized using DAB chromagen according to the manufacturer's instructions (Dako). Slides were counterstained with hematoxylin.Expression of PD-L1 in ESCC tumor specimens was assessed by IHC with an Autostainer Plus (Dako-Agilent Technologies), utilizing a proprietary PD-L1 antibody  at 1:1000 dilution. Human tonsil and muscle tissue served as positive and negative control tissue, respectively. Both CD8 and Foxp3 antibodies were purchased from Abcam and used at 1:200 dilution. Sections were baked for 60 min at 60°C in a dehydration oven and dewaxed and rehydrated using xylene and graded alcohol washes. Antigen retrieval and deparaffinization were carried out using the EnVisionPD-L1 expression on the membranes of tumor cells and TIL/histiocytes was scored blindly. Frequency of PD-L1+ cells and staining intensity were analyzed and every tumor specimen was scored according to the intensity of PD-L1 staining . In tumor cells, PD-L1+ by a 1% cut off was defined as specimen with ≥1% membranous expression on tumor cells with ≥1+ intensity, PD-L1+ by a 5% cut off was defined as specimens with ≥5% membranous expression on tumor cells with ≥1+ intensity, and PD-L1+ by a 25% cut off was defined as specimens with ≥25% membranous expression on tumor cells with ≥2+ intensity. Positive PD-L1 expression on lymphoid cells and stromal cells was defined as ≥1% positive cells with membranous staining at ≥1+ intensity. The degree of infiltration by TILs was evaluated from the H&E-stained specimens and scored from 0 to 3: 0 = none; 1 = rare; 2 = moderate and focal infiltration; and 3 = prominent and diffuse infiltration (high). Quantitative evaluation of CD8+ and Foxp3+ cells was carried out using 5 to 10 areas with the most abundant distribution within the tumor using an ocular grid at X200 magnification in a blinded manner. The average numbers of cells were used for statistical analysis.Disease-free survival (DFS) time was defined as the period from the date of surgery to confirmed tumor relapse date for relapsed patients or from the date of surgery to the date of last follow-up for nonrecurrent patients. Progression-free survival (PFS) time was defined as the period from the date of initiation of first-line chemotherapy to the date of tumor progress or last follow up for patients without tumor progression. All patients who did not experience the event of interest were censored at the last follow-up date.The distributions of patient and clinical characteristics between PD-L1+ and PD-L1- were compared using Chi-square test, Wilcoxon rank-sum test, or two-sample T test, when appropriate. The correlation between PD-L1 expression and other factors  was analyzed using Spearman's rank correlation. Survival curves were estimated by the Kaplan-Meier method and compared using the log-rank test. The stepwise Cox-regression model was used to identify factors significantly related to OS and DFS (PFS). P<0.05 (two-tailed) was considered to indicate statistical significance. All statistical analyses were performed using SAS 9.3.


Different anti-PD-1 and anti-PD-L1 antibodies bind different epitopes. However, whether the results from the SP142 and 22C3 immunochemistry (IHC) assays can be interchanged to determine patient eligibility for immunotherapy remains largely unknown. Histologic sections from 135 tumor samples were probed with both 22C3 and SP142 antibodies. The concordance of PD-L1 expression determined by the two assays was assessed. Additionally, we evaluated the association of PD-L1 expression detected by different assays with clinicopathological features and prognosis. In total, 105 (77.78%) of 135 samples evaluated by the 22C3-IHC platform produced the same results with the SP142-IHC platform . In addition, 69 (51.11%) of 135 samples evaluated by the SP142-IHC platform produced the same results with the 22C3-IHC platform . PD-L1 expression based on the 22C3-IHC assay was significantly correlated with smoking status, whereas that based on the SP142-IHC assay was correlated with smoking status, sex, and histology. Compared to the SP142-IHC assay, the 22C3-IHC assay usually resulted in an underestimation of PD-L1 expression in tumor cells and immune cells. Thus, the results from the two assays cannot be interchanged. Our data also suggest that the use of different reagents may account for inconsistencies in the literature regarding the association between PD-L1 expression and clinicopathological features. Tumor cells (TCs) can evade immune responses through the upregulation of PD-L1 and blockade of the PD-1/PD-L1 interaction via monoclonal antibodies and can produce a durable clinical response in patients with NSCLC. The anti-PD-1 antibody pembrolizumab and the anti-PD-L1 antibody atezolizumab are now approved by the US Food and Drug Administration (FDA) for treating patients with advanced NSCLC4. For example, the results from the phase II/III KEYNOTE-010 study showed that compared with docetaxel, pembrolizumab prolonged overall survival (OS) in previously treated advanced NSCLC patients with positive PD-L1 expression determined by immunohistochemistry (IHC) on at least 1% of TCs5. Data from patients with advanced NSCLC in a phase III study (KEYNOTE-024) demonstrated an objective response rate (ORR) with pembrolizumab in 44.8% of patients with at least 50% PD-L1-positive TCs compared with the ORR of 27.8% with chemotherapy. The median progression-free survival (PFS) was 10.3 months in the pembrolizumab group versus 6.0 months in the chemotherapy group6. Based on these data, pembrolizumab was approved for use in conjunction with a companion diagnostic, the Dako 22C3 PD-L1-IHC platform. In the phase II POPLAR study, previously treated NSCLC patients (in a second- or third-line setting) were stratified by PD-L1 expression on tumor-infiltrating immune cells (ICs) and TCs assessed by IHC with the SP142 antibody and were randomized to receive atezolizumab or docetaxel treatment. Improved efficacy with atezolizumab was observed with increasing PD-L1 expression. Additionally, in the subgroup of patients with the highest PD-L1 expression , the OS was 15.5 months and 11.1 months (hazard ratio (HR) 0.46, p = 0.070), the PFS was 7.8 months and 3.9 months (HR 0.57), and the ORR was 38% and 13% for atezolizumab and docetaxel, respectively. In contrast, patients with the lowest PD-L1 levels  did not appear to benefit from atezolizumab7.There is data indicating that the PD-L1 expression status might help guide therapy8, and various commercially available anti-PD-L1 antibodies have been used to determine PD-L1 expression in TCs and/or ICs9. This raises the following questions: Can different assays yield identical results for maximizing the therapeutic benefit and avoiding unnecessary toxicities? Is the use of different reagents one of the reasons for inconsistent results reporting the association between PD-L1 expression and clinicopathological features?Currently, clinical trials have utilized different PD-L1 assays and testing platformsTo better understand the differences in the results of two IHC assays for PD-L1, we assessed the conformance of PD-L1 expression between the 22C3-IHC and SP142-IHC assay results in surgically resected tumors from NSCLC patients. Moreover, we investigated the association between clinicopathologic characteristics and PD-L1 expression measured by the two assays.All samples were obtained with signed consent for the use of tissues under an approved protocol from the Ethical Committee of Fujian Cancer Hospital. All patients provided written informed consent for the study. