Introduction: Sarcoidosis is a granulomatous disease with unknown cause that can vary from an asymptomatic condition. Almost half of the patients with sarcoidosis have no symptoms. In this article, we describe a sarcoidosis patient with lung and liver engagement; it may be confused with metastasis.Case report: A 39-year-old man, with known as hypothyroidism who had come to the emergency ward with dyspnea and coughing after exposure to detergents in a closed environment. The patient smoked for 10 years (3 pack/year). No other findings were found in clinical examinations except for wheezing in the right lung. The patient's chest radiography was shown a mass. For further investigation, spiral CT scan was performed. Large lymph nodes on the right side of the trachea, measuring about 23 mm and a mass of 70 × 77 mm in the vicinity of the right lung hilum and a hypodense nodule in the posterior part of the liver with malignancy suspicious were reported. After several biopsy results was shown chronic granulomatous inflammation, the most important differential diagnosis is tuberculosis (TB) and sarcoidosis. Sputum smear, culture, and PCR were performed for tuberculosis. Also, the level of angiotensin-converting enzyme (ACE) was measured for sarcoidosis. the results ruled out TB and shown a higher level of ACE (ACE = 88 IU/L).After diagnosis treatment started with prednisolone. Now, the patient is in the follow- up.Conclusion: In hilar lymphadenopathy of lung sarcoidosis is the importance differential diagnosis that should be considered. Almost half of the patients with sarcoidosis have no symptoms. In some cases, with the help of chest radiography findings looking for other pathologies, it is diagnosed. Since the lungs are often involved, patients usually come to the clinic with lung complaints 3. Some of the clinical manifestations of sarcoidosis have a poor prognosis, including treatment-resistant lung sarcoidosis , cardiac sarcoidosis, neurosarcoidosis, and multiple organ sarcoidosis. These clinical manifestations are often not diagnosed until the end stage of the disease, and their response to treatment is low. The incidence of severe sarcoidosis is the most common cause of death4. On time diagnosis, treatment and follow-up of patients reduces mortality. In this article, we describe a sarcoidosis patient with lung and liver involvement, which may be misdiagnosed as cancer metastasis.Sarcoidosis is a granulomatous disease with unknown cause that can vary from an asymptomatic condition to being life-threateningA 39-year-old man, taxi driver, known to have hypothyroidism (being treated with levothyroxine) presented to the emergency ward with dyspnea and coughing after exposure to detergents in a closed environment. The patient was smoker (3 pack/year). The only in clinical examinations except for wheezing in the right side of chest. The patient's chest radiography identified a mass. For further investigation, a spiral computerized tomography (CT) scan was performed. Lymph nodes were enlarged on the right side of the trachea, measuring about 23 mm with a mass of 70 × 77 mm, in the vicinity of the right lung hilum. A hypodense nodule in the posterior part of the liver, suspected to be malignant, was also reported . The les5. Diagnosis is based on medical history, granuloma manifestations in at least two different structures, staining and negative culture for acid-fast bacilli, a lack of occupational and internal exposure to toxins and absence of drug-related illnesses. It is difficult to determine the prevalence and incidence of sarcoidosis since the number of patients who have sarcoidosis but do not have symptoms is unknown. In countries whereMycobacterium tuberculosis is common, sarcoidosis may not be detected7. The first evaluation of patients suspected of sarcoidosis include; cell blood count, serum biochemistry including creatinine, calcium, liver enzymes, alkaline phosphates, urine analysis, serum protein electrophoresis, inflammatory markers, lactate dehydrogenase, level of enzyme (ACE), and Complete Pulmonary Function Tests that should be performed in patients with respiratory symptoms or abnormalities of the lung parenchyma. In all cases,Mycobacterium and fungal disease should be considered, as it has a similar history (chronic cough) and clinical image (pulmonary lymphadenopathy)8. A few clinical case report has shown that sarcoidosis is less common in smokers10. Sarcoidosis has many clinical manifestations and affects all organs of the body. The lung is involved in at least 90% of sarcoidosis patients. Skin, eyes, liver and peripheral lymph nodes, with a frequency of 10 to 30% for other involved organs10. Cardiac involvement occurs in 25% of cases, but only causes clinical problems in 5% of cases, although it may suddenly be fatal, so it is important to examine all patients for cardiac sarcoidosis12. All patients should also be screened for eye involvement as it can cause visual impairment13. Sarcoidosis, like drugs, poisons, viral infections, and flukes induce liver dysfunction15. Up to 35% patients have abnormal liver function tests that are not related to the degree of disease17. Serum levels of ACE are increased in 60% of patients and have been shown to correlate with the level of disease activity. This test is non-invasive and is highly effective because the enzyme is produced by epithelial granuloma cells and its serum level reflecting the entire granulomatous activity in the body18. Fatigue was reported in over 50% of patients, which has a major effect on the quality of life. Pain was reported in 70% of patients. Arthralgia is the most common type of pain; a headache and chest pain also being reported19. Sarcoidosis treatment, such as rheumatologic diseases and vasculitis, is based on corticosteroid. In terms of treatment, Corticosteroids are a selective therapeutic drug, and methotrexate and hydroxychloroquine are often alternative drugs23. Our patient had no abnormal blood counts, liver test dysfunction, and non-specific inflammatory markers abnormalities, only an increased titer of ACE with addition liver symptoms that included itching, jaundice, fever and abdominal pain.Sarcoidosis occurs mainly in people aged 20 – 40 years and more common in femalesIn hilar lymphadenopathy of lung, sarcoidosis is an importance differential diagnosis that should be considered and prior to biopsy of lymph nodes and any invasive procedures, ACE enzyme levels should be measured.The data referenced by this article are under copyright with the following copyright statement: Copyright: © 2018 Jafari B et al.Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).No data is associated with this article.Written informed consent was obtained from the patient for the publication of the patient’s clinical details and accompanying images. Considering the authors respond to major comments, I think the manuscript is now acceptable for indexing.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The authors present a case of pulmonary sarcoidosis arising in a country where tuberculosis may be more common.A 39-year-old man, known as hypothyroidism who had come to the emergency ward with dyspnea and coughing after exposure to detergents in a closed environment A 39-year-old man, known as hypothyroidism who had come to the emergency ward with dyspnea and coughing after exposure to detergents in a closed environment." There is an error in the Abstract where a sentence is repeated "with known hypothyroidism..." In the abstract, the authors should revise the sentence "A 39-year-old man, known as hypothyroidism..." this probably should be revised to "A 39-year-old man On page 3, please revise the sentence "The incidence of severe sarcoidosis is the most common cause of death." On page 3, the authors should provide information from the bronchoscopy procedure report as to whether there was airway narrowing visualized during the bronchoscopy. This is of particular interest since the patient's main radiographic abnormality is enlarged thoracic lymph nodes without any notable lung infiltrates (stage I chest x-ray). On Page 4, please consider revising "It is difficult to determine the prevalence and incidence of sarcoidosis without clinical symptoms." Perhaps rephrase to clarify that "It is difficult to determine the prevalence and incidence of sarcoidosis since the number of patients who have sarcoidosis but do not have symptoms is unknown."I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. This case study presents a man with cough and dyspnea following exposure with irritant agent. His chest imaging showed a right hilar mass. Although this is not an unusual presentation of sarcoidosis but confirms that not all mass is malignant.What do you mean by "The incidence of severe sarcoidosis is the most common cause of death"? As Swigris et al. had published, did you mean "The underlying cause of death in most patients with sarcoidosis was the disease itself."Would you please clarify and rephrase this sentence: "The only in clinical examinations except for wheezing in the right side of chest" The authors please add the bronchoscopic findings; was there any mass or narrowing there. Please add the coronal view of chest CT; considering chest physical finding, wheeze, the reader would like to see if there is any narrowing in right main and/or intermediate bronchus.Please add the unit of measurement of ACE level.Please add the histologic slide of biopsy to the manuscript. My comments:I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.


