Diachasmimorpha longicaudata entomopoxvirus. Phylogenetic sequence analysis of all Poxviridae F9 and L1 orthologs revealed the SGPV genome to contain the most distantly related F9 and L1 sequences compared to the vaccinia proteins studied here. The structural differences between F9 and L1 suggest functional adaptations during evolution from a common precursor that underlie the present requirement for each protein.The poxvirus F9 protein is a component of the vaccinia virus entry fusion complex (EFC) which consists of 11 proteins. The EFC forms a unique apparatus among viral fusion proteins and complexes. We solved the atomic structure of the F9 ectodomain at 2.10 Å. A structural comparison to the ectodomain of the EFC protein L1 indicated a similar fold and organization, in which a bundle of five α-helices is packed against two pairs of β-strands. However, instead of the L1 myristoylation site and hydrophobic cavity, F9 possesses a protruding loop between α-helices α3 and α4 starting at Gly90. Gly90 is conserved in all poxviruses except Salmon gill poxvirus (SGPV) and  The MV consists of a nucleoprotein core surrounded by a single lipoprotein membrane thought to be derived from the endoplasmic reticulum; the WV contains two additional membranes acquired from the trans-Golgi network; during exocytosis the outer WV membrane is lost forming the EV, which has two membranes4. To enable replication, the viral core harboring the double-stranded DNA genome is released into the cytoplasm, which defines the entry step5. MVs can enter the cell at neutral pH at the plasma membrane or through an endocytic route at low pH7. EVs shed their outer membrane at the cell surface allowing MV-like particles to fuse with the plasma or endosomal membrane8 following macropinocytosis9. The fusion apparatus is embedded in the MV membrane and consists of eleven proteins namely A1610, A2111, A2812, F913, G314, G915, H216, J517, L118, L519, and O320, which comprise the entry fusion complex (EFC). These proteins were identified in vaccinia virus (VACV) based on the inability of mutants to mediate entry and their interaction to form a complex. All Poxviridae share orthologs of these proteins with the apparent exception of Salmon gill poxvirus (SGPV), for which homologs of G3, L5, and O3 were not identified21. Three heterodimeric sub-complexes, A16/G9, A28/H2, and G3/L5, have been identified in the VACV EFC24. Interactions between these and other components of the EFC remain under investigation. The EFC proteins have N- or C-terminal hydrophobic domains but lack signal peptides and are unglycosylated, suggesting that they do not normally traffic through the ER and likely insert directly into the viral membrane. The EFC proteins are not required for the attachment of virus particles to cells, which is mediated by other viral proteins that interact with glycosaminoglycans and chondroitin sulfate27.Poxviruses are large enveloped DNA viruses that replicate in the cytoplasm and exist in three distinctive infectious forms: mature virions (MV), wrapped virions (WV), and extracellular virions (EV)17, but the complex has not been physically characterized. Since each EFC component has a putative transmembrane domain, they may be organized in a two-dimensional network on the MV membrane. Except for L1 and F9, each of the EFC proteins is required for assembly or stability of the complex. For this reason, L1 and F9 are sometimes referred to as EFC-associated proteins and the others as core components18. L1 and F9 have approximately 20% amino acid identity. Nevertheless, each is required for entry suggesting that they arose from a common ancestral gene but evolved distinct functions13. Knock-down of F9 has a greater effect on the hemifusion step of entry than knock-down of L128. In addition, the binding of a neutralizing antibody to a conformational epitope on L130 reduces core entry but not the hemifusion step28. Polyclonal antibodies induced by immunizing rabbits with soluble F9 protein neutralize infectivity but were not tested in a hemifusion assay13. Recently, a human monoclonal antibody against F9 was isolated and characterized but did not neutralize virus in a plaque reduction test31.The association of the 11 EFC proteins was demonstrated by immunoaffinity