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. All experiments were performed in accordance with relevant guidelines and regulations.th TNM staging system10, and lung tumor histology was recategorized according to the 2015 World Health Organization (WHO) classification system for lung tumors11.We retrospectively collected 135 samples from patients with pathologically confirmed adenocarcinoma or squamous cell carcinoma at the Fujian Cancer Hospital in China. All surgeries were conducted between January 2008 and December 2010. These patients were not treated with PD-L1 axis therapies or targeted therapy. The pathological TNM stage was reassigned according to the 8Each tumor sample was pathologically examined using six 5-µm serial sections that were sent to two institutions for analysis as follows: three sections were sent to the University of Hong Kong for analysis with a 22C3-IHC on an Autostainer Link 48 with the murine 22C3 anti-human PD-L1 antibody  according to the manufacturer’s protocol – one section was stained with hematoxylin and eosin (HE); another section was stained for PD-L1; and a third section was used as the negative control. The other three sections were sent to Fujian Cancer Hospital for IHC analysis using the SP142 antibody on the Ventana Benchmark platform. Two board-certified pathologists (Chao Li and W Zhu) jointly evaluated all stained slides for PD-L1 membrane staining. All areas in each tissue section were observed for the appropriate evaluation of the expression of PD-L1 on TCs as an estimate of the percentage of TCs exhibiting partial or complete membranous staining and on ICs as an estimate of the percentage of the tumor area occupied by PD-L1-expressing ICs. Determination of PD-L1 expression was performed without considering any cut-off value, and the staining intensity was not part of this evaluation.Scoring was performed based on the standards for the 22C3-IHC and SP142-IHC assays. For the 22C3-IHC assay, PD-L1 expression was determined by using a Tumor Proportion Score (TPS), which is the percentage of viable TCs showing partial or complete membrane staining. PD-L1 expression was scored at three levels, namely, TPS < 1%, TPS 1 to 49% and TPS ≥ 50%. For the SP142-IHC assay, baseline PD-L1 expression was scored using the number of PD-L1 TCs as a percentage of total TCs, as follows: TC3 ≥ 50%, TC2 ≥ 5% and < 50%, TC1 ≥ 1% and < 5%, and TC0 < 1%. Additionally, tumor-infiltrating ICs were expressed as the percentage of the tumor area as follows: IC3 ≥ 10%, IC2 ≥ 5% and < 10%, IC1 ≥ 1% and < 5%, and IC0 < 1%. To compare the overall agreement of the two assays, we interchanged various cut-off values on each set of slides stained by the two assays.2 test. The OS of patients with PD-L1-positive and PD-L1-negative tumors was estimated using the Kaplan–Meier method, and differences were compared using the log-rank test. All tests were two-sided. Statistical significance was set at p < 0.05. Statistical analyses were performed using SPSS 21.0 software.The weighted kappa (κ) coefficient and McNemar-Bowker test were used to evaluate the agreement and inconsistency between the results using each assay. A κ coefficient of 0.75 or less indicated poor to fair agreement, and a value greater than 0.75 indicated almost perfect agreement. Additionally, clinicopathological characteristics of PD-L1-positive tumors according to the interpretation of the 22C3-IHC or the SP142-IHC assay standards were compared with those of PD-L1-negative tumors using the χA total of 135 patients were eligible for the study, and their characteristics are summarized in Table ). One sample demonstrated 1% PD-L1-positive TCs based on the SP142-IHC assay, but it was negative based on the 22C3-IHC assay. The variability in the staining pattern of ICs between samples was greater than that for TCs . In a total of 62 discordant samples, compared to the 22C3-IHC assay, the SP142-IHC assay resulted in the underestimation and overestimation of PD-L1 expression in ICs in 53 (39.2%) and 9 (6.7%) samples, respectively (p < 0.001). With the adoption of the 22C3-IHC assay scoring algorithm for TCs, 20 (14.8%), 25 (18.5%), and 90 (66.7%) of the 135 samples were classified into groups that were strongly positive, weakly positive, and negative for PD-L1, respectively. Moreover, 105 (77.78%) of 135 samples produced the same results with the SP142-IHC reagent assay . The PD-L1 levels on TCs as detected by the 22C3-IHC assay were underestimated by the SP142-IHC assay in 29 samples. Only one sample with negative PD-L1 expression based on the 22C3-IHC assay was misclassified as having weak positive PD-L1 expression based on the SP142-IHC assay  . In 60 (91.0%) of 66 discordant samples, PD-L1 status determined with the SP142-IHC assay was overestimated in the 22C3-IHC assay. In contrast, the PD-L1 status was underestimated by 22C3-IHC assay in six cases  , 14 (10.4%), 37 (27.4%) and 74 (54.8%) of 135 samples evaluated using the SP142-IHC the assay were classified into groups that were strongly positive, moderately positive, weakly positive, and negative for PD-L1 expression, respectively. However, only 69 (51.11%) of 135 samples produced the same results with the 22C3-IHC assay .We determined the association of clinicopathological characteristics with PD-L1 expression based on each assay. PD-L1 expression based on the 22C3-IHC assay was significantly correlated with smoking status. However, the results from the SP142-IHC assay were different, indicating that PD-L1 expression was significantly correlated not only with smoking status but also with sex and histology  indicated that the assay using the SP142 antibody differed from the others, as it detected significantly lower PD-L1 expression in TCs and ICs. The limitation of the study is that the authors used the SP142 antibody on a Ventana platform not identical to the platform now approved by the FDA14. In addition, the analysis was based on the average value as the gold standard to determine the sensitivity and specificity of various reagents. We used the standard for the 22C3-IHC as the gold standard with a 50% and 1% cut-off values and evaluated the sensitivity and specificity of PD-L1 labeling. We learned that the sensitivity with the SP142-IHC assay is poor but that the specificity is better. Thus, if the SP142-IHC assay results are negative, the 22C3-IHC assay results will also be negative. Because the details of antibodies are proprietary information, we can only speculate that the poor concordance of the results with the two antibodies may be due to different antibody affinities, limited specificity, or distinct target epitopes.The Blueprint study was designed to evaluate the agreement of results from four PD-L1 IHC assays, namely, Dako 28-8, Dako 22C3, 29 Ventana SP263, and Ventana SP142. Thirty-eight non-interventional NSCLC cases were enrolled into the study, and each tumor was probed for PD-L1 using all four assays then scored by three pathologists. For TC staining, Dako 22C3, Dako 28-8 and Ventana SP263 assays demonstrated similar analytical performance, whereas Ventana SP142-IHC assay consistently labeled fewer TCs. Similar to our results, the SP142-IHC assay was associated with significantly lower levels of PD-L1 detection than was the 22C3-IHC assayIt is important to note that we employed standardized platforms and reagents that are now approved by the FDA for this study and that this is currently the largest clinical study assessing the concordance between SP142-IHC and 22C3-IHC results in NSCLC.16. The reason for these discrepancies may be multifactorial17. Differences in distributions of histologic subtypes, antibodies and cut-off values may account for some of these discrepancies18. In our study, PD-L1 expression based on the 22C3-IHC assay was significantly correlated with smoking status. However, these results were different those obtained using the SP142-IHC assay, which indicated correlations of PD-L1 expression with sex, smoking status, and histology. Additionally, PD-L1 expression based on either IHC systems was not associated with patient prognosis. Our data represent the most direct evidence that demonstrates differences between two reagents and may partly explain the conflicting results regarding the correlation of PD-L1 expression and clinicopathological characteristics in other studies.The prognostic impact of PD-L1 protein expression and its association with clinicopathological features have not been conclusively elucidatedOur study has several limitations. First, all samples were obtained from patients who had not undergone PD1 or PD-L1 therapy. We did have access to therapeutic outcome data, and we could only evaluate the assay results by themselves without clinically meaningful comparisons of PD-L1 assays. Second, the vast majority of specimens were archived samples and not biopsies; these samples may not reflect the real situation in the clinic.In conclusion, we identified that the use of different reagents for testing PD-L1 expression can be misleading; the two reagents cannot be interchanged with each other. Our findings also suggest that different reagents may account for inconsistent associations between the expression of PD-L1 and clinicopathological features.