Our previous data demonstrated that the endonuclease complex ERCC1-XPF, which cleaves a 3′ DNA flap structure, limits L1 retrotransposition. Although the ERCC1-XPF endonuclease participates in several different DNA repair pathways, such as single-strand annealing, or in telomere maintenance, its recruitment to DNA lesions is best characterized in the nucleotide excision repair (NER) pathway. To determine if the NER pathway prevents the insertion of retroelements in the genome, we monitored the retrotransposition efficiencies of engineered L1 elements in NER-deficient cells and in their complemented versions. Core proteins of the NER pathway, XPD and XPA, and the lesion binding protein, XPC, are involved in limiting L1 retrotransposition. In addition, sequence analysis of recovered de novo L1 inserts and their genomic locations in NER-deficient cells demonstrated the presence of abnormally large duplications at the site of insertion, suggesting that NER proteins may also play a role in the normal L1 insertion process. Here, we propose new functions for the NER pathway in the maintenance of genome integrity: limitation of insertional mutations caused by retrotransposons and the prevention of potentially mutagenic large genomic duplications at the site of retrotransposon insertion events.Long interspersed elements 1 (L1) are active mobile elements that constitute almost 17% of the human genome. They amplify through a “copy-and-paste” mechanism termed retrotransposition, and  ERCC1-XPF is a structure-specific endonuclease that nicks double-stranded DNA 5′ of a DNA lesion . ERCC1-XPF has a global function in cellular DNA damage repair, notably in the removal of the DNA flap structures during single-strand annealing (SSA) repair of DSBs  were grown in Eagle’s Minimal Essential Medium (EMEM) supplemented with 10% Fetal Bovine Serum, 0.1 mM nonessential amino acids (Life Technologies), and 1 mM sodium pyruvate (Life Technologies) at 37° in a 5% carbon dioxide environment. The following cell lines were obtained from the Coriell Cells Repository: XPC-SV40-transformed fibroblast (GM15983), XPD-SV40-transformed fibroblast (GM08207), the stably complemented version of XPD-cell line (XPD+) (GM15877) and the isogenic pair of XPA-SV40-transformed fibroblasts (GM04312), and the stably complemented version (XPA+) (GM15876). XPC-, XPD-, and XPA- cell lines were grown in EMEM supplemented with 10% Fetal Bovine Serum and 0.1 mM nonessential amino acids (Life Technologies) at 37° in a 5% carbon dioxide environment. The XPD+ cell line was grown in the same conditions with the presence of 500 μg/ml G418 (Life Technologies). The XPA+ cell line was grown in Dulbecco’s Modified Eagle Medium supplemented with 10% Fetal Bovine Serum (Life Technologies).mblastI indicator cassette cloned in pCEP4  promoter upstream of the L1.3 element, deleted for L1 5′UTR, and the mblastI indicator cassette cloned in pCEP4 (TAMD702A/L1.3 derives from TAM102/L1.3 and contains the reverse transcriptase-deficient mutant of the L1.3 element and the mblastI indicator cassette cloned in pCEP4 (TAMH230A/L1.3 derives from TAM102/L1.3 and contains the endonuclease-deficient mutant of the L1.3 element and the de novo L1 inserts was previously described (The synL1_neo vector used for the recovery of CH_blast rescue vector is a pBS-L1PA1CH plasmid (de novo L1 inserts. (Addgene 69611)The pBS-L1PA1pCMV6-XPC (ORIGENE) consists of XPC cDNA driven by the CMV promoter.BglII (nt 753) and BamHI (nt 1095) sites, in the XPC cDNA to create a defective deletion product.pCMV6-XPCΔ, control vector, was constructed by deleting 342 bp, between pIRES2-EGFP (Clontech) contains a neomycin resistance gene expressed from a SV40 promoter that allows for the selection of transfected cells, which is used as a toxicity control. The vector also contains a multi-cloning site downstream of a CMV promoter and upstream of an IRES and eGFP marker.pCMV-Bsd (Life Technologies) contains a blasticidin resistance gene.Both plasmids above were used in parallel in the retrotransposition assays as a combined control for transfection and growth variations between the cell lines used.All plasmid DNA was purified by alkaline lysis and twice purified by cesium chloride buoyant density centrifugation. DNA quality was also evaluated by the visual assessment of ethidium bromide-stained agarose gel electrophoresed aliquots.6 cells were seeded in T75 flasks. Cells were transfected the next day at about 90% confluence using Lipofectamine 2000 (Life Technologies) following the manufacturer’s protocol. For L1 retrotransposition assays, cells were transfected with 1 or 3 μg of L1.3 or L1.3-RT (-) construct tagged with the mblast retrotransposition cassette (TAM102/L1.3 or TAMD702A/L1.3) in T75 flasks. Next, 3 µg of XPC expression vector were used to transiently complement XPC-deficient cells. Cells were selected for the presence of the L1 plasmid in selective medium containing 200 µg/ml hygromycin (Life Technologies) for 5 days. The selection was removed and cells were grown for 7 days in growth medium. After the growing period, hygroR cells were trypsinized and reseeded in six-well plates in triplicate at serial dilutions, from 106 to 103 cells, and selected for transposition events in medium containing 10 µg/ml blasticidin (InvivoGen). After 15 days, cells were fixed and stained with crystal violet solution . The number of blastR or neoR colonies was counted in each well and the relative number of colonies per 106 transfected cells was determined.For cells with lower transfection efficiencies such as XPA-, XPA+, XPD-, XPD+, and XPC- fibroblasts, L1 retrotransposition assays were performed as described in 6 cells were seeded in T75 flasks. Cells were transfected the next day at about 90% confluence using Lipofectamine 2000 (Life Technologies) following the manufacturer’s protocol. Cells were transfected with 3 μg of L1.3, L1.3-RT (-), or L1.3-EN (-) construct tagged with the mblast retrotransposition cassette . To transiently complement XPC-deficient cells, 3 µg of XPC expression vector were used. Cells were selected for the presence of the L1 plasmid in selective medium containing 200 µg/ml hygromycin (Life Technologies) for 14 days. The cells were then fixed and stained with crystal violet solution . The number of hygroR colonies were counted in each flask.L1 toxicity and colony formation assays were performed using the L1 episomal vectors. Briefly, 5 × 106 cells were seeded in T75 flasks. Cells were transfected the next day at about 90% confluence using Lipofectamine 2000 (Life Technologies) following the manufacturer’s protocol. Cells were transfected with 3 μg of L1.3 construct tagged with the mblast retrotransposition cassette (TAM102/L1.3). To allow for the expression of the vector, total RNA was extracted the next day from each flask using TRIzol Reagent (Life Technologies). We then carried out chloroform extraction and isopropanol precipitation. RNA was suspended in 80 µl DEPC-treated water. The cDNA was synthesized using the Reverse Transcription System , following the manufacturer’s protocol. Briefly, 1 µg total RNA was denatured at 75° for 5 min. Reverse transcription reaction was primed with Oligo(dT)15 primers and incubated at 42° for 1 hr in a thermocycler . The enzyme was then heat-inactivated at 99° for 5 min. To avoid contamination with L1 DNA from the genome or the expression vector, and to make sure that only the L1 transcript was quantified, we analyzed the spliced mblast reporter cassette in L1 RNA using the TAQMAN qPCR approach. The PCR reaction  was performed in a qPCR thermocycler  using 1 µl of cDNA and the blasticidin primers at 250 mM final concentration each: Primer F (5′-GCAGATCGAGAAGCACCTGT-3′), Primer R (5′-TGGTGTCAATGTATATCATTTTACTGG-3′) and the 5′-FAM-labeled, ZEN internal quencher, Iowa-Black Fluorescent Quencher (IDT) probe at 150 mM final concentration (5′−/56-FAM/AGGTTGCCA/ZEN/GCTGCCGCA/3IABkFQ/-3′) as follows: initial denaturation at 95°, 45 cycles of 95° for 10 sec, 60° for 20 sec. The probe was designed to span the splice junction of the mature RNA so that it would not anneal to the unspliced DNA. The fluorescence was read during the annealing/elongation step . For normalization, predeveloped TaqMan Assay Reagents for Human β-actin  were used. Reactions were done in triplicate. The values of the Cq for each PCR reaction were determined using Bio-Rad CFX Manager software (Bio-Rad).First, 5 × 10De novo L1 insert recovery was performed as previously described with slight modifications, as noted below . Cells were selected with 200 µg/ml hygromycin for 5 days, and then put under 400 µg/ml of G418 or 10 µg/ml of Blasticidin for 14 days to allow for colony formation. NeoR and blastR cells were harvested by trypsinization and genomic DNA was extracted using a QIAGEN DNeasy Blood and Tissue kit . Genomic DNA was digested with 100 U HindIII (NEB) overnight at 37°. The following day, digested genomic DNA was self-ligated using 1200 U T4 DNA ligase (NEB) in a volume of 1 ml overnight at room temperature. DNA was purified and concentrated using centrifugal filters . Purified DNA was transformed by electroporation into competent DH5α Escherichia coli (Life Technologies). Individual kanamycin- or blasticidin-resistant colonies were grown and plasmid DNA was harvested using a SV Wizard miniprep kit (Promega). The 5′ end of the de novo L1 insert was sequenced using primers specific to the L1 rescue plasmid and primer walking until the 5′ end of the insert was recovered as described in StuI (NEB) to relax supercoils, and then sheared by sonication using a Bioruptor . Sheared plasmid DNA was primer extended using an oligo specific to the 3′ end of the L1 rescue plasmid (3′_rescue_1: 5′-ATATATGAGTAACCTGAGGC-3′ or 3′_rescue_1_secondpA: 5′-GTGGGCATTCTGTCTTGTTC-3′). Duplexed T-linkers were ligated using 10 U T4 DNA ligase and PCR was performed using the primers: linker-specific (5′-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′) and 3′_rescue_1 (or 3′_rescue_1_secondpA) primers as follows: initial denaturation at 94°, 20 cycles of 94° for 30 sec, 60° for 1 min, 72° for 1 min, and a final extension for 10 min at 72°. PCR reactions were run on a 1% agarose gel and a light smear between 400 and 700 nt was gel extracted with the Qiaquick gel extraction kit (QIAGEN). Gel-extracted DNA (1 μl) was subject to an additional 15 cycles of PCR amplification as described above using linker-specific and nested 3′ rescue vector primers (3′_rescue_2: 5′-TGAGTAACCTGAGGCTATGCTG-3′ or 3′_rescue_2_secondpA: 5′-TTCTGTCTTGTTCCGGTTCTTAAT-3′) primers. The nested PCR product was run on a 1% agarose gel and the resulting smear was gel extracted and cloned into TOPO-TA (Life Technologies). Cloned PCR products were Sanger sequenced using M13 forward and reverse primers to determine 3′ end junctions. Samples were sent for sequencing to Elim Biopharmaceuticals . Lasergene 10 SeqBuilder software was utilized for sequence analysis. Flanking regions were mapped on human reference genome hg19 (build 37) using the Blat tool (https://genome.ucsc.edu/cgi-bin/hgBlat). We verified the presence of the 5′ and 3′ flanking sequences within a L1 rescue plasmid with a combination of PCR between the two regions . Sequences from rescues are included in File S1.To evaluate expression of NER proteins in the cells, XPA-, XPA+, XPC-, XPD-, and XPD+ cells were haverested in 300 µl of lysis buffer . After 10 min of sonication , lysates were clarified by centrifugation for 15 min at 4° at 13,000 rpm and the protein concentration was determined by Bradford assay (Bio-Rad). 40 µg of proteins were loaded on 4–12% bis-tris polyacrylamide or 3–8% tris-acetate polyacrylamide gels (Life Technologies). Proteins were transferred to a nitrocellulose membrane using an iBlot gel transfer system from Life Technologies (manufacturer’s settings). The membrane was blocked for 1 hr at room temperature in PBS (pH 7.4), 0.1% Tween 20 , 5% skimmed milk powder (OXOID), and then incubated overnight at 4° with an anti-XPC monoclonal antibody , anti-XPD monoclonal antibody , or anti-XPA polyclonal antibody  diluted at 1:1000 in PBS, 0.1% Tween 20, and 3% nonfat dry milk. Membrane was then incubated for 1 hr at room temperature with the secondary donkey anti-rabbit HRP-conjugated antibody  diluted at 1:100,000 in PBS, 0.1% Tween 20, 3% nonfat milk. Signals were detected using Super Signal West Femto Chemiluminescent Substrate .To determine XPC protein expression, 24 hr after transfection with 3 μg of XPC expression vector or control vector, XPC-deficient cells and untransfected HeLa cells were harvested in 300 µl of lysis buffer . After 10 min of sonication , lysates were clarified by centrifugation for 15 min at 4° at 13,000 rpm and the protein concentration was determined by Bradford assay (Bio-Rad). 