purification32. The L1 structure exhibits a bundle of α-helices leaning against a pair of two-stranded β-sheets and a disorganized C-terminal tail that is proximal to the transmembrane domain. A hydrophobic cavity capable of accepting and shielding a conjugated myristic acid is located near the N-terminus32. Myristoylation of L1 is required for infectivity but not for assembly of the EFC and incorporation into viral particles33. The amino acid motif for adding a myristate moiety is conserved in all poxvirus orthologs of L1, except in Salmon gill poxvirus (SGPV)21. In contrast, none of the F9 orthologs have a predicted myristoylation site.Based on their primary structures, none of the EFC proteins resemble type 1, 2 or 3 entry proteins of other viruses. Until now, L1 was the only EFC component with a determined atomic structure32. Here, we report the 2.10 Å structure of F9 and demonstrate a fold similar to L1 but without a hydrophobic cavity. A novel feature, not found in L1, is a loop protruding from the F9 surface. We also analyzed the phylogeny of F9 and L1, which likely arose from a common ancestral gene, within the family Poxviridae. Homology based three-dimensional models of SGPV F9 and L1, the most distant orthologs relative to VACV, were generated and compared to the atomic structures of VACV F9 and L1.F9, the subject of this study, is a 23.8 kDa protein with a putative transmembrane domain near the C-terminus. Based on the amino acid sequence, F9 was predicted to have a structure similar to that of L134 of 2.93 nm suggested that F9 may not have been monomeric at high concentration  of 2.06 Å calculated over 120 α-carbons  revealed conserved amino acid residues and residues with conservative and semi-conservative changes in F9 and L1. The degrees of conservation were used to color the surface images of F9 and L1 in a heat map display or color ramp . The identity between the full length F9 and L1 of VACV was 20%.When F9 and L1 orthologs were compared to each other, the highest identity (24%) was found in Goatpoxvirus ‘Pellor’ . Entomopoxvirinae F9 orthologs to F9 are between 1.30 and 1.69 substitutions per site. Interestingly, the distance of F9 to SGPV097, the L1 ortholog, is only 1.53 substitutions per site. In case of VACV L1, the distance to SGPV097 is 1.17 substitutions per site. VACV L1 and its orthologs in Entomopoxvirinae have a distance of 0.88 to 1.03 substitutions per site.A phylogenetic tree was constructed with the neighbor joining method and 1,000 bootstraps using the conserved sites (90 amino acid residues). However, tree calculations including all gap free sites (177 amino acid residues) resulted in the same phylogeny with slightly increased distance values. The root was forced between F9 and L1, which form two distinctive clusters in an unrooted tree Fig. . Clades 39. The protein sequences were aligned and cysteines in SGPV092 were presumed to form disulfide bonds as in F9. Distances between relevant cysteines forming disulfides were restricted to 2.00 Å in iTASSER. We then superimposed both structures in PBDeFold . In the present study, we determined the protein structure of F9, one of the eleven EFC proteins. We demonstrated through X-ray crystallography that the overall structure of F9 is a bundle of α-helices leaning towards and packed against two pairs of β-sheets. Three disulfide bonds covalently link the helices and sheets and are not solvent accessible, which was anticipated due to the relatively reducing cytoplasmic environment where F9 is expressed. F9 exhibits a distinct protein fold not found in structures other than L1 as determined using the DALI server32. The DALI comparison of F9 and L1 (PDB code: 1ypy) has a Z-score of 15. Despite their similarity, F9 and L1 are each required for fusion and entry of the viral core suggesting distinct functions35. Within the EFC, there are two groups of sequence-related proteins: A16, G9, J5, and F9, L1. These two groups of related proteins are present in all members of both the Chordopoxvirus and Entomopoxvirus subfamilies suggesting that their sequences and functions diverged early in poxvirus evolution. With regard to the divergence of F9, its differing orientation of helix α3, its absence of a myristoylation site, and its lack of a hydrophobic cavity