Visuospatial processing requires wide distribution or narrow focusing of attention to certain regions in space. This mechanism is described by the zoom lens model and predicts an inverse correlation between the efficiency of processing and the size of the attentional scope. Little is known, however, about the exact timing of the effects of attentional scaling on visual searching and whether or not additional processing phases are involved in this process.Electroencephalographic recordings were made while participants performed a visual search task under different attentional scaling conditions. Two concentric circles of different sizes, presented to the participants at the center of a screen modulated the attentional scopes, and search arrays were distributed in the space areas indicated by these concentric circles. To ensure consistent eccentricity of the search arrays across different conditions, we limited our studies to the neural responses evoked by the search arrays distributed in the overlapping region of different attentional scopes.Consistent with the prediction of the zoom lens model, our behavioral data showed that reaction times for target discrimination of search arrays decreased and the associated error rates also significantly decreased, with narrowing the attentional scope. Results of the event-related potential analysis showed that the target-elicited amplitude of lateral occipital N1, rather than posterior P1, which reflects the earliest visuospatial attentional processing, was sensitive to changes in the scaling of visuospatial attention, indicating that the modulation of the effect of changes in the spatial scale of attention on visual processing occurred after the delay period of P1. The N1 generator exhibited higher activity as the attentional scope narrowed, reflecting more intensive processing resources within the attentional focus. In contrast to N1, the amplitude of N2pc increased with the expansion of the attentional focus, suggesting that observers might further redistribute attentional resources according to the increased task difficulty.These findings provide electrophysiological evidence that the neural activity of the N1 generator is the earliest marker of the zoom lens effect of visual spatial attention. Furthermore, evidence from N2pc shows that there is also a redistribution of attentional resources after the action of the zoom lens mechanism, which allows for better perform of the search task in the context of low attentional resolution. On the basis of the timing of P1, N1, and N2pc, our findings provide compelling evidence that visuospatial attention processing in the zoom lens paradigm involves multi-stage dynamic processing. One of the most notable characteristics of the human attentional system is the limited amount of resources available to it. When performing visual search tasks, the amount of visual information pertinent to a given search situation often exceeds the maximal amount of information that can be processed by the attentional system at any given time. In this case, top-down attention control plays a key role in rationalizing the use of such limited resources for attention processing. Through this mechanism, an observer can choose to process a small number of spatial stimuli while ignoring information associated with other locations. This mechanism was described by the spotlight model, which assumes that attention can be shone over the attended area as a spotlight . InformaIn realistic situations, however, one may not be able to determine the location of a target, even when the target’s features are known in advance. At this point, a better strategy would be to adjust the scope of attention based on the current situation, aiming to include the possible location of the target. This attentional control mechanism is well captured by the zoom lens model . The zooBy recording and analyzing event-related potentials (ERPs) in high temporal resolution, In the current study, we further evaluated the multiple processing stages associated with the zoom lens mechanism. Observers were asked to search for a target in searching scopes that had different sizes. Different searching scopes appeared alternately. In this setting, some search arrays would be spread over the overlapping region of two searching scopes. We were only interested in these search arrays, since they belong to different attentional scopes but have perfectly matched physical properties (same eccentricity and number of distractors). Under this scenario, target-elicited neural activity could only be modulated by the different sizes of the attentional scopes, rather than by the physical properties of the search arrays.In addition, for the first time, we also examined the redistribution of the attentional resources driven by changes in the attentional scopes. The zoom lens model suggests that widening the attentional scope can reduce the density of processing resources in this region, and therefore can increase the difficulty of search tasks . HoweverData collection conformed to the Declaration of Helsinki and the ethics committee of Zhongshan hospital affiliated to Dalian university approved the research protocol . Verbal informed consent was obtained from all the study participants as agreed by the review board.Nineteen paid participants from the Liaoning Normal University community participated in this experiment. One participant was excluded from the analyses owing to poor performance (32% errors). Another two were excluded because of excessive alpha activity. The remaining sixteen participants  reported normal or corrected-to-normal vision, and had no known neurological or visual disorders.Stimuli were presented on an LCD monitor  at a viewing distance of 70 cm. In the experiment, a gray central fixation cross (0.26°) always appeared at the center of the monitor . The search array was a circular array of eight squares , arranged equidistantly. There were two different color squares, green  and blue . In each trial, the color of one square differed from that of the other squares, serving as a target, and the remaining seven squares served as distractors. The color of the target square was consistent within each block and was balanced within the observer. The target square was shown randomly at one of three possible locations on each side of the visual field . Each sqStimuli and procedure were controlled via E-Prime 2.0 routines . At the beginning of each trial, a central fixation cross was presented for 500 ms, followed by the concentric circles, which were displayed for 500–900 ms. Then, a search array was presented for 200 ms. Observers were instructed to respond as quickly and accurately as possible to press the number keys to the right of the keyboard using their right thumb and index finger. When the line within the target square was horizontal, the observers were asked to press the number key “1,” while when a vertical line appeared, the observers were asked to press the number key “2.” The concentric circles disappeared after the observers reacted, or disappeared automatically after 2,000 ms, and were replaced by a solid background, preparing the observers for the next trial.For each size of the attentional scope, search arrays were randomly distributed over one of the four virtual rings within a limited attentional scope (a detailed description was provided in the previous section). The overlapping region of the two attentional scopes was a virtual ring with a radius of 3.27°. In the current study, we were primarily interested in the search arrays distributed over this overlapping region. These search arrays had exactly the same physical properties (the eccentricity and the number of distractors), while being within the sizes of both attentional scopes. Search arrays that appeared outside of the overlapping region were not included in our analysis owing to their unmatched eccentricities.The entire experiment contained a total of 24 experimental blocks, with a total of 1,440 test trials. Each block contained 60 test trials, followed by a minimal break of 30 s. Within each block, 24 search arrays were distributed in the overlapping region, and 36 search arrays were distributed in other non-overlapping region. Therefore, for each size of the attentional scope, the number of search arrays distributed in the overlapping region was 288. Each