20 µg of proteins for transfected cells and 40 µg of proteins for HeLa cells were loaded on a 3–8% tris-acetate polyacrylamide gel (Life Technologies). Proteins were transferred to a nitrocellulose membrane using an iBlot gel transfer system from Life Technologies (manufacturer’s settings). The membrane was blocked for 1 hr at room temperature in PBS (pH 7.4), 0.1% Tween 20 , 5% skimmed milk powder (OXOID), and then incubated overnight at 4° with an anti-XPC polyclonal antibody  diluted at 1:1000 in PBS, 0.1% Tween 20, and 3% nonfat dry milk. The membrane was then incubated for 1 hr at room temperature with the secondary donkey anti-rabbit HRP-conjugated antibody  diluted at 1:100,000 in PBS, 0.1% Tween 20, and 3% nonfat milk. Signals were detected using Super Signal West Femto Chemiluminescent Substrate .5 cells were seeded in 6-cm plates and grown in growth medium for 24 hr. The growth medium was removed and the cells were irradiated in the presence of 1 ml of 1 × PBS with a bactericidal UVC lamp  at 0, 3, 6, 9, and 12 J/m2 UVC dose. The PBS was removed and replaced with growth medium. After 4 days, cells were counted with a hemocytometer to determine cell survival. Cell survival was calculated as the percent of live cells in the irradiated sample relative to the untreated sample. To determine the efficiency of the XPC transient complementation, 3 × 106 XPC-deficient cells were transfected with 3 μg of XPC expression vector or the control vector in T75 flasks using Lipofectamine 2000 (Life Technologies) and were reseeded in 6-cm plates the next day in the conditions described above. The UV assay was then performed as mentioned above.The protocol was adapted from Cells from confluent T75 plates were harvested and fixed for over 24 hr with ice-cold 70% ethanol at −20°. After fixation, cells were washed once with PBS and incubated in PI/Triton X-100 staining solution  Triton X-100, and 0.2 mg/ml RNAse A in PBS) for at least 2 hr. Flow cytometry of the stained cells was carried out on a Becton Dickinson LSRII using DiVA Software (Louisiana Cancer Research Consortium FACS Core) and 50,000 events were collected. The data were analyzed using Modfit Software (Verity Software House).The authors state that all data necessary for confirming the conclusions presented in the article are represented fully within the article.mblast) retrotransposition cassette . In addition, immunoblot analysis verified that XPA and XPD protein expression in the complemented XPA- and XPD-deficient cell lines was restored . A UV sensitivity assay . The increase in L1 retrotransposition in NER-deficient cells does not seem to be explained by the growth rate of the different cell lines, because the cell cycle analysis by FACS revealed a slightly longer cycle for NER-deficient cells, as a few more cells accumulated in G2 phase . A longer cell cycle does not seem to be a significant factor of a retrotransposition rate increase in our studies, because previous publications have shown that, although transiting the cell cycle does not seem to be required for retrotransposition  or TAMD702A/L1.3, RT(-)] . In parallel, to rule out any bias engendered by various transfection efficiency, growth rates, and L1-caused toxicity, we verified that L1 expression did not alter colony formation in a manner that could cause retrotransposition rate differences in any of the cells used in the experiment .As a control, we tested for the possibility that NER-deficiency would promote the retrotransposition of L1 elements defective in the endonuclease (EN) as has been seen in other DNA repair defects , while the cell survival rates of stably complemented XPA+ and XPD+ cell lines and HeLa cells were about 50% for the same UV dose . However the cell survival of transiently complemented XPC- cells (XPC+) using a vector expressing XPC cDNA (pCMV6-XPC), which expresses a large amount of XPCp  (Monitoring XPC protein (XPCp) expression level and repair efficiency of the UV-induced DNA damage, we determined that the transient transfection of XPC-deficient cells resulted in a partial complementation of the deficiency . In fact of XPCp , was sigr trend) . Showingr trend) . These rde novo L1 inserts. We extracted the genomic DNA of XPC-, XPA-, and XPD-deficient cells and in the stably complemented partner, XPD+, expressing the marker of retrotransposition. We then sequenced de novo inserts and the flanking regions to evaluate the features of the inserts, such as the length of the insert, the length of the polyA tail, the size of the target site duplication (TSD), and the sequence of the L1 endonuclease cleavage site. The principle of the method is presented in Figure S6A. The previously published method . Because the stably complemented cell lines were resistant to neomycin, we could not use the previously described synL1_neo vector . Except for the insert size that depends on the length of the reporter cassette, the characteristics of de novo L1 inserts obtained with the new L1PA1CH_blast rescue vector in HeLa cells were similar to those previously published in .Because complementation of NER-deficiency leads to a decrease in the apparent retrotransposition rate, suggesting that NER proteins inhibit retrotransposition events, we wished to investigate whether mutations in NER proteins impact the characteristics of de novo L1 inserts in XPD-deficient cells (XPD-), 9 in XPA-deficient cells (XPA-), and 26 in XPC-deficient cells (XPC-) using the synL1_neo vector and 17 in stably complemented XPD-deficient cells (XPD+) using the L1PA1CH_blast rescue vector . In comparison with data obtained in HeLa cells, we detected no significant difference in the median length of L1 inserts in NER-deficient and proficient cells lines . Therefore, NER proteins do not seem to influence the size of the de novo L1 inserts.We recovered 29 e vector . We partde novo L1 inserts occurred at typical L1 endonuclease cleavage sites . Therefore, endonuclease-independent insertion of L1 in NER-deficient cells does not seem to explain these events, and these atypical inserts may be the results of recombination events between L1 and genomic sequences occurring at the insertion site during the insertion process.The majority of ge sites  in XPA-dge sites , and 3 oge sites  occurredde novo L1  in XPC-deficient cells   in XPC- cells, and 22 out of 28 (78.6%) in XPD- cells presented TSDs over 100 bp in length  and of these, 18 (38%) had TSDs of over 1 kb in length. Only three out of nine (33%) events presented TSDs over 100 bp in the stably complemented cells (XPD+), a difference that is statistically significant . It seems possible that the slightly elevated levels of longer TSDs in the complemented cells relative to previous studies in HeLa, for instance, may be due to the complementation not necessarily bringing the NER pathway to maximum efficiency. Therefore, our findings seem to imply that the expression of NER proteins is required to prevent large genomic rearrangements at the L1 insertion site.One area in which NER-deficient L1 inserts differed from those in NER-proficient cells is the size of the target size duplication. In NER-deficient cells, TSDs ranged from 5 bp to 5.3 kb in size with a median length of 1495 bp, while in XPD+ cells, TSDs ranged from 2 bp to 1.6 kb in length with a median of 14 bp . TSD lenL1 elements have a tremendous capacity to cause damage to the human genome, potentially causing disease, cell death, and aging . This suppression seems likely to be the result of an altered ability to complete the retrotransposition process successfully, rather than an ability to make longer inserts in NER deficiency, given that we detected no significant differences in insert length .Our data demonstrate that the entire global NER pathway represents an additional mechanism regulating L1 activity. The ERCC1-XPF complex, previously identified as an L1 regulator, is involved in many DNA repair pathways and is more essential for life than the other NER components. All three NER proteins tested in this study (XPAp, XPDp, and XPCp, It seems likely that the NER pathway is primarily responsible for the inhibition of L1 retrotransposition by ERCC1-XPF, as the magnitude of the effect of XPA, XPD, and ERCC1 deficiencies on L1 retrotransposition were very similar. However, this does not rule out the possibility that some of the other sensors and pathways that recruit ERCC1-XPF to DNA lesions may also be involved, to a lesser extent, in the control of L1 insertion. In particular, TCR, the other NER subpathway, may also contribute to the sensing of L1 insertion events. However, because TCR only protects the portion of the genome that is being actively transcribed in that specific cell type (Saccharomyces cerevisiae (Previously, the Garfinkel laboratory reported that Ssl2 and Rad3, two DNA helicases of the TFIIH complex and homologs of human XPB and XPD proteins, respectively, can inhibit Ty1 retrotransposition in In addition to the role of the NER factors in limiting the insertion of a new copy of L1, our L1 insert recovery data suggest that the NER pathway may also be involved in the less well-defined last step of the L1 TPRT process, as a high number of extremely large TSDs was observed in NER-deficient cell lines. These results suggest that the NER factors present at the site of L1 insertion may influence the formation or selection of the second nick necessary for the completion of the TPRT process at a location close to the cleavage site of the L1 endonuclease . Althougde novo L1 insertions, these “tailless” inserts seem to indicate some other undefined aberrant processes during the insertion.In addition to the long TSDs in these L1 insertions, the only other unusual feature is an unusually high number of events that do not look like they went through the normal TPRT process on the polyA tail. Although most of the inserts have the typically long A tails expected of The extremely large TSDs found in NER-deficient L1 insertions fall within the size range of genomic copy number variations . This la


The chemical vapor deposited monolayer graphene was transferred onto the synthesized ITO to enhance its electrical properties and it was capable of reducing sheet resistance over 12% while preserving the bi-film surface smoother. The ITO films contain the In2O3 phase only and exhibit the polycrystalline nature of cubic structure with 14.35 ± 0.5 nm crystallite size. The graphene/ITO bi-film exhibits reproducible optical transparency with 88.66% transmittance at 550 nm wavelength, and electrical conductivity with sheet resistance of 117 Ω/sq which is much lower than that of individual sol-gel derived ITO film.Indium tin oxide (ITO) still remains as the main candidate for high-performance optoelectronic devices, but there is a vital requirement in the development of sol-gel based synthesizing techniques with regards to green environment and higher conductivity. Graphene/ITO transparent bi-film was synthesized by a two-step process: 10 wt. % tin-doped ITO thin films were produced by an environmentally friendly aqueous sol-gel spin coating technique with economical salts of In(NO Tin (Sn) doped indium oxide, also known as indium tin oxide (ITO), is still employed in TCO-based industry, and offers the best combination of high optical transmittance and low electrical resistance, which properties are sensitive to the synthesis methods12. The aqueous precursor sol-gel technique has recently become of interest compared with the other synthesis methods of ITO, such as rf and dc sputtering, spray pyrolysis, vacuum evaporation, and pulsed laser deposition, due to the simple and low synthesis cost for bulk-scale production, possibility to change the film microstructure, chemical stoichiometry, and easy introduction of a dopant15. However, the use of flammable and environmentally harmful organic solvent and additives as stabilizers and binders during the preparation process of organic based sol-gel is not suitable for mass industrial production16. Further, the aqueous sol-gel method, which uses water as the solvent, has become more convenient for mass production17. On the other hand, oxygen plasma treatment can be used to enhance the surface free energy (SFE) of the substrate, because of the wettability improvement of the substrate through the SFE enhancement19. This correlation between the SFE of the glass substrate and its wettability can be used to prepare a continuous high-quality thin ITO film on the glass substrate through the aqueous sol-gel spin coating technique. However, the lowest sheet resistance obtained through sol-gel spin coating