likely contribute to F9 functional differences from L1. Nevertheless, the comparable parts of the α-helices of F9 and L1 align with an RMSD of 1.77 Å. The best overlap between F9 and L1 was found within the β-sheet region with an RMSD of 1.68 Å over all four β-strands.F9 resembles the structure of L141. These regions are also highly hydrophobic and therefore might be shielded from aqueous solvent by binding other members of the EFC. However, there is no evidence that F9 and L1 are interacting directly with each other in the EFC18. Although the large interaction surface between the β-sheets areas of adjacent F9 molecules  and L1 molecules  in the crystals suggest a homodimer formation, their biological relevance, if any, still has to be proven. In F9, the area predicted to interact with proteins comprises α4. A stabilization within that surface would also provide a stable environment for the emerging loop but allow flexibility within the α1-α3, and β1 and β2 area.Some residues have been predicted to be involved in protein-protein interactions. This hydrophobic surface area might interact with other VACV EFC proteins for quaternary structure formation. Larger hydrophobic interfaces have been described for obligate complexes42 indicated that the three-dimensional structure of proteins is evolutionary more conserved than expected by sequence homology. The phylogenetic analyses suggested that the F9 ortholog in SGPV, SGPV092 (2.0504 substitutions/site), is even more distant to VACV WR F9 than the subfamily of Entomopoxvirinae (1.3044 to 1.6919 substitutions per site). The results are also supported by the identities calculated with ClustalX43. Differences in the structures of the VACV and SGPV orthologs of F9 are predicted within the loop region between α3 and α4. Assuming all disulfide bonds are formed, the protruding loop formed in F9 would be significantly shorter and partly defined in a helical formation in SGPV092. Furthermore, there is no Gly in SGPV092 corresponding to Gly90 in F9 that might allow the formation of a longer protruding loop by breaking the α-helix.A database search to determine the conservation of full length F9 and L1, revealed a higher degree of conservation in L1. We chose to model the most distant orthologs of F9 and L1 to estimate structural conservation. Analyses by Kaczanowski and ZielenkiewiczDiachasmimorpha longicaudata entomopox. The L1 ortholog in SGPV (SGPV097) does not exhibit a predicted N-terminal myristoylation site. The threaded structural model shows a small cavity not large enough to incorporate a myristic moiety. In addition, the Phe108 (L1) which is located at the end of the hydrophobic cavity might have a counterpart in Phe105 (SGPV097) that is modeled at the N-terminal end of α4 and would, therefore, not be facing the inside of the hydrophobic cavity. Phe108 of L1 is conserved in all other poxvirus orthologs and shown to be essential for conformation and complete disulfide bonding33. The N-terminal modification by myristate and the formation of a hydrophobic cavity in L1 for burying the myristic acid leads to a higher degree of conservation in L1 because of these constraints on sequence changes.The only other F9 ortholog without a Gly in this area is from 21. We also screened the SGPV genome for the presence of the O3 protein, another component of the EFC. No related sequence was found by alignment nor was a putative O3-like hydrophobic protein identified within the haplotype block. With the differences in F9 and the absence of G3, L5, and O3, Salmon gill poxvirus (SGPV) may have developed a modified version of the poxvirus EFC.It should be noted that no orthologs for G3 and L5 were found in the SGPV genomeE. coli and synthesized by Blue Heron Biotech in the expression vector pNAN44. The plasmid was transformed into NiCo21(DE3) cells (New England BioLabs). Cultures were grown until OD600nm of 0.5–0.6 and the T7 promoter-driven protein expression was induced with the addition of 1 mM isopropyl β-D-thiogalactoside (IPTG). Cells were harvested by centrifugation and the cell pellet washed once with resuspension buffer ). The cell pellet was suspended in resuspension buffer and stirred for 1 h. Five volumes of lysis buffer , 5 mM MgCl2) with 1 mg/ml lysozyme and 750 U benzonase were