size of the attentional scope lasted six continuous blocks and the order was well-balanced. To make the observers familiar with the task requirements, one training block with 12 trials was offered at the beginning of the experiment. The observers were only allowed to enter the formal experiment when their correct rate exceeded 75%. On the search task, the observers were asked to maintain a central fixation, while minimizing their head and eye movements.The ANT Neuro EEGO system was used to record electroencephalographic (EEG) signals using an array of 64 electrodes mounted using a cap with 10/20 montage. Horizontal electrooculograms, which recorded bipolar signals from the outer canthi of the eyes, were used to measure horizontal eye movements. Vertical electrooculogram, which recorded from the FPz site, was used to detect eye blinking. The CPz site was used as the online reference. Electrode impedances were kept below 5 kV with a sampling rate at 500 Hz for on-line recording.www.neuro.uestc.edu.cn/rest , while the statistical power was executed by G-Power 3.1 . The accuracy of the data also suggested that observers behaved worse for the larger attentional scope (90% ± 2%) compared with the smaller scope scenario (93% ± 1%), (t (15/2) = −2.77, p = 0.014, Cohen’s d = 0.70). These results were in line with the predictions of the zoom lens model, indicating that as the scope of attention narrowed, the observers were able to identify the feature of the target faster and more accurately.Trials with mean reaction time (RT) below or above two standard deviations were excluded . Analysis of RT data revealed faster responses to targets for the smaller attentional scope (424.06 ± 59.15 ms) compared with the larger one (444.82 ± 77.19 ms), (t (15/2) = 2.20, P1. p = 0.681) and the electrode sites  = 1.70, p = 0.21) were not significant. Besides, the interaction between the electrode sites and the attentional scope was also not significant  = 0.43, p = 0.54).N1. As shown in p = 0.01, ηp2 = 0.36). However, the main effect of the electrode sites  = 0.69, p = 0.43) and the interaction between the electrode sites and the attentional scope  = 3.20, p = 0.09) were not significant. Further examination of the main effects of the attentional scope showed that the targets for the smaller attentional scope (−8.43 ± 2.26 μV) elicited a more negative N1 compared with the larger attentional scope (−7.95 ± 2.48 μV), (t (15) = −2.921, p = 0.01, Cohen’s d = 0.73), indicating that as the size of the attentional scope decreased, the neural generators of P1 became more active.N2pc. Grand-average ERPs at the electrode sites PO7/PO8 as well as N2pc difference waves are shown in p = 0.005, Cohen’s d = 0.82).The present study investigated the different stages of attentional processing in the zoom lens paradigm. More specifically, we attempted to examine the effects of changes in the attentional scope on early visual evoked potentials (P1/N1) and N2pc, reflecting the redistribution of attentional resources. In agreement with previous studies , during The P1 component originating in the extrastriate cortex represents the earliest stage in the visual processing modulated by spatial attention. In the current study, we found that the amplitude of P1 did not change with the scaling of spatial attention, suggesting that the scale effect of visual spatial attention does not modulate the exogenous-like P1 component. Unlike the current results, previous studies have found that the amplitude of P1 was modulated by different sizes of attentional scopes. Different from P1, we found that the lateral occipital component N1 with a temporal lag could be sensitive to changes in the size of the attentional scope. Specifically, the amplitude of N1 increased with decreasing the size of the attentional scope. This is consistent with the findings of According to the zoom lens model, it is unlikely that the scale effect of visual spatial attention is driven by bottom-up factors; rather, it should be the result of a top-down modulation. Some recent studies have explored the involvement of higher brain regions in flexible modulation of the scale effect of spatial attention. In addition, we also explored the modulation of the late N2pc components by the scale effect of the attentional scope. We found that, contrary to the earlier occurring N1, the amplitude of N2pc increased with increasing the scale of spatial attention. N2pc is generally used as a neurological indicator of attentional spatial selection , and itsIt has been demonstrated that the amplitude of N2pc is also sensitive to the redistribution of attentional resources that is modulated by the task difficulty . In the It was shown previously that top-down modulation associated with the right frontal parietal network forms the neural basis of the zoom lens mechanism. In light of the above discussion, this modulation was likely to occur before the redistribution of attentional resources reflected by N2pc, approximately within the N1 delay period after the search arrays were presented. Based on this evidence, we speculated that the scaling effect of spatial attention during visual search may operate in at least three different stages. The first stage is the earliest visual spatial attention processing. At this stage, search arrays for different sizes of the attentional scope are firstly perceived with no difference. In the subsequent stages, the spatial gradient of the attention system is modulated by the zoom lens mechanism, as indicated by changes in the activity of the N1 generator. Before the final behavioral response, there is a redistribution of attentional resources. This process is reflected in the modulation of N2pc and is probably driven by reduced attentional resolution as the size of the attentional scope increases.In summary, utilizing high temporal-resolution ERP technology, we provided solid evidence to support the view that the modulation of the zoom lens effect on visual processing during search tasks involves multiple dynamic processing stages. Our results suggest that lateral occipital N1 might be the neurophysiological marker of the attentional scaling effect during visual search. In addition, there was also a redistribution of attentional resources after the action of the zoom lens mechanism, indicating that top-down control mechanisms are involved in the post-attentional scaling process.10.7717/peerj.4538/supp-1Supplemental Information 1Click here for additional data file.


Graphene nanoribbons (GNRs) are ultra-narrow strips of graphene that have the potential to be used in high-performance graphene-based semiconductor electronics. However, controlled growth of GNRs on dielectric substrates remains a challenge. Here, we report the successful growth of GNRs directly on hexagonal boron nitride substrates with smooth edges and controllable widths using chemical vapour deposition. The approach is based on a type of template growth that allows for the in-plane epitaxy of mono-layered GNRs in nano-trenches on hexagonal boron nitride with edges following a zigzag direction. The embedded GNR channels show excellent electronic properties, even at room temperature. Such in-plane hetero-integration of GNRs, which is compatible with integrated circuit processing, creates a gapped channel with a width of a few benzene rings, enabling the development of digital integrated circuitry based on GNRs. et al. have used a hexagonal boron nitride template to grow narrow, integrated graphene nanoribbons with small bandgaps.Graphene nanoribbons are promising candidates for 2D material electrical interconnects; however, the top-down fabrication of nanoribbons has remained a challenge. Here, Chen  Ideal graphene nanoribbons (GNRs) have been shown to exhibit extreme chirality dependence as metals or semiconductors24591113144 at room temperature and carrier mobility values of ∼750 cm2 V−1 s−1. For the narrowest GNRs, the band gaps extracted from the electrical transport data are >0.4 eV.Here, we demonstrate the successful growth of GNRs directly on h-BN substrates with smooth edges and controllable widths via templated growth using chemical vapour deposition (CVD). Transistors made with sub-10-nm GNRs demonstrate large on–off ratios of more than 10The nano-trenches exhibited predominant separation angles of 60° relative to each other with no obvious correlation to