technique is comparatively higher than that of ITO film produced by expensive methods, such as rf and dc sputtering and pulsed laser depositions, because the high porous structure and fine grain size of sol-gel derived ITO thin film is a significant disadvantage.Transparent conductive oxide (TCO) is one of the most important and extensively studied classes of advanced functional materials, with applications such as smart windows, flat panel displays, liquid crystal displays (LCD), organic light emitting diodes (OLED), solar cells, and touch screens21. This atomically thin two-dimensional material with an anomalous quantum Hall effect, massless Dirac electronic structure can be synthesized through exfoliation of graphite, epitaxial growth on electrically insulating surfaces, chemical reduction of graphite oxide, arc-discharge method, chemical vapor deposition (CVD) on a catalyst metal substrate, and so on22. Out of the above-mentioned methods, the CVD technique, since it was first reported in 2008, has become the most frequently used promising method for large-area high-quality graphene synthesis23. The graphene synthesis via CVD consists of chemical reaction of CH4, and deposition on a catalytical surface, such as Cu and Ni, at high temperature 24.Graphene consists of a single layer carbon atoms arrangement in a hexagonal lattice, and its exceptional micromechanical and electron mobility properties, even at room temperature, make it a promising material for practical applications. Consequently it has received much attention in scientific studies26. The surface of this kind of combined materials shows lower sheet resistance than that of the individual surfaces, due to the improvement of surface carrier concentration in bi-film materials25. Further, high surface area semiconductive metal oxides, such as sol-gel derived ITO (high surface roughness promotes the high surface area), which does not have better electrical conductivity, can be used with graphene to improve the electrical performance more than that of individual sol-gel derived ITO, while keeping the optical transmittance at a desired level27.Recently, it has been reported that hybrid bi-film, such as conductive metal oxide-carbon complexes, is an interesting new scientific field to achieve outstanding electrical and optical properties in an effective wayHerein, the graphene/ITO conductive bi-film was synthesized to obtain 12.03% electical conductivity improvement of the sol-gel derived ITO via a two-step process: 10 wt.% tin-doped, ITO thin films were produced by an environment-friendly aqueous sol-gel spin coating technique on SFE enhanced glass substrate, and graphene was synthesized on Cu foil by a CVD method, and then transferred onto sol-gel derived ITO on glass substrate. The characteristics of sol-gel derived ITO, CVD graphene, the optical and electrical performances of graphene/ITO bi-film, and the effect of ITO on the Raman shift of CVD graphene will be addressed.3.xH2O, 99.99% trace metals basis, Aldrich) and tin(IV) chloride  were used as initial materials for the synthesis of ITO. The hydration number of indium nitrate was determined by thermogravimetrical analysis (TGA), prior to mixing the materials. Indium tin solution was prepared by dissolving 1.938 g of In(NO)3.H2O and 0.324 g (145.55 µl) of SnCl4 in 15 ml of deionized water, and the resultant solution was refluxed at 40 °C for two hours. The concentration of Sn ion in the solution was adjusted to obtain Sn 10 wt. % for the final ITO film. The obtained sol was aged for 48 hours to obtain gelation, before going to the coating process.Indium(III) nitrate hydrate (In(NO)2 gas flow. Prior to the spin coating, the cleaned glass substrates were oxygen plasma treated to improve the wettability and surface free energy of the surface, which helps to make continuous ITO film on the surface. The glass substrates were placed in the inductively coupled plasma (ICP) machine, and the chamber was vacuumed. The treatment was done by using oxygen gas with 85 sccm for 5 min under 150 W source power and 3.8 × 10−2 torr pressure. SFE was calculated by measuring the contact angles, and using the “Acid-Base (Van Oss-Chaudhury-Good)“ method.Square glass substrates of 20 × 20 mm size were used to deposit ITO by spin coating. The glass substrates were washed ultrasonically with acetone, isopropanol, and deionized water, respectively, and dried by using NFrom Equations  and 2),,2), and A sufficient amount of prepared ITO gel was used to cover the glass substrate, and then it was spun at 3,000 rpm for 30 seconds. The coated substrates were dried at 100 °C for 15 min after each step. Spinning and drying cycles were repeated to get the desired thickness for the final film. Finally, coated substrates were annealed at 600 °C for 1 hour in low vacuum conditions about 0.15 Torr to complete the crystallization of ITO.2 gas was introduced into the chamber at 600 °C under 10 sccm flow rate, and the chamber pressure was maintained at 0.13 ± 0.01 Torr. At 1,000 °C, high quality pure CH4 gas was supplied into the chamber with 30 sccm flow rate with the continuous supply of H2 gas, and the chamber pressure was maintained at 0.5 ± 0.1 Torr. After 1 min time of CH4 gas supply, the supply of both gases, and the heater system were turned off, and the chamber system was cooled down to room temperature28.Graphene synthesis was performed under a CVD method by using over 99.99% pure and 25 µm thick Cu foil as the catalytic substrate. After being cut to 3 × 10 cm sizes, the Cu foil was treated with 10% diluted 48–51% HF at room temperature for 10 s to obtain Cu oxide free surface prior to graphene synthesis. The treated Cu foil was then placed in the CVD chamber, and heated up to 1,000 °C. 99.999% pure H29. The PMMA  film was removed by using acetone and deionized water, respectively, and dried by using N2 gas flow. The graphene/ITO bi-films on glass substrates were then heated up to 100 °C for 30 min in normal atmosphere conditions. To compare the Raman signals of graphene on the synthesized ITO, a part of the prepared graphene were transferred onto SiO2 (300 ± 5 nm) /Si substrate, which was used as a reference substrate.The synthesized graphene was transferred onto ITO using a spin-coating of Poly(methyl methacrylate) (PMMA) - based indirect transfer methodα radiation with 1.54060 Å wavelength). The Scherrer equation was used to determine the crystallite size. Detailed information about the chemical composition of the ITO surface of the thin film was determined by XPS, and the elemental composition of the film was determined by EDX coupled with FE-SEM.The crystal structure and crystalline phases of ITO thin films were analyzed by XRD with monochromatic Cu anode  observation of both ITO thin film and graphene were done by using FE-SEM. The optical transmission and sheet resistance were measured by UV-Vis spectroscopy and four point probe head at room temperature, respectively. Electrical properties were also measured by using V-I characteristics.The quality of the synthesized graphene was characterized by Raman spectroscopy with 532 nm excitation laser beam, and data were collected at room temperature and normal atmosphere in the case of both SiO−2. The SFE increased with the oxygen plasma treatment time, and it reached a maximum value of 97.698 mJm−2 after 5 min. Figure 31. In fact, at 5 min of oxygen plasma treatment, the glass was severely damaged, causing a high surface root mean squared (RMS) roughness of about 213 pm, with respect to the oxygen plasma un-treated glass with a surface RMS roughness of about 172 pm. Moreover, slight SFE reduction of the glass surface with further oxygen plasma treatment was also observed, and after 6 min, it reduced to 93.012 mJm−2. This SFE reduction may occur during continuous oxygen plasma treatment (after 5 min of time), due to surface smoothness through the initiation of the splitting of hills, which occurred at the initial stage of oxygen plasma treatment of the glass substrate30.Figure −2) produced discontinuous, highly dispersed, agglomerated ITO islands, instead of continuous layer. Continuous ITO film was not obtained, even after a number of multiple coatings of ITO sol-gel on oxygen plasma untreated glass substrate, and Fig. Figure 2O3 has [100] direction orientation, and obtained (111) orientation indicates that doped tin substitutionally replaces indium in the lattice structure32. The peaks of (211), (222), (400), (431), (440) and (622) were found at 21.13°, 30.52°, 35.42°, 45.73°, 50.95° and 60.63°, respectively. The absence of characteristic peaks at 26.5° and 33.2°, which correspond to SnO2 and SnO respectively, reveals that one single phase of In2O3 is present, without any single phase of Sn in the ITO film. The interplanar spacing of (222) was obtained as 2.926 Å, which shows good agreement with 2.921  Å, which is the interplanar spacing of (222) according to the joint committee on powder diffraction standards (JCPDS) card. The obtained crystal quality parameter I222/I400 (intensity ratio of (222) and (400)) was 3.301. Furthermore, the crystallite sizes of ITO were determined from the line broadening nature of the XRD pattern according to the Scherrer formula33, and the obtained crystallized sizes for (222) and (400) peaks were 14.35 ± 0.5 and 14.37 ± 0.5 nm respectively .Figure 13. When the content of Sn is increasing, all the Sn atoms cannot participate in substitution for In atom, and Sn ions closely pack to each other, which makes a defect complex with average electrical charge of + 3, due to the tendency of Sn(IV) conversion to Sn(II). These Sn ions in the defect complex do not participate in the electrical conductivity6. The bulk chemical composition present in the ITO film was determined by energy dispersive X-ray spectrometry (EDX) along with FE-SEM, as shown in Fig. 2O3 lattice. A peak relevant to the Si was obtained in EDX, due to the use of glass as a substrate for ITO coating.It is very important to maintain the Sn doping value within a certain level limit to obtain better electrical properties of ITO film, and previously reported research found that 10 wt. % Sn content ITO shows the minimum sheet resistance34. The peak positions of In3d5/2 and Sn3d5/2 corresponding to the metallic In and Sn occur at 443.6 ± 0.2 and 484.5 ± 0.2 eV, respectively35. The peak positions of In3d5/2 and Sn3d5/2 for the prepared ITO film are around 444.28 and 486.7 eV, respectively. The chemical shifts of In3d5/2 and Sn3d5/2 from metallic form to the prepared ITO film were 0.68 ± 0.2 and 2.2 ± 0.2 eV, respectively, which clearly reveals the prepared ITO has only the oxide forms of In and Sn, and is free of metallic compounds.Figure 4+ to Sn2+34. The peak positions of Sn4+3d5/2 and Sn2+3d5/2 would be at 486.8 and 485.6 eV, respectively, and the 1.2 eV peak shift would be associated with the reduction of Sn4+34. However, no peak at around 485.6 eV was observed, which revealed that during the preparation of ITO sol-gel and the annealing process of ITO film, there was no Sn4+ reduction to Sn2+. Figure 2- lattice ions in In2O3, namely the oxygen-deficient region, and non-deficient region6.On the other hand, to know the oxidation state of Sn is very important, because the electrical conductivity reduces with reduction of Sn36. Fig. 2 (300 ± 5 nm)/Si substrate /Si was used as a reference substrate), the G and 2D bands of synthesized graphene on SiO2/Si appeared at 1,582.58 and 2,688.69 cm−1 respectively, with a very low intense D peak at 1,348.35 cm−1. The FWHM of the 2D band was around 28 cm−1, which can be used as a quantitative analysis of the number of layers of graphene, and the FWHM of G band was around 19 cm−1. The determined I2D/IG (intensity ratio of 2D to G) and ID/IG (intensity ratio of D to G) of graphene on SiO2/Si were 2.11 and 0.098, respectively. Figure 2/Si, and the blue graph represents the graphene on ITO/glass. These Lorentzian curves can be used along with the I2D/IG to represent the number of layers in graphene40. The single Lorentzian feature of the 2D band at room temperature, as shown in Fig. I2D/IG indicate the existence of monolayer graphene, since at room temperature, the 2D band for monolayer graphene shows a single Lorentzian feature, while bilayer graphene shows four Lorentzian features. The same single Lorentzian feature was also observed after graphene was transferred to the ITO/glass; however, in contrast, the G and 2D bands were broader, and a comparatively high D peak was observed, as shown in the blue graph of Fig. −1, respectively, when the CVD graphene was transferred to the ITO spin-coated glass. In other words, on average, the positions of D, G, and 2D peaks were red shifted by 