added. Samples were stirred for 1 h at room temperature. Further cell lysis was achieved by three cycles of freeze-thawing. The inclusion body fraction was harvested by centrifugation, then washed and homogenized three-times in lysis buffer without MgCl2. After three washes in wash buffer  inclusion bodies were denatured overnight at 4 °C in 100 mM Tris-HCl, pH 8.0, 6 M guanidine-HCl, 10 mM DTT, 1 mM EDTA. Insoluble material was removed by centrifugation at 10,000 × g for 1 h at 4 °C. Inclusion bodies were stored at −80 °C until use.DNA encoding the first 176 amino acid residues of vaccinia virus (VACV) Western Reserve F9, lacking the transmembrane segment, and containing nucleotides for an N-terminal 6xHis tag, spacer and TEV protease cleavage site was codon optimized for −1cm−1. To remove the N-terminal tag, F9 protein was incubated with TEV protease (10:1 F9 to protease by weight) overnight at room temperature. Uncleaved F9 and recombinant protease were removed by binding to Ni-NTA. Highly pure F9 was obtained by SEC using a Superdex-200 column and 10 mM Tris-HCl, 50 mM NaCl, pH 8.0 running buffer. The monomeric fraction was collected and concentrated to 10 mg/ml with centrifugal filter units (EMD). After removing precipitated material by high-speed centrifugation, the integrity of the concentrated protein was monitored by dynamic light scattering. The purity of the protein was >95% as assessed by SDS-polyacrylamide gel electrophoresis.Approximately 10 µmol F9 inclusion bodies were refolded by rapid dilution into 1 l of 50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 500 mM arginine, 2 mM EDTA, 40 mM sucrose, 10 mM DTT, 5 mM cystamine-HCl. After refolding at 4 °C overnight, 50 mM cystamine-HCl was added to react with free cysteines. The solution was dialyzed twice against 50 mM Tris-HCl, 50 mM NaCl, pH 8.0. After filtration through a 22 µm filter membrane, F9 protein was loaded onto a Ni-NTA column (GE) equilibrated with 50 mM Tris-HCl, 50 mM NaCl, pH 8.0. The column was washed with three column volumes of equilibration buffer. Bound F9 was eluted with a linear increasing imidazole gradient. Fractions containing eluted protein were combined and dialyzed against 50 mM Tris-HCl, 50 mM NaCl, pH 8.0 overnight to remove imidazole. The concentration was measured spectrophotometrically at 280 nm using a molar extinction coefficient of 10,805 Met al.45. Briefly, the TEV protease was produced as a fusion with maltose binding protein (MBP), which cleaves itself from the fusion in vivo. The resulting soluble TEV protease was released from the cells by sonication and purified with Ni-NTA chromatography. Eluted TEV protease was dialyzed and subsequently loaded onto a Superdex column. Concentrated TEV protease was flash frozen in liquid nitrogen and stored at −80 °C until use. The purified protease was concentrated to ~1.5 mg/ml and flash frozen in liquid nitrogen.Plasmid pRK793 (Addgene) encoding recombinant 6 × His-TEV(S219V)-5 × Arg protease was transformed into NiCo21(DE3) cells. Production and purification was done according to Tropea Native crystals were obtained in hanging drops by vapor diffusion with two buffer conditions. One mother liquor contained 3.0–3.2 M NaCl and 0.1 M 2-(N-morpholino)ethanesulfonic acid (MES). For cryo-protection and phasing the crystals were incubated in 3.5 M NaCl, 0.1 M MES, 1 M NaI for 30 s at room temperature. Crystals were also obtained from a solution containing 25% PEG 8000, 10% ethanol, 1–5% cocktail mixture of low molecular alcohols, 30% sucrose at pH 8.5 buffered with Tris in the presence of 50 mM NaCl. The drops were set up with 1 µl protein and 1 µl mother liquor. For X-ray data collection, crystals were harvested and flash frozen in liquid nitrogen.47 were collected on a RIGAKU MicroMax-002 + high-intensity microfocus sealed tube X-ray generator equipped with an R-Axis IV++ image plate detector using Cu radiation. The data processed with XDS48 showed the presence of a strong anomalous signal . AutoSol located nine iodide sites, and anomalous difference maps confirmed that the space group was P4122. Auto building of the F9 protein model was able to locate two F9 molecules per asymmetric unit, which was consistent with the prediction based on the calculation of the Matthews