the direction of the gas flow, indicating anisotropic etching along the different crystallographic directions . SeparatThe existence of the giant Moiré pattern indicates that the graphene was highly crystalline and precisely aligned with the h-BN underneath. It was noticed that the Moiré pattern appeared to be stretched along the GNR, while it appeared relaxed laterally. This trend differs from regular hexagons with a periodicity of ∼14 nm, which have always been observed with well-aligned graphene domains on h-BN1617−1, which was attributed to the Raman-active LO phonon of h-BN (ref. −1) as well as a single-Lorentzian-shaped 2D-band , which were expected for monolayer graphene. Compared to that of the pristine graphene domain−1 in the G-band position indicates the existence of a tensile strain of ∼0.6% in the GNR20−1. At 1,617.5 cm−1 (D′-band), a tiny shoulder appears on the right side of the G band. Both the Raman D- and D′-bands may have primarily originated from the lattice distortions and disorder at the GNR/h-BN boundary. The 2D-band exhibits a characteristic of I(2D)/I(G)>1, also indicating the single-layer nature of the GNR, where I(2D) and I(G) represent the intensity values of the 2D and G-bands, respectively. In addition, the observed broadening of the full width at half-maximum for the Raman G- and 2D-bands may have been due to strain variations at the nanometre-scaleRaman measurements were carried out to investigate the structural and electronic properties of the nanoribbons. -BN ref. . For the-BN ref. . The Ram2 ; then, it gradually switched on, exhibiting a relatively high G=0.7 × e2 × h−1 at room temperature for Vgate=−40 V. The scattering mean free path was estimated to be ∼50 nm. The mobility of the GNR was estimated to be ∼765 cm2 V−1 s−1 at 300 K. Accurately measuring the off-state resistance of the narrowest GNR FET became challenging due to noise and set-up limitations. It was found that the conductance near the on-state exhibited an exponential relationship with inverse temperature from 200 to 300 K. This result indicates that a Schottky barrier dominated the conductance of the GNR FET because of the high work functions of Pd and Ni (refs Eg) of the narrow GNRs by fitting the conductance near the on-state kB is Boltzmann's constant and T is temperature. The extracted band gap value for the 5-nm GNR was 489.4±19.0 meV ∼α/(w+β) (w is in units of nm), where parameter α≈1.99 eV nm and parameter β=−1.28 nm.To investigate the electrical properties of the GNRs, field effect transistor (FET) devices were produced using p-type doped Si/SiOodel see , the valodel see . The fit Ni refs , 23. We e–e interactions. It is noted that our narrow GNRs with similar widths exhibited relatively larger band gaps than those reported in previous literature9In the GNR samples, the band gap exhibited a strong dependence on the width of the ribbons. In particular, the sub-10-nm GNRs exhibited an electronic band gap of ∼0.5 eV. It is important to understand the possible origins of the band gap. Normally, the electronic structure of GNRs is strongly dependent on the edge of the GNRs. For this study, it was challenging to characterize the exact geometry of the narrow ribbons obtained . However, the GNRs were generally parallel to the ZZ pattern of the h-BN and free of detectable edge roughness. Therefore, it is likely that most segments of the GNR edges followed a ZZ pattern. Previous studies in the literature predicted that GNRs with pristine ZZ edges would always be metallic because of their peculiar flat-band edge states localized near the Fermi level24264. Our results demonstrate that it is possible to resolve the fundamental gapless limitation of graphene, paving the way for the realization of graphene-based digital electronics that can operate at room temperature.The applicability of graphene for future digital devices is often questioned due to its intrinsic gapless nature. Nanoribbons offer a potential solution, but both the width and edges must be precisely controlled. In-plane graphene–BN hetero-structured films have been reported, however, only on metal surfaces313233342 flow (850:150 sccm) to remove the tape residue. After the heat cleaning process, a solution of NiCl2:H2O at a concentration of 0.01 mg ml−1 was spun at 1,800 r.p.m. for 100 s onto the substrate surface and then baked for 10 min at 80 °C on a hot plate to evaporate the solvent. This NiCl2-treated sample was then submitted to a two-step process under an Ar:H2 flow (850:150 sccm): annealing at 500 °C for 20 min, which resulted in Ni nano-particle formation, and etching at 1,200 °C for 60–180 min. The optimal etching pressure was found to be ∼150 Pa.To prepare h-BN samples for nano-particle-assisted etching, multiple cleaning steps were adapted to ensure the reliable production of the cuts. Before depositing the h-BN flake, quartz substrates were cleaned with acetone and isopropyl alcohol and then annealed at 600 °C for 30 min to remove organic contaminants. Subsequently, the h-BN was mechanically exfoliated using semiconductor-grade tape. Next, the h-BN samples were heated in a quartz tube at 500 °C for 15 min under an Ar:H2H2 flow and a mixture of silane and Ar (5% mole ratio of silane to Ar) were introduced into the system for GNR growth. The ratio of C2H2 to silane was ∼1:1. The pressure was maintained at 5 Pa during the growth process, and the growth time was ∼5 min. After growth, both the C2H2 and silane/Ar flow were turned off, and the system was cooled to room temperature with flowing Ar. The samples of GNRs on h-BN grown on a quartz surface were moved to a highly p-type doped silicon wafer with a 300-nm-thick SiO2 capping layer for electrical transport studies.Before graphene growth, etched h-BN flakes were cleaned separately in HCl solution, DI water and acetone. The substrate with the h-BN flakes was then loaded into a growth chamber. GNR growth was carried out in a low-pressure CVD furnace at 1,280 °C under an Ar flow of 10 sccm, corresponding to 15 Pa; the samples were then annealed for 5 min. Next, the Ar flow was turned off, and a Ck of the cantilever tips was in the range of 0.05–0.5 N m−1. The scan rate was set to 10–60 Hz to minimize any noise from thermal drift. The integral gain and set point were adjusted to be as low as possible during the measurement. Several hours of pre-scanning were carried out to warm up the scanner to obtain good stability during imaging. To ensure a highly accurate atomic-resolution image, scanners with a travel range of <10 μm along the x and y directions were used. Calibration at atomic resolution was performed with newly cleaved highly ordered pyrolytic graphite before measurement.Cleaned samples were characterized using one AFM , while atomic-resolution images were taken by another AFM  under ambient conditions. AFM measurements were acquired in contact mode to obtain height and friction images. SNL-10 AFM tips from Bruker, which possess a nominal tip radius of <10 nm, were used in all measurements. The use of friction contrast was necessary because this mode gives clear information about the super-structure and atomic lattice. For atomic-resolution scanning, the force constant Raman spectra were obtained with a commercially available confocal Raman instrument: model Alpha 300R from WITec. The Raman data were recorded using a laser wavelength of 488 nm 2. Next, the devices were annealed in a hydrogen atmosphere at 200 °C for 3 h to remove the resist residues and to reduce the contact resistance between the GNRs and metal electrodes before electrical measurements because the thickness of the h-BN flakes on the silicon wafer was ∼15 nm. Electrical transport measurements were carried out using a physical property measurement system  via a Keithley 4200 semiconductor characterization system.GNR devices were created by a standard electron beam lithographic technique with Ni or Pd as the source and drain contacts on p-doped silicon wafers with 300-nm-thick SiOThe authors declare that the main data supporting the findings of this study are available within the article and its How to cite this article: Chen, L. et al. Oriented graphene nanoribbons embedded in hexagonal boron nitride trenches. Nat. Commun.8, 14703 doi: 10.1038/ncomms14703 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Table, Supplementary Notes and Supplementary References.