2, 9, and 22 cm−1, respectively. Figure 2/Si).Raman spectroscopy was used to characterize the synthesized graphene because of its non-destructive nature, and the possibility of providing not only a wide variety of information, such as disorder, the number of layers, grain boundaries, and doping, but also the mechanical, electrical and optical properties44. Moreover, it was also known that when the graphene is in strain conditions due to the elongation of carbon – carbon bond, the breaking of symmetry and anisotropy nature of the graphene would strongly affect the redshift of Raman peaks46. The stress/strain might be initiated due to the lack of surface smoothness of the sol-gel derived ITO  coated glass. The heating effect during Raman spectra analysis may not have affected the shift of G peak position, since data was collected at room temperature, and all the observed Raman red-shifts were more than 2 cm−1, which is the previously reported red shift due to the heating effect48. In addition, the G peak position is known to not be changed, but would be split when the glass surface affects the Raman phenomena of the graphene50. However, that kind of glass surface-induced splitting of G peak was not observed in the above Raman spectrum, even after the graphene transference onto the ITO coated glass substrate. According to the obtained results, the most interesting fact is that this red shift highly influenced the 2D band more than the G band, and the red shift of the 2D peak is around 2.4 times higher than that of the G peak. Further, the D peak of graphene on ITO/glass was intense, while both G and 2D peaks were reduced, when compared with those peaks in the case of graphene on SiO2/Si. The ID/IG of graphene on the ITO/glass was 0.392, and it represents that the concentration of defects is higher than that of graphene on SiO2/Si (ID/IG of graphene on SiO2/Si was 0.147). This defective nature occurs during the transference of graphene onto the rough surface of synthesized ITO. The graphene transfer process tends to form wrinkles in graphene; moreover, the high surface adhesion in rough substrate helps to increase the number of wrinkles51. Fig. 2/Si substrate and graphene/ITO bi-film, respectively. The continuous graphene layer can be easily observed on the SiO2/Si substrate. However, while it is difficult to use the optical microscope to observe the continuity of CVD graphene on ITO in bi-film, even the graphene edges can be easily identified on the ITO. Figure The peak positions depend on not only the material structure, but also the external factors, such as doping level, stress/strain conditions, temperature, and environmental effects; the substrate-induced charge transfer by n-type doping affects red-shift, while p-type doping affects the blue shift of both 2D and G bandsFigure ITO) film according to the number of coating times, which was calculated by the following equation, where T is the optical transmission at 550 nm wavelength, and Rs is the electrical sheet resistance52.ITO was obtained from six times coated ITO film, indicating that the transmittance of the bi-film is highly dependent on the properties of ITO film.Figure 16. The thickness of the ITO film for 6 times coating was 100 ± 2 nm, which indicates that the thickness of one single coating of ITO is approximately 16.5 nm. The sheet resistance of the synthesized graphene was around 700 Ω/sq. The most interesting observation is that the sheet resistance of bi-film was 117 Ω/sq, which is much better electrical performance improvement of ITO (12.03%) compared to the previously reported value (2.39%) of graphene on ITO film by Liu J. et al. The improvement of electrical conductivity of the sol-gel derived ITO after the introduction of graphene on it occurs due to carrier mobility and surface carrier concentration improvement through graphene. Since the conductivity is proportional to both the carrier concentration and the carrier mobility, the same conductivity could be achieved from a large value of carrier concentration with a small value of carrier mobility, or a small value of carrier concentration with a high value of carrier mobility53. Graphene has high carrier mobility and of higher value than that in ITO22. However, even though the carrier density of graphene is lower than in ITO, it could be improved through the introduction of n/p doping from a foreign material, such as ITO in the present study. Therefore, by using graphene in contact with ITO, both the carrier concentration and carrier mobility of the bi-film could increase more than the values of the individual ITO. Furthermore, although the intrinsic carrier mobility of graphene would be degraded, due to scattering caused by charged impurities located near graphene/ITO interfaces, and contact resistance between graphene and ITO, the value remains higher than in individual ITO22. As both the carrier concentration and carrier mobility in bi-film show more increased values than those values in individual ITO, the bi-film shows better conductivity or current than does individual ITO. Moreover, graphene provides surface smoothness to the bi-film, which helps to enhance the electrical performances of high surface rough sol-gel derived ITO22. Fig. Simultaneously, the sheet resistance of the ITO film was also reduced up to six times of coating, with film thickness and sheet resistance of 100 ± 2 nm and 133 Ω/sq, respectively. As with the light transmittance, the sheet resistance of the bi-film is also dependent on the thickness of the ITO film that was used to produce the bi-film. After six times of ITO coating, a slight increment of sheet resistance was also observed. This behavior occurs in the sheet resistance of the ITO film due to the initiation and propagation of defects, the increment of micro-cracks, agglomeration of materials, and lower interaction within each layer and with the surface, after a certain thickness of the ITO film55. Additional resistivity is induced due to diffuse scattering of conduction electrons on the rough surface, and effect of surface roughness on electron scattering increases with increase of surface roughness. Conduction electrons impact each other during transferring and this acts as a barrier for free electron movement due to the effect of surface roughness scattering57. Effect of surface roughness on electron transfer can be explained in the following ways. Surface roughness introduces a variation of film thickness, causing fluctuations in electrostatic potential of the film and thus in the electron sub-band profile. Surface roughness changes the thickness of the film, introducing external variations in the sub-band energy, as well as fluctuations in the wave function shape56. Moreover, the Born approximations can be used to express the conductivity of film when surface roughness contributes to electron scattering57. In the framework of this theory, surface roughness scattering is becoming more critical due to rapid variation of film thickness and resulting in reduction of electrical conductivity55. Electron scattering can be reduced through improvement of surface smoothness, resulting in improvement of electrical properties through improvement of electron mobility. The conductivity of sol-gel derived ITO with rough surface can be improved by introducing graphene on ITO, since graphene acts as a bridge on the rough ITO surface, which facilitates a smooth path for electron transfer. In addition to the surface smoothness improvement of ITO from graphene, the carrier mobility of bi-film is improved over that of individual ITO22. So the surface carrier density improvement through electron doping from ITO and the carrier mobility improvement from graphene facilitate higher conductivity or lower sheet resistance to bi-film, when compared with the individual ITO. ITO film obtained from this study reveals high surface root mean squared (RMS) roughness of about 14.398 nm and mean roughness of 12.522 nm that comparatively much higher than reported roughness values of ITO from other synthesis method such as radiofrequency (RF) magnetron sputtering59. Graphene/ITO bi-film with surface RMS roughness of approximately 9.387 nm and mean roughness of 7.322 nm, indicating that surface roughness of ITO film can reduce by coating graphene on it  method  can be determined by using the plot of (αhν)2 versus hν, where α is the absorption coefficient, hν is the incident photon energy, and α can be calculated as follows:64g is related to α and hν as described in Tauc’s relationship, as follows:65Figure 2 versus hν plot of graphene/ITO bi-film, and the obtained optical band gap of the bi-film was around 3.82 eV. The Eg is highly dependent on the carrier concentration, and the wide Eg is related to the increment of the concentration of carrier charges, which can be explained by the Moss-Burnstein effect66.where, All data generated or analysed during this work are included in this article.Supplementary figures and table


Through our study, high carrier density induced from doping in both graphene and SWCNT leads to low contact resistance. This development of contact engineering, namely modulation of carrier density in the junction and contact length (Lcon) scaling shows the potential for all-carbon based electronics.In this work, we present the experimental investigation on the contact resistance of graphene/single-walled carbon nanotube (SWCNT) junction using transfer length method with the simple equivalent circuit model. We find that p–n like junctions are formed in graphene/SWCNT transistors, and the contact resistance in the junction is observed to be ~ 494 and ~ 617 kΩ in case of metallic SWCNT (m-SWCNT) and semiconducting SWCNT (s-SWCNT), respectively. In addition, the contact resistance increases from 617 to 2316 kΩ as VThe online version of this article (10.1186/s40580-017-0130-1) contains supplementary material, which is available to authorized users. Therefore, CNTs and graphene are perspective electrical materials for future devices due to their high mobility, high on/off ratio and flexibility, with the sizes down to < 10 nm  as a supporting layer, similar to the graphene transfer method.Aligned SWCNTs were grown on the pre-annealed  ST-cut quartz  substrate using CVD method. First, catalyst deposited quartz substrate  was inserted into a quartz tube and then heated up to 920 °C under ambient pressure with flowing Ar gas (1000 sccm) for pre-annealing of 1 h. After the pre-annealing, mixture of Ar (50 sccm), H2 (10 sccm) and CH4 (150 sccm) gas was injected to reaction chamber for 20 min for SWCNTs growth. The quartz substrate was rapidly cooled down to room temperature under Ar flow (1000 sccm) followed by the SWCNT growth [2 substrate in which silicon and silicon dioxide served as back-gate electrode and dielectric, respectively. To isolate each sets of TLM test structures, the pattern was formed by depositing a positive photoresist (AZ5214) and then etching by oxygen plasma. After removing the photoresist with acetone, the samples were annealed in a furnace under vacuum (10−4 torr) with Ar flow (500 sccm) at 400 °C for 3 h to remove PMMA and photoresist residue. The Cr/Au (~ 3–50 nm) pad electrode was e-beam evaporated on the top of graphene electrode in a vacuum with a pressure of 10−7 torr.A mask aligner system  was used for the patterning of graphene, SWCNTs, and metal pad lines on the Si/SiOThe microstructure of the graphene/SWCNT TFTs were observed with a scanning electron microscope (SEM) (Hitachi S4800) and atomic force microscopy (AFM). We analyzed the electrical characteristics using a simple equivalent circuit model in which we assume diffusive transport in the channel. The quality of graphene/SWCNT TFTs were characterized by Raman spectroscopy . All electrical properties were measured by a probe station (Keithley 4200-SCS).2 (100 nm) substrate. The single layer graphene was synthesized on the Cu foil with a conventional CVD method, as reported in our previous work [2 substrates by using PMMA as a supporting layer [ch of the devices was defined by various spacing of patterned graphene  and overlapping length and width  with aligned SWCNTs. The additional geometric information of graphene/SWCNTs device was shown in Additional file chs of aligned SWCNTs with the graphene electrodes and probing pads at each device. In previous reports, it was determined that the diameter of individual SWCNTs is an important factor in the device behavior, a result attributed to the band gap parameter of the SWCNTs [2 substrate was measured by AFM as shown in Fig. −1 and average diameter distribution at ~ 0.87 nm. Moreover, for the differentiation of the SWCNTs chirality in this sample, AFM images were used