coefficient52. The auto-built model was 60% complete. This partial model was further improved by employing cycles of refinement using the programs CNS53 and Phenix followed by manual model rebuilding using the programs O54 and Coot55 into new electron density maps calculated after each refinement cycle until no further improvement of the model could be achieved at the 3.20 Å maximum resolution of the P4122 data set. This model was subsequently used as a search model for obtaining a molecular replacement solution in a 2.10 Å data set from an F9 crystal that belonged to the P212121 space group and contained four molecules in the asymmetric unit. Multiple cycles of refinement with stepwise increase in the resolution followed by manual rebuilding using the same programs as above produced the final model that includes residues 6–164 for all four molecules with an Rwork/Rfree of 22.57/26.85 and good geometry. The F9 structure as accession code 6CJ6 was submitted to the RCSB Protein Data Bank at www.rcsb.org.X-ray data on F9 crystals rapidly soaked with NaIal Table  and were36. The precision was set on ‘highest’ to calculate the RMSD and the structural alignment. The output pdb-files were used to visualize the alignment in Chimera56. Protein-protein interaction sites for comparison of both structures and for comparison to contact areas within the crystals were predicted with meta-PPISP37.The F9 chain A was set as query and the chain A of L1 database entry 1ypy was used as the target in PDBeFold57 and full-length F9 or L1 from VACV WR as query sequence, we set searching parameters to BLOSUM45  and organism Poxviridae. Partial sequences were removed from the sequence data sets. Sequences of L1 orthologs recovered together with the F9 sequence set were identified by their myristoylation site and removed and vice versa. The collected sequences were aligned in MAFFT38 with BLOSUM45 and ‘leaving gappy regions’. The MSA was used to identify the conservation state of each residue. Using a proportional gradient, the surface conservation was visualized with Chimera56. The MSA was also entered into ClustalX43 to calculate the pairwise % sequence identity scores. We used the sequence identity to estimate the relationship between F9 and L1 orthologs within one virus species. Phylogenetic trees were constructed by the Neighbor joining method. The confidence values of nodes were calculated with 1,000 bootstrap replications. The SGPV L1 ortholog SGPV097 did not present a predictable myristoylation site. SGPV F9 homolog SGPV092 and SGPV097 were present in both dataset and used as outgroups to root the respective trees. After combining both datasets and aligning F9 and L1 orthologs together, the root of the phylogenetic tree was forced between F9 and L1 orthologs. Dendroscope58 (www.dendroscope.org) was used for visualization.Using BLASTpet al.35 have shown that missing disulfide bonds destabilize the protein and thus impair its function. VACV F9 and SGPV092 as well as VACV L1 and SGPV097 were superimposed by their structures using PBDeFold with ‘highest’ precision. The VACV proteins were defined as query and the SGPV sequences were defined as target. The three-dimensional structural alignments were collected in a RMSD distance matrix.Atomic structures of F9 and L1 were used as query structures to obtain a model of the respective SGPV proteins using iTASSER. The most likely model 1 was processed. In the case of F9, both proteins were aligned using BLOSUM45 and distances of Cys in SGPV092, which correspond to disulfide bonds in F9, were restricted to 2.00 Å. More constraints for modeling SGPV092 on F9 were necessary to obtain closed disulfide bonds. Senkevich Supplemental MaterialSupplemental table 1Quality report


Radiology images are an essential part of clinical decision making and population screening, e.g., for cancer. Automated systems could help clinicians cope with large amounts of images by answering questions about the image contents. An emerging area of artificial intelligence, Visual Question Answering (VQA) in the medical domain explores approaches to this form of clinical decision support. Success of such machine learning tools hinges on availability and design of collections composed of medical images augmented with question-answer pairs