Programmed cell death 1 (PD-1) and programmed cell death-ligand 1(PD-L1) inhibitors have captured our attention as new therapeutic options for several tumor types. Nonetheless, the differences in efficacy between PD-1/PD-L1 inhibitors and conventional treatments (chemotherapy or targeted therapy) in pretreated advanced cancer patients remain unclear.A systematic literature search was conducted to identify phase III randomized controlled trials (RCTs)-based investigations of PD-1/PD-L1 inhibitors (atezolizumab) against pretreated advanced cancer. We evaluated these trials for inclusion, assessed each study’s risk of bias and selected relevant data for analysis.P < 0.001) and median month difference . The progression-free survival HRs favored PD-1/PD-L1 inhibitors over conventional treatment , whereas median month difference was just the opposite .The eligibility criteria were met by 5,093 patients from 8 phase III RCTs. PD-1/PD-L1 inhibitors significantly extended overall survival relative to the conventional treatment, expressed as hazard ratio [HR] , were associated with improvement in overall survival (2.83 months) but not progression-free survival. These findings will be important in considering PD-1/PD-L1 inhibitors in the treatment of pretreated advanced cancer and have implications for future study design. Cancer, especially advanced cancer, is still a pressing worldwide health issue . AlthougImmune checkpoint inhibitors, especially programmed cell death 1(PD-1) and programmed cell death-ligand 1(PD-L1) have captured our attention as new therapeutic options for patients with selected advanced cancer for which no effective treatment yet existed . PD-1 reUntil now, several randomized controlled trials (RCTs), which compared the efficacies of PD-1/PD-L1 inhibitors to conventional treatments (chemotherapy or targeted therapy) against various pretreated advanced cancer, have been conducted –23. HoweClinicalTrials.gov databases was performed from their inceptions to February 20, 2017. The search was conducted using the following keywords: “nivolumab,” “pembrolizumab” or “atezolizumab.” We also searched the American Society of Clinical Oncology conference proceedings, the European Society for Medical Oncology conference proceedings and Google Scholar to ensure that no eligible studies were overlooked. After the titles and abstracts were screened by two independent reviewers (H. H and Q. Z), the full texts from potentially relevant studies were retrieved to confirm the eligibility criteria. We also reviewed reference lists of original articles, review articles, and relevant books. When a duplicate publication of the same trial was found, the study with the most complete, recent, and updated report was included. All procedures were performed per the preferred reporting items for systematic reviews and meta-analyses statement , which estimated the percentage of total variation across studies due to heterogeneity rather than chance, with I2 >50% indicating significant heterogeneity. The sources for heterogeneity were explored by conducting predefined subgroup analyses and meta-regression (in cases where the percentage of never smokers was given): treatment type (PD-1 inhibitors vs PD-L1 inhibitors), tumor type (NSCLC vs others), and PD-L1-positive status (unselected vs selected). A two-tailed P value of less than 0.05 was considered statistically significant. Funnel plots were performed to assess the potential publication bias. The Duval and Tweedie trim-and-fill test and the classic fail-safe N test were applied to define the extent of publication bias [A general variance-based method was used to estimate the summary HR and their 95% CIs, which were calculated to assess the benefit with respect to OS and PFS. When median survival times or median months of PFS were available, median differences were generated and combined. Random effects models were used to compute all the outcome measures investigated , 31 and ion bias . When puion bias  and Eggeion bias  were useAs shown in Figure 7 trials used PD-1 inhibitors including 5 trials with nivolumab –20, 2 wiAll trials recruited patients with a performance status of 0 through 2 in the Eastern Cooperative Oncology Group (ECOG), apart from 1 trial , which iAll trials used commonly recommended second- or later-line therapeutic regimen as the control group  and median month difference . Moreover, there was no publication bias for OS expressed as HR and median month difference , whereas PD-1/PD-L1 inhibitors suggested shorter median PFS expressed in months  . However, a significant statistical heterogeneity was noted in the analysis of median month difference in PFS (I2 = 65.3%). No or minimal publication bias for the outcome PFS expressed as HR and median month difference was noted  Figure . No signas noted . The DuvP values for subgroup difference: type of treatment, 0.35; and type of tumor, 0.58, respectively). However, the subgroup result of PD-L1-positive status appeared to be discordant: trials recruiting selected PD-L1-positive patients showed no significant difference in median month difference of PFS between PD-1/PD-L1 inhibitors with control group . On the other hand, in trials recruiting patients with unselected PD-L1 expression, PD-1/PD-L1 inhibitors demonstrated a shorter median PFS  compared with control treatment. PD-L1-positive status can partly explain the heterogeneity between the trials, but the subgroup difference did not yet reach the level of statistical significance (P = 0.11). In a meta-regression analysis regarding smoking status, there was no correlation between the percentages of never smokers and the standardized mean difference for PFS (P = 0.29); Subgroup analyses were carried out to explore the heterogeneity in the terms of median month difference for PFS because the significant heterogeneity existed within included studies Table . There wThe result of risk of bias assessment is provided in Figure We performed the meta-analysis with a focus on investigating the efficacy difference between PD-1/PD-L1 inhibitors and conventional therapies in pretreated advanced cancer patients. This study included all published 8 well-conducted, good-quality, phase 3 RCTs incorporating 5,093 patients with pretreated advanced cancer. Considering PD-1/PD-L1 inhibitors blockade PD-1/PD-L1 pathways by activating an immune response directed against cancer, it exhibit antitumor activity in the form of a cytostatic, rather than a cytotoxic effect . Thus, PPresent pooled analysis suggested that PD-1/PD-L1 inhibitors prolong OS (2.83 months) compared with conventional treatments, however, the corresponding reduction of median PFS with PD-1/PD-L1 inhibitors were 0.69 months. The apparent discordance between PFS and OS have been commonly noted , 22, 23,PD-1 inhibitors block the PD-1 interaction with both PD-L1 and PD-L2. However, PD-L1 inhibitors block interactions between PD-L1 and both PD-1 and CD80 whereas the latter interaction may down-modulate T-cell responses . MoreoveEfforts to find the appropriate biomarker to identify which patients would benefit most from PD-1/PD-L1 inhibitors is a crucial personalized-medicine approach to increase response rates and many different predictive biomarkers are evaluated. Of these, the relationship between PD-L1 expression on tumor cells and outcome to PD-1/PD-L1 inhibitors has been extensively investigated but are still inconclusive, which could partly be due to the function of complex interactions between tumors and the immune system , 42, 49.PD-1/PD-L1 inhibitors have shown promising activity against many tumor types, whereas only a subset of patients can respond to such mono-therapy , 10. TheTwo meta-analyses have tried to explore the efficacy of PD-1/PD-L1 inhibitors. One meta-analysis included only 3 trials of PD-1/PD-L1 inhibitors  and the Inclusion of well-conducted, good quality, phase 3 RCTs are the strengths of our study. The lack of heterogeneity in the assessment of OS represents a well selection of studies. Furthermore, subgroup analyses were all pre-planned and limited to a minimum. Inclusion of large number of patients and recent studies are other strengths. However, our study encountered several limitations relevant to this meta-analysis. First, the analysis was based on extracted data and not on individual patient data. Therefore, the interpretations of the results needed to be carefully performed, especially for the specific relationships in the subgroup analysis. Further studies using the individual patient data will be necessary to assess outcomes, such as the primary outcomes and subgroup interactions. Second, this meta-analysis involved the different treatment schedules between the trials. In two trials, patients received pembrolizumab per the following schedules: 2 mg/kg, 10 mg/kg  or 200 mIn conclusion, our meta-analysis identified that in selected pretreated advanced cancer patients, OS was significantly longer (2.83 months) with PD-1/PD-L1 inhibitors than with conventional treatments, whereas median PFS was just the opposite (−0.69 months). These findings provide important and clinically useful information with respect to PD-1/PD-L1 inhibitors as novel treatment strategies to improve patient overall survival, as well as could be useful for the design of future studies.