with Raman spectra of SWCNTs to measure the diameter distribution. Consequently, we assume that the ratio of semiconducting SWCNTs to metallic SWCNTs is 63:37, and both the SWCNTs (channel) and graphene (electrode) were observed to have few defects in Fig. m, Ns , Figure ous work . The higng layer . Detaile2 00 nm sub2 00 nm sub2 0 nm subs2 00 nm sub2 00 nm sub2 00 nm subd–Vd output characteristics of the graphene/SWCNTs transistors with typical Lch . The Lcon and channel width were 25 and 100 μm, respectively. The overall drain current responses linearly with drain biases, indicating an ohmic-like contact behavior of graphene/SWCNTs junctions. From the transfer characteristics of graphene-SWCNTs transistors, measured with the source grounded, the drain voltage held at − 0.01 V, and gate voltage swept between ± 30 V  and 56 kΩ μm−1 (s-SWCNT), respectively. Also, the contact resistance between graphene and SWCNTs at fixed contact area (25 by 100 μm) was estimated to be around 494 kΩ (m-SWCNT) and 617 kΩ (s-SWCNT), respectively. As the Vg in the channel is increased from − 30 to − 10 V with steps of 4 V, the contact resistance increases from 617 to 2316 kΩ  graphene/SWCNT junction TFTs. Figure cons. In the same manner as above, contact resistances were extracted and plotted on Fig. con shortened, the resistance increased. Some studied have suggested that transport between a metal and a SWCNT occurs only at the contact edge, with no Lcon dependence [Lpendence . This diT), which is the length along a contact at which the applied voltage drops to 1/e of its value, is extrapolated from line intercepts from the x-axis of the TLM plot. The best fit to the data using equation below is shown by solid curve. The determined LT from fitted line are 8.74 and 9.75 μm for m-SWCNT and s-SWCNT, respectively. The experimental data agree with the fitting model. This suggest that the contact resistance depends significantly on Lcon even in micrometer regime due to the long transfer length.The transfer length (Lcon can alter the contact resistance in graphene/SWCNT junctions. Moreover, further development of contact engineering such as investigation of SWCNT chirality and lowering the defect density of graphene shows the potential for all-carbon based electronics.In conclusion, we have explored the contact resistance in graphene/SWCNT junctions using TLM with a simple equivalent model. The Schottky barrier between graphene and SWCNT leads to a p–n like junction. We investigated the contact resistance modulation of graphene/SWCNT junctions, which is controlled by back-gate bias. From our measurement, the contact resistance in the junction was found to be ~ 494 and ~ 617 kΩ in the case of m-SWCNT and s-SWCNT. We confirmed that the work function tuning of graphene and SWCNT induced by doping and controlling the LAdditional file 1: Figure S1. Additional geometric information of graphene/SWCNT devices.


PDL1 mRNA was measured using RNA in situ hybridization (RISH). The overall prevalence of PD-L1 protein expression was 25.2% in tumor cells and PDL1 mRNA expression was 11.9%. There was a strong positive correlation between PD-L1 IHC and RISH results . In adenocarcinoma, PD-L1 protein and mRNA expressions significantly correlated with poorly differentiated histologic subtype . PD-L1 expression was also associated with genetic alteration in adenocarcinoma. High PD-L1 expression level was associated with EGFR-naïve and KRAS-mutant subgroup . With a 1% cut-off value, PD-L1 protein expression showed a short overall survival duration in early stage adenocarcinoma with marginal significance . Our study revealed that PD-L1 expression varied with histologic subtype and genomic alteration status in lung adenocarcinoma, and activation of the PD-L1 pathway may be a poor prognostic factor especially in early stage lung adenocarcinoma. In addition, PDL1 RISH showed promising results in predicting PD-L1 protein expression in NSCLC.In this study, we present the clinicopathological features associated with PD-L1 protein and mRNA expression in a large Asian cohort of patients with non-small cell lung cancer (NSCLC) and assessed the prognostic implications of PD-L1 expression, particularly in early stage NSCLC. We retrospectively analyzed 687 NSCLC specimens (476 adenocarcinoma and 211 squamous cell carcinoma) using tissue microarray. PD-L1 immunohistochemistry (IHC) was performed using Dako 22C3 pharmDx assay and  Researchers have recently become interested in developing immunotherapies for the treatment of non-small cell lung cancer (NSCLC), particularly monoclonal antibodies targeting the programmed cell death-1 (PD-1) receptor and its ligand (PD-L1) . InteracBesides acting as a predictive biomarker, PD-L1 shows inconsistent results among various studies as a prognostic biomarker. Studies investigating the prognostic role of PD-L1 and its association with clinicopathological features and driver mutations in NSCLC have yielded quite different results –13. ThisPDL1 mRNA exhibited a positive nonlinear relationship with PD-L1 protein using this assay, suggesting the potential applications of the RNAscope assay in future clinical studies [The variety of PD-L1 immunohistochemical (IHC) assays, involving the use of different antibodies and interpretation criteria, may also contribute to the lack of consistent results . Given t studies ,16.In this study, we evaluated the clinicopathological features associated with PD-L1 protein and mRNA expression in a large Asian cohort of patients with NSCLC and investigated the prognostic implications of PD-L1 expression, particularly in early stage NSCLC.th edition of the American Joint Committee on Cancer staging manual [Our cohort consisted of 687 patients with NSCLC, including 476 with adenocarcinoma (ADC) and 211 with squamous cell carcinoma (SqCC) who underwent surgical resection between May 2003 and December 2012 at Seoul National University Bundang Hospital. None received pre-operative chemotherapy or radiation therapy. Clinicopathological information was obtained from clinical records and pathology reports. The pathologic staging was based on the 7g manual . The stuAll resected tumor specimens were fixed with formalin and then stained with hematoxylin and eosin (H&E). All H&E slides were carefully reviewed by two of the authors (H. Kim and J.H. Chung) to confirm the original diagnosis and classify the histological subtype. ADC in situ and minimally invasive ADC samples were excluded from the study. All other invasive ADC samples were categorized as lepidic, papillary, acinar, micropapillary, solid, or invasive mucinous according to the 2015 World Health Organization Classification of Lung Tumors . These hThe slides were independently reviewed by two pathologists (H. Kim and J.H. Chung) to select the most representative sections. The most representative tumor area was carefully marked on the H&E-stained slide of each sample tissue. A TMA was constructed using 2-mm-diameter cores derived from the representative tumor areas selected at random of the FFPE tissue blocks from each case by SuperBioChips Laboratories .TMAs were sectioned at a thickness of 4-μm and stained using the Dako pharmDx assay. Briefly, the slides were stained with anti-PD-L1 22C3 mouse monoclonal primary antibodies with the EnVision FLEX visualization system on a Dako Autostainer Link 48 instrument , along with negative control reagents and cell line run controls, as per the manufacturer’s instructions. The IHC slides were scored independently by two pathologists (H.J. Kwon and H. Kim). PD-L1 was considered positive in tumor cells only in cases of at least 100 viable tumor cells, if membranous staining alone or membranous and cytoplasmic staining together was present. Membranous staining in tumor cells directly adjacent to immune cells was not considered positive if the surface touching immune cells was the only stained part. The percentage of stained cells in the overall area of the tumor (Tumor Proportion Score) was scored regardless of intensity . Cases wPDL1 mRNAs were measured using RNAscope assays  following the manufacturer’s instructions [ructions . BrieflyPD-L1 mRNA signals were in the tumor compartment or mesenchyme, as visualized by red dotted or clustered patterns. No standard scoring criteria for PD-L1 mRNA expression in NSCLC had been determined; therefore, we adopted the RNAscope system scoring guidelines (“RNA scope score”): 0 (no staining or < 1 dot per 10 cells); 1 (1–3 dots per cell); 2 (4–9 dots per cell); 3 (10–15 dots per cell); and 4 (> 15 dots per cell and > 10% dots in clusters) [PD-L1 RNA scope and proportion scores showed a linear correlation , cases showing either an RNA scope score of 1 or more or an RNA proportion score of 1 or more were designated as PD-L1 mRNA positive.lusters) . We alsoEGFR mutations in exons 18–21 and KRAS mutations at codons 12, 13, and 61, as described previously [ALK gene was assessed using fluorescence in-situ hybridization with an ALK probe  and a 15% cut-off value, as described previously [Polymerase chain reaction and DNA sequencing with FFPE tissue samples were used to analyze eviously . Rearraneviously .p values of less than 0.05.Statistical analysis was carried out using Stata Statistical Software version 14  and R program . Spearman’s test and logistic regression were performed to compare assays and determine appropriate cut-off values. Cohen’s ĸ coefficient of agreement was obtained to cross-check the results. A Kaplan-Meier analysis was performed to construct survival curves, and statistical significance was assessed using log-rank tests. A multivariate analysis was performed by Cox proportional hazards regression modeling. All statistical tests were two sided, and statistical significance was accepted for Clinicopathological characteristics are summarized in p < 0.05). PD-L1 mRNA expression was detected in 11.9% (82/687) of patients. In subgroup analysis, SqCC showed higher mRNA positivity than ADC . The overall prevalence of PD-L1 protein expression in tumor cells was 25.2% 173/687; . With a PD-L1 mRNA expression . As the TPS of PD-L1 protein expression increased, PDL1 mRNA expression was observed frequently  pairwise between assays at two PD-L1 IHC cut-off values (1% and 50%). OPA with 1% and 50% IHC cut-off values were 80.1% and 91.1%, respectively . Positivp < 0.001 and p = 0.002, respectively; EGFR-mutated group than in the EGFR-negative group , PD-L1 protein expression was higher in the KRAS-mutated group than in the KRAS-negative group (p = 0.017). Smoking history, pathological stage, and ALK status were not associated with PD-L1 status. In SqCC, only tumor size was associated with PD-L1 protein expression with marginal significance .Next, we investigated the associations between PD-L1 status and clinicopathological parameters in ADC and SqCC. In ADC, both PD-L1 protein and mRNA expression were correlated with histologic subtype (p = 0.02)  in multivariate analysis after adjusting for the conventional clinicopathological covariates .We performed survival analysis to investigate the prognostic role of PD-L1 protein and mRNA expression in ADC and SqCC. In ADC, lymphovascular/perineural invasion and pathologic TNM stage were independent poor prognostic factors for disease-free survival (DFS) and overall survival OS; . PD-L1 e = 0.02)  and tendvariates . For PD-PD-L1 mRNA expression and compared PD-L1 protein and mRNA expression; the results showed that PD-L1 mRNA was a potential surrogate marker, with a positive correlation with protein expression.In this study, we demonstrated that PD-L1 expression was significantly associated with histologic grade and genetic alteration status in lung ADC. We also found that PD-L1 protein expression was an adverse prognostic marker for OS in patients with early stage lung ADC. We also assessed Our study showed that PD-L1 protein and mRNA expression levels were significantly associated with high histologic grade and solid subtype of ADC. Our results are in line with the results of several studies which reported the correlation between PD-L1 expression in tumor cells and poor differentiation and solid histology –12. ThisFrom biological point of view, elevated expression of PD-L1 poorly differentiated lung ADCs compared with well-differentiated ADCs might account the inactivation of effector-immune cells through PD-1 receptor signaling which could ultimately enhance tumor progression. This relationship supports the results of our study that ADCs with high expression of PD-L1 are associated with poorly differentiated histology and poor prognosis.EFGR mutation status and PD-L1 expression in NSCLC is still unclear. Although preclinical studies have suggested that EGFR-driven NSCLC inhibits antitumor immunity through activation of the PD-1/PD-L1 pathway in an intrinsic manner, epidemiological studies have suggested that EGFR-mutant NSCLC is more likely to exhibit decreased PD-L1 expression. Two recent pooled analyses have provided further support for this inverse relationship. In one study, patients harboring EGFR mutations were more likely to have decreased PD-L1 expression  [EGFR wild-type status  [EGFR mutations and PD-L1 expression could be the variability, including the assessment of biomarkers from a single lesion site at a single time point, which often provides poor insights into spatiotemporal dynamics. For example, PD-L1 expression has been shown to fluctuate during EGFR tyrosine kinase inhibitor (TKI) and post-progression [EGFR TKI treatment, which could exclude the temporal heterogeneity of PD-L1 status. PD-L1 protein and mRNA expression were lower in the EGFR-mutated group than in the EGFR-negative group. Low PD-L1 expression in EGFR-mutated ADC may be related to the lower prevalence of PD-L1 in the Asian population than in Western populations. In our cohort, PD-L1 expression was observed in 16.2% of patients with a 1% cut-off and in only 6.1% of patients with a 50% cut-off. Several reports have shown low PD-L1 prevalence in lung ADC of Asian patients [EGFR mutation prevalence may be one of the causes of ethnic differences in PD-L1 expression.The relationship between 10–2.93) , and in  = 0.01) . One reagression . In our patients . Thus, tThe prognostic impact on PD-L1 expression in NSCLC is still controversial ,27, but In this study, PD-L1 expression was found as frequently in stages I and II  as in stages III and IV , indicating that aberrant expression of this ligand may be an early event. Patients with early stage NSCLC may have a more intact immune system and the potential for long-lasting immune priming against micrometastases . TherefoPDL1 mRNA expression. Further investigation of the mechanism of PD-L1 protein expression bypassing mRNA expression, and minimizing the analytic variables of RISH assay is necessary to reduce discrepancy between the two assay.Finally, we assessed PD-L1 expression using IHC and RISH. Many studies have attempted to assess PD-L1 expression using different techniques, including RISH ,16. The The lack of response data to anti-PD-1/PD-L1 drugs is the major limitation of our study. The issue on utilizing PD-L1 mRNA assay should be made based on data from clinical trials of the drug under consideration and further studies are needed with the therapeutic responses.EGFR-naïve status in lung ADC. In addition, PD-L1 expression may be a prognostic marker in patients with early stage lung cancer.In conclusion, elevated PD-L1 expression was associated with poorly differentiated histology and S1 Table(XLSX)Click here for additional data file.


Most theories of lie detection assume that lying increases cognitive load, resulting in longer response latencies during questioning. However, the studies supporting this theory are typically laboratory-based, in settings with no specific validity in security contexts. Consequently, using virtual reality (VR), we investigated how response latencies were influenced in an ecologically valid environment of interest to security professionals. In a highly realistic airport security terminal presented in VR, a security officer asked participants yes/no questions about their belongings. We found that liars actually responded more quickly to questions on which they were lying than to questions on which they were telling the truth. A control group, who answered the same questions but were not lying, answered equally quickly for all questions. We argue that this decrease in response time is possibly an unconscious reaction to questions on which individuals must answer deceptively. These results call into question the generalizability of previous research and highlight the importance of ecological validity when researching lie detection. These findings also uncover a new potential tool for enhancing lie detection in real-world scenarios. Due to increases in the number of terrorist attacks internationally this century , and the effect sizes were very heterogeneous across studies. The response latency effect was also affected by several moderators. The differences between truth tellers and liars were larger when people were discussing their feelings and facts than when they were only discussing facts. Giving participants time to prepare their statement reduced the difference between truth tellers and liars to nonsignificance, whereas motivating participants to do well increased it. The effect of response latency was larger in studies using within-subjects designs than in studies using between-subjects designs. Sporer and Schwandt concluded that the meta-analysis of response latencies supported the cognitive-load model and not the attempted-control model. DePaulo et al.’s (d = 0.02), but agreed on the high heterogeneousness of the studies in this area. Like Sporer and Schwandt, they found that the difference between truth tellers’ and liars’ response latencies was affected by preparation. In unplanned deception tasks, liars’ latencies tended to be longer, but for planned deception, this effect was reduced.Sporer and Schwandt’s  meta-anaet al.’s  meta-anar = .18). Colwell et al.  technology, we can now create realistic, immersive environments that individuals experience as similar to their counterparts in the real world, even in forensic settings . All participants were native or fluent English speakers. All had normal or corrected-to-normal vision to participate in the VR scenario, and all provided informed consent.Forty undergraduate students from Lancaster University were recruited to take part in this study, using opportunity sampling and the Lancaster University participant pool  by 2 (Question Type) by 8 (Question) mixed design. The deception variable was varied between subjects: Half of the participants’ bags contained nonrestricted as well as restricted items, which they were instructed to lie about (deception condition). The other participants’ bags contained all nonrestricted items (nondeception condition). The questions type variable was varied within subjects: Half of the questions required those in the deception condition to lie , but they responded truthfully to the others (control questions). In the nondeception condition, participants responded truthfully to all 16 questions. Question was also a within-subjects variable: All participants were asked eight questions per question type. The dependent measure was the response latency—the time between the end of the question and the start of the answer. Responses were recorded using Audacity software, which allowed for millisecond precision. We also assessed immersion using a standard immersion questionnaire  used was the Oculus DK2, which provided both position and orientation tracking of head movements.An empty backpack was provided for participants to pack specific items, depending on their assigned condition. These items included the items prohibited through airport security—a large bottle of liquid, a hammer, scissors, and lighter fluid—as well as the control items—a chocolate bar, a pair of glasses in their case, a box of paracetamol, a t-shirt, and a notebook. This aspect of the experiment, packing a bag with items about which the deception questions were asked, was adapted from Mullins et al. .A 3-D model of an empty industrial building was used to simulate the airport security VE, using the Unity3D software program. Objects typically found at airport security checkpoints, such as a metal detector and baggage X-ray machine, were put into this empty room. A stationary, armed airport security guard avatar was also present in the airport environment. The security guard avatar had his mouth covered throughout, so as to avoid issues with mouth movements not properly corresponding with the questions being asked. The security guard was also carrying a rifle, in an attempt to counteract the difficulty of mimicking the imposing atmosphere of real airport security checkpoints in this VR scenario. The VE was displayed through an HMD (Oculus DK2), which also had tracking technology enabled, allowing for participants’ head movements to correspond with visual changes in the VE.Using text-to-speech technology, a set of 16 questions, comprising eight control questions and eight experimental questions  requiring responses to participants’ experience in the airport environment was administered. Six of the seven questions used a 7-point Likert scale, whereas the final, open-ended question required a general response that (1) helped enhance the realistic nature of the airport environment for future experiments, but (2) reduced the realism of the scenario. The open-ended question was merely used to gauge participants’ experiences, to improve the scenario in the future. The answers from this question were not included in the immersion analysis. The overall scores, which were calculated from the sum of the six Likert responses, were used to measure participants’ immersion in the VR environment.This questionnaire was adapted from a similar immersion questionnaire developed by Slater, Usoh, and Steed , to see After going through the informed-consent procedure, participants packed the bag with the items they were to take through the VE. Participants then donned the HMD and were immersed in a VE resembling an airport security checkpoint, complete with metal detectors and baggage scanners see Fig. . Each paSDs from the mean. Another participant’s data were not included due to a technical error with recovering the recorded data; thus, their immersion responses were not used. Immersion scores were quite high, M = 26.87, SD = 5.58, and did not differ between conditions, p = .410.Trials on which participants did not respond, answered the question incorrectly, or did not hear/understand the question were removed from the analysis, which accounted for 2.6% of the data. One participant’s data were removed due to immersion scores that were –4 p = .052) nor that of deception condition (p = .525) was significant. However, the Question Type × Condition interaction was significant, F = 4.83, p = .034, ηp2 = .12; see Fig. t tests for the deceptive and nondeceptive groups, with question type as the within-subjects variable and mean latency as the dependent measure. For the deception condition, we found that the mean response latency scores were significantly longer for the control questions, M = 0.95, SD = 0.23, than for the experimental questions, M = 0.84, SD = 0.16, t(20) = 2.82, p = .011, d = 0.61; see Fig. M = 0.93, SD = 0.18, and experimental questions, M = 0.93, SD = 0.18, p = .89.We conducted a repeated measures analysis of variance, with condition as the between-subjects variable (control vs. deception), questions type  and question number (eight questions per type) as within-subjects variables, and response latency as the dependent measure. Neither the main effect of question type p = .05 nor thatt(37) = –1.62, p = .11, nor were the experimental question response latencies in the deception group significantly different from those latencies in the nondeception group, t(37) = –1.69, p = .099. This lack of differences was likely due to power, because the significant difference between the experimental and control questions in the deceptive group was a within-subjects comparison, whereas these t tests were performed between subjects. However, the within-subjects comparison was what could be used as a cue to deception in applied settings, not the group-level comparison. Therefore, the lack of significance at the group level was of less interest.For completeness, we directly compared the response latencies to the experiment and control questions between the deception and nondeception groups. Experimental question response latencies in the deception group were not significantly different from the control question response latencies in the nondeception group, when they were lying, whereas truth tellers responded equally quickly to both groups of questions. These results suggest that in this ecologically valid scenario, liars answer more quickly when lying, not more slowly, as has previously been thought. Additionally, because no difference was found between the latencies of the experimental and control questions in truth tellers, we can be reasonably sure that these results are not merely a consequence of the difference in content between the experimental and control questions.The results were opposite to those in the previous literature: Liars showed shorter latencies when lying. Interestingly, liars did not respond