Heightened attentional bias to emotional information is one of the main characteristics of disorders related to emotion dysregulation such as anxiety, depression, and substance abuse. Although reappraisal, an emotion regulation strategy, is known to effectively modulate subjective experience of emotions, it remains unknown whether reappraisal can alter attentional biases to emotional information. In the current research, we investigated the influence of instruction-induced state reappraisal (Study 1) and trait reappraisal (Study 2) on attentional biases to happy and angry faces. In Study 1, healthy young women were recruited and randomly assigned to one of the three groups: up-, down-, and no-regulation. Participants were instructed to reappraise their emotions to increase and decrease emotional experience while viewing an emotionally negative film clip. Attentional bias was assessed with a dot-probe task with pictures of angry and happy facial expressions. In Study 2, a separate group of healthy young men and women participated. Participants’ trait reappraisal and suppression as well as state and trait anxiety were assessed. A dot-probe task was completed by all participants. Statistical tests in Study 1 revealed that participants who reappraised to decrease negative emotions while viewing an emotionally negative film clip had reduced attentional bias to subsequently presented angry faces compared to participants who reappraised to increase negative emotions. Multiple regression analyses in Study 2 revealed that trait reappraisal predicted slower orienting toward angry faces, whereas state anxiety predicted slower disengagement from angry faces. Interestingly, trait suppression predicted slower disengagement from happy faces. Taken together, these results suggest that both instruction-induced state reappraisal and trait reappraisal are linked to reduced attentional bias to negative information and contribute to better understanding of how everyday emotion regulation styles contribute to attentional processing of emotional information. Individuals have a biased tendency to attend more to emotionally meaningful versus ordinary information . AlthougIn fact, dysfunctional attentional biases to emotional information have been linked to various emotion dysregulation disorders including anxiety, depression, and substance abuse. For example, patients with anxiety show biased attention to threat-related information, and substance abusers also attentional biases to substance-related information . AttentiGiven the critical association between emotion dysregulation and increased attentional bias , one mayIn addition, the momentary effect of reappraisal may accumulate over time if reappraisal is habitually used for daily emotional events, and this could produce a systematic difference in attentional responses to emotional information. Indeed, individual differences in trait-level reappraisal have been associated with daily positive and negative emotional experiences . Neural In the current research, we investigated the effects of momentary, instruction-induced reappraisal and habitual reappraisal on attentional biases in two studies. Attentional bias was assessed with a dot-probe task , which iIn Study 1, we investigated the effect of instruction-induced reappraisal on attentional bias to subsequently presented faces. Healthy young women were recruited and randomly assigned to one of the three reappraisal groups: up-regulation, down-regulation, and no-regulation. First, emotional states were induced by having participants watch a film clip showing a female victim of physical violence. While watching the film, participants were instructed to use reappraisal to increase or decrease emotions, or did not reappraise their emotions. This was followed by a dot-probe task with angry, happy and neutral faces. For Study 1, only women participants were recruited because naturally occurring initial emotional reactions to a female victim may differ across men and women. Our main hypothesis was that reappraisal to increase negative emotions would lead to a greater attentional bias to angry faces, whereas reappraisal to decrease negative emotions would lead to a reduced attentional bias to angry faces. We also examined attentional biases to happy faces. A previous study reported no changes in attentional bias to positive words after negative mood induction . As partIn Study 2, we further tested whether habitual daily uses of reappraisal and suppression predict attentional biases to angry and happy faces. Healthy young men and women performed a dot-probe task as in Study 1. Individual differences in reappraisal and suppression were assessed with the Korean version of the Emotion Regulation Questionnaire ERQ; . State aFifty-seven healthy young women (mean age = 22.30 ± 2.65 years) were recruited through campus flyers. The sample size was determined based on pilot studies and previous studies demonstrating the effect of reappraisal using between group design . All volFor the dot-probe task, we selected faces with angry , happy , and neutral  expressions from a standardized database of face photographs . ValenceAfter signing a consent form, participants completed the Beck Anxiety Inventory BAI;  and EmotNext, participants received emotion regulation instructions according to group assignment . ParticiNext, participants watched a 5-min film segment selected from the movie “One on One (2014)” directed by Kim Ki-Duk. This film segment depicted a man’s violent behavior against his girlfriend. An independent group of 16 participants (nine men and seven women) rated the film segment as having an emotional valence of 2.1  and emotional arousal of 5.2 . Immediately after watching the film, participants again rated their mood on the VAS (VAS-post). Participants also rated how successful they were in following the reappraisal instructions on a VAS . Finally, participants performed the dot-probe task. There were eight practice trials composed of face pairs that were not used in the main task before the experimental trials. After completing the task, participants were debriefed and compensated for their time.A total of 1.1% of the total data were excluded due to incorrect responses (0.4% of the data) and outlier responses, defined as trials with reaction times less than 200 ms or greater than three standard deviations above the individual mean (0.7% of the data). Attentional bias scores were calculated by subtracting mean reaction times to dot probes that replaced emotional faces from mean reaction times to dot probes that replaced neutral faces. Positive attentional bias scores indicate greater attention to emotional relative to neutral faces, and negative attentional bias scores indicate avoidance of emotional faces. Attentional bias was calculated separately for happy and angry faces. We further assessed two subcomponents of attentional bias: orienting and disengagement. Orienting was calculated by subtracting mean reaction times to probes replacing emotional faces in emotion-neutral pairs from mean reaction times to probes replacing neutral faces in neutral–neutral pairs. Greater positive scores indicate faster orienting to emotional faces. Disengagement was calculated by subtracting mean reaction times to probes replacing neutral faces in neutral-neutral pairs from mean reaction times to probes appearing in the location of the neutral expression in emotion-neutral pairs. Greater positive scores indicate slower disengagement from emotional expressions. Statistical analyses were performed using SPSS 19 .Table 1. There were no groups differences in age , reappraisal , suppression , and BAI  scores (Table 1).Descriptive statistics for each group are presented in F = 8.37, p = 0.001] and time  and the group × time interaction, , were significant. Overall, negative mood increased post-film  compared with pre-film . Overall the up-regulation group reported greater negative mood  compared with the down-regulation group . To further examine the interaction effect, follow-up t-tests were conducted. Results showed no group differences in negative mood before the film (ts < 1). However, a significant group effect was found post-film, F = 26.33, p < 0.001. The down-regulation group reported lower negative mood than the up-  and no-regulation groups . The up-regulation group reported greater negative mood than the no-regulation group, . A one-way ANOVA on self-ratings of regulation success with group as a factor revealed no group differences, F = 1.44, p = 0.246.Negative mood scores were calculated by summing all VAS scores (depressed-happy scores were reverse-coded). A two-way ANOVA with time (pre versus post) as a within-subject factor and group as a between-subject factor was conducted. The main effects of group, . Follow-up t-tests showed that the increase in negative mood scores was greater in the up-regulation group than no-regulation group  and down-regulation groups . The increase in negative mood was significantly smaller for the down-regulation group compared to no-regulation group .To specifically examine whether mood change after watching the film differed across groups, we calculated mood change scores by subtracting pre-film scores from post-film scores. A one-way ANOVA on mood change scores with group as a factor revealed a significant main effect . Follow-up t-tests showed that the attentional bias to angry faces was reduced in the down-regulation group  compared to the up-regulation group , t(36) = 2.43, p = 0.020, but not to the no-regulation group , t(36) = 0.85, p > 0.250. There was a marginally significant increase in attentional bias to angry faces in the up- compared to no-regulation group, t(36) = 1.72, p = 0.094 . A one-way ANOVA with group as a factor did not reveal any group differences in orienting to or disengagement from angry faces, F = 0.80, p = 0.455, and F = 2.20, p = 0.120, respectively.A one-way ANOVA on attentional biases to angry faces with group as a factor revealed a significant effect of group, . No significant group effects were observed for orienting to happy faces, F = 0.59, p = 0.556, and disengagement from them, F = 1.15, p = 0.325.A one-way ANOVA on attentional biases to happy faces with group as a factor revealed no group differences, [Forty-seven right-handed college students  were recruited through campus flyers. The sample size was determined based on pilot studies and previous studies . ParticiThe dot-probe task was similar to Study 1, except that the faces were selected from a different database and all conditions had the same number of trials. A total of 16 angry , 16 happy , and 64 neutral  faces were selected from a standardized database of face photographs . Valencer = -0.19, p = 0.197).After signing the consent form, participants completed Spielberger State-Trait Anxiety Inventory STAI; . This sct-tests were performed comparing attentional bias scores against zero. We also performed multiple regression analysis to identify variables predictive for each angry and happy bias index . As predictor variables, we entered trait reappraisal, trait suppression, trait anxiety (STAI-T), state anxiety (STAI-S), as well as individual variables such as age and gender. Statistical analyses were conducted using SPSS 19 .As in Study 1, trials with incorrect responses (0.7% of the data) and response outliers (1.2% of the data) were excluded from the analyses. Attentional bias, orienting, and disengagement scores were calculated separately for angry and happy faces. One-sample t-tests revealed a statistically significant bias toward angry faces, t(46) = 3.89, p < 0.001, but no significant bias toward happy faces, t(46) < 1, p > 0.250, which is consistent with what we found from Study 1. Multiple regression analyses revealed that overall attentional bias toward angry faces were predicted by trait reappraisal  and state anxiety . Interestingly, reappraisal negatively predicted orienting to angry faces  and state anxiety positively predicted disengagement from angry faces . On the other hand, overall attentional bias toward happy faces was predicted by suppression . However, no predictive factors were observed for orienting. A marginal level of relationship between suppression and happy disengagement was found .One-sample In this research, we investigated whether reappraisal modulated attentional biases to angry and happy faces in two studies. In Study 1, we found that self-reported negative mood, induced by a short film clip with unpleasant content, diminished in participants who reappraised to reduce negative emotions while watching the film clip as compared to those who reappraised to increase negative emotions and those who naturally viewed the film without reappraisal. More interestingly, participants who reappraised to reduce emotions also showed smaller attentional biases to subsequently presented angry faces compared to those who reappraised to increase emotions. There were no group differences in attentional biases to happy faces. In Study 2, we found that individuals more prone to using reappraisal in regulating everyday emotions were slower in orienting toward angry faces, whereas those more prone to using suppression were slower in disengaging from happy faces. Together, these results suggest that instructed and habitual patterns of emotion regulation are associated with attentional processes toward emotional information.Across Studies 1 and 2, we found that both instruction-induced, state reappraisal and trait reappraisal contribute to attentional responses to angry faces. Reappraisal to reduce negative emotions during film viewing induced less vigilant attention to subsequently presented angry faces compared with reappraisal to increase negative emotions. Notably, participants were not instructed to reappraise emotions during the dot-probe task. Their explicit goal was to respond to the dot-probe as quickly as possible. Therefore, it appears that altered emotional mood states due to reappraisal while viewing the film clip were transferred to the experimental task and modulated attentional processes to subsequently presented angry faces. Alternatively, participants may have continued to reappraise during the dot-probe task, which could have directly influenced overt attention. However, a previous report showed that people tend to direct overt attention to the emotion source when they have an explicit reappraisal goal . TherefoAttentional bias did not differ between the down- and no-regulation groups. Therefore, it remains unclear whether this reduced attentional bias was truly due to decreased attentional bias in the down-regulation group or increased attentional bias in the up-regulation group. There was a trend toward a significant increase in attentional bias in the up- versus no-regulation group. Although the interpretation of these results is limited, the current findings still have important implications for clinical applications. Given that patients with emotional disorders have heightened attentional biases to task-irrelevant emotional information, our findings suggest that reappraisal may be useful for reducing a maladaptive level of attentional bias. More studies are needed to precisely elucidate the role of appraisal on attentional bias.It has been repeatedly demonstrated that reappraisal has acute effects on affective states; however, its long-term cumulative effect still has to be determined . The resInterestingly, multiple regression analyses in Study 2 revealed that attentional biases to happy faces were predicted by trait suppression. More specifically, individuals with greater tendency to suppress emotions tended to disengage more slowly from happy faces even if they were task-irrelevant. However, the underlying mechanisms of the association between habitual suppression of emotions and attentional bias toward happy faces are still unclear. Although it remains speculative, longer attention time to dwell on happy faces signaling acceptance and safety might be instrumental in accomplishing an internally driven goal of managing emotions. Habitual suppression has been associated with heightened anxiety and increased sympathetic activation , which cIt is notable that no measurable overall attentional bias to happy faces was observed. This lack of attentional bias to positive expressions is interesting given that quick and elaborate processing of positive information is as important and evolutionarily adaptive as quick and elaborate processing of negative information . A previAlthough the results of the current research are revealing, several limitations are also identified. First, in Study 1 we used a short film clip depicting a female victim of violence to induce negative emotions and found differences in attentional biases to angry faces following reappraisal. However, we did not observe differences in happy faces. It would be interesting and worthwhile to see whether reappraisal of positive emotions would result in changes in attentional biases to happy faces. Second, Study 1 tested only female participants in order to control sources of variability such as initial emotional responses to women victim and types of reappraisal strategies. A bigger scale of study with both male and female participants is warranted for better understanding of factors influencing attentional biases to emotional information. Third, in Study 1 we did not assess initial baseline levels of attentional biases before the exposure to the film clip. Repeated assessment of attentional biases before and after treatment may have the potential to increase demand characteristics bias. Despite that understanding, a baseline level of attentional bias measure for each subject would have allowed to draw stronger conclusions in relation to the influence of instructed reappraisal on attentional biases. Finally, in Study 2 we did not evaluate current mood states although we assessed current state anxiety. The inclusion of both current positive and negative affect as predictive factors in our analyses might have been more revealing, especially regarding the relationship between positive affect and attentional biases to happy faces.To conclude, the main finding of the current research revealed that both instruction-induced state reappraisal and trait reappraisal are linked to reduced attentional bias and slower orientating to task-irrelevant angry faces. These results contribute to our understanding of how reappraisal regulates negative emotions and provides insight relevant to the development of treatment strategies for emotional disorders.SAK and SHK conceived and designed the study. SAK conducted the study and collected the data. SAK and SHK analyzed the data. SAK, HK, and SHK interpreted the data. SAK drafted the first version of the manuscript. HK and SHK revised the manuscript. All authors approved the final version of the manuscript.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.