faster than truth tellers overall. Liars only responded more quickly faster when lyingThese findings may mean that in an airport setting, the cognitive-load aspect of lying is not as prominent as it is in laboratory situations. Because the “right answer” is known, the cognitive load induced by passing through a security check is likely lower for both truth tellers and liars than is answering the types of questions used in laboratory settings. The added cognitive load of lying is also reduced, because there is no need to actively create a new answer. This overall reduction in cognitive load, plus the reduction in load added by lying, likely means that the difference between truth tellers and liars in cognitive load is close to eliminated. However, this only explains why liars are not slower than truth tellers, not why liars are It could be that this reduction in response latencies is an effect of arousal or fear. In a more ecologically valid situation in which they are being questioned by an imposing figure, participants might experience more arousal or fear. Greater arousal is associated with faster response times in non-lie-detection fields of psychology (Eason & Harter, Alternatively, liars’ faster latencies may be a result of them either consciously or subconsciously responding more quickly when lying, to appear more confident in an attempt to deceive the officer. The main point supporting this interpretation is the finding that liars are only faster when lying. If liars were trying to be as fast as possible in order to appear more confident when engaging in deception, they would likely do so only for those questions. This decrease in response latencies could possibly also be an unconscious strategy. The response latencies were all under 1 s, and were even lower when participants engaged in deception. Therefore, it is unlikely that participants had time to deliberately contemplate answering more quickly to only the questions they answered deceptively, while answering less quickly to questions they answered truthfully. One possibility is that hearing a question in which one must answer deceptively triggers an impulse to respond as quickly as possible, especially for yes/no questions that the liar is anticipating. Future research should differentiate between the conscious and unconscious mechanisms. Following increased investigation, these findings may show a potentially powerful tool in deception detection.Interestingly, the majority of the lie detection literature has shown longer response latencies, but only when lying about arbitrary stimuli and using open-ended responses. These results highlight the notion that certain cues to deception do not provide a “one size fits all” solution. The context and types of questions matter in terms of how people engage in deception. Perhaps these differences as a consequence of context and question type are some of the reasons why the research on deception detection has been so divided. Likely, reliable cues to deception exist, but different cues are used that depend on the task and environment. Thus, for research on deception to be useful, it should focus on investigating cues to deception in the explicit scenario and on the types of questions in the environment of interest.A second area of future research should be expansion of the use of VR in lie detection research. Our findings that participants experienced high immersion and that truth tellers and liars did so equally mean that VR may be a solution to the problem of ecological validity in lie detection research. This problem is longstanding and well-known, but so far there has been little progress on a solution (see, e.g., DePaulo et al., Our research has uncovered a new, potentially useful cue to deception. Additionally, our results call into question the generalizability of previous research on response latencies as a cue to deception. They highlight the importance of ecological validity when researching lie detection, and uncover a new potential tool for enhancing lie detection in real-world scenarios.


Zostera marina throughout its range in the Northern Hemisphere. By contrasting host microbiomes with those of surrounding seawater and sediment, we uncovered the structure, composition, and variability of microbial communities associated with eelgrass. We also investigated hypotheses about the assembly of the eelgrass microbiome using a metabolic modeling approach. Our results reveal leaf communities displaying high variability and spatial turnover that mirror their adjacent coastal seawater microbiomes. By contrast, roots showed relatively low compositional turnover and were distinct from surrounding sediment communities, a result driven by the enrichment of predicted sulfur-oxidizing bacterial taxa on root surfaces. Predictions from metabolic modeling of enriched taxa were consistent with a habitat-filtering community assembly mechanism whereby similarity in resource use drives taxonomic cooccurrence patterns on belowground, but not aboveground, host tissues. Our work provides evidence for a core eelgrass root microbiome with putative functional roles and highlights potentially disparate processes influencing microbial community assembly on different plant compartments.Plant-associated microorganisms are essential for their hosts' survival and performance. Yet, most plant microbiome studies to date have focused on terrestrial species sampled across relatively small spatial scales. Here, we report the results of a global-scale analysis of microbial communities associated with leaf and root surfaces of the marine eelgrass IMPORTANCE Plants depend critically on their associated microbiome, yet the structure of microbial communities found on marine plants remains poorly understood in comparison to that for terrestrial species. Seagrasses are the only flowering plants that live entirely in marine environments. The return of terrestrial seagrass ancestors to oceans is among the most extreme habitat shifts documented in plants, making them an ideal testbed for the study of microbial symbioses with plants that experience relatively harsh abiotic conditions. In this study, we report the results of a global sampling effort to extensively characterize the structure of microbial communities associated with the widespread seagrass species Zostera marina, or eelgrass, across its geographic range. Our results reveal major differences in the structure and composition of above- versus belowground microbial communities on eelgrass surfaces, as well as their relationships with the environment and host. The health and performance of plants are often modulated by their associated microbiomes. The colonization of above- and belowground plant tissues by microorganisms from surrounding environments initiates interactions that are essential for plant productivity , 2, fitn5–8–Zostera marina, or eelgrass, in particular, provides a habitat for ecologically diverse and economically important ecosystems along coasts throughout much of the Northern Hemisphere  at the rarefaction depth considered herein. In contrast to belowground sediment communities, which exhibited higher taxonomic diversities than root communities on average , we did not detect a difference in Shannon diversity between eelgrass leaf and seawater microbial communities (P = 0.838)    on eelgrass host surfaces. Bacterial community α-diversity analyses revealed a higher taxonomic diversity  on below= 0.838) . A largeironment  and d. A< 0.001) . By contonships) .P = 0.005) (P = 0.185) (P = 0.96) and root microbiomes that were globally less variable compared with leaf communities (P < 0.001).The taxonomic compositions of leaves and seawater microbiomes were more similar within seagrass beds than between them (= 0.185) , which iF3,311 = 106.4, P < 0.001) when rarefied to a common sampling depth. We did not detect a difference in PD between eelgrass leaf and seawater microbial communities (P = 0.991), a result that recapitulates those from analyses of taxonomic community diversities , whereas root communities were phylogenetically more distinct from sediment communities . However, in contrast to results from taxonomic diversity analyses, eelgrass leaves were not more phylogenetically similar to seawater communities from the same seagrass bed in comparison to seawater communities from other seagrass beds, indicating that seawater harbors globally phylogenetically similar communities that can assemble onto seagrass leaves despite differences in the specific OTUs present in different locations.Analyses of rarefied Faith's phylogenetic diversity (PD)  and norm compare  and e. L compare   on the Z. marina leaves relative to seawater , of which most of the cultured isolates are sulfide oxidizers  to identify OTUs that were significantly enriched or depleted on hosts relative to their mean normalized abundance in the adjacent putative colonization source. We observed 33 enriched and 116 significantly depleted OTUs on seawater , reveali classes , consistP = 0.141) (P = 0.008) (P = 0.009), suggesting that predictions from metabolic modeling were not simply recapitulating the phylogenetic relationships between taxa  . However= 0.008) , suggesteen taxa .Zostera marina eelgrass microbiome revealed a high degree of similarity between leaf and seawater communities in comparison to those of root surfaces, whose taxonomic and phylogenetic compositions were less heterogeneous than, and more distinct from, the surrounding sediment  under different biotic and abiotic conditions to uncover the relative contributions of host- versus mutualism-based strategies by vascular plants in marine sediments for tolerating toxic sulfide concentrations. Our results indicate that this will be an important enterprise for future research.Prior research has documented a positive relationship between seagrass biomass production rates and the density of sulfide-consuming lucinid clams in seagrass beds, owing to the hypothesized am gills . Howeveram gills . Thus, uam gills  certainlZ. marina seagrasses by contrasting host samples with those of their surrounding environments across the entire Northern Hemisphere. We hope that this will encourage researchers to study the microbiomes of other plant hosts across their geographic ranges, as such large-scale studies produce the empirical knowledge needed to develop a deeper understanding of microbial roles in the ecology and evolution of plants and the ecosystems that depend on them.Seagrasses and their ecosystems have been the subject of a great amount of research covering topics including ecology and biogeography , evolutiZostera Experimental Network (ZEN), a global network of seagrass scientists , using the methods described by Meadow et al.  + G + H + E, where Y is the diversity index, B1 is a regression coefficient, G and H are random effects of the seagrass bed, to account for the fact that multiple plants were measured from the same bed, nested within a spatial block (ZEN location), and E is a vector of errors. The normality of residuals was confirmed in all models using quantile-quantile plots. All models were fitted using the nlme package in the statistical programming environment R  were calculated at this sampling depth using the Shannon and Faith's PD  indices,onment R . Pairwispost hoc test for pairwise contrasts.Community taxonomic and phylogenetic compositional dissimilarities  between host and environmental samples were calculated using the Canberra and normalized unweighted UniFrac  distanceTo test the hypothesis that eelgrass-associated microbiomes were more similar to their adjacent environmental communities than to others , we analyzed community dissimilarities of host and environmental samples at the scale of the seagrass bed using a Monte Carlo bootstrapping approach following ordination  on rareP < 0.01), as these taxa represent portions of the microbiome that were most likely to be actively selected for by the host  method , which wthe host .ijc, representing the Jaccard distances between the seed sets of OTUs i and j. The relationship between cooccurrence dissimilarity (measured as Jaccard distances of OTU abundance profiles) and OTU resource dissimilarity (C) matrices were assessed for leaf- and root-enriched taxa using Mantel tests with 1,000 matrix permutations. If enriched taxa are more likely to cooccur when they utilize similar predicted metabolic resources, then we would expect a positive correlation between the cooccurrence and C dissimilarity matrices. Such patterns are consistent with a community assembly mechanism whereby organisms that require a particular set of resources tend to cooccur in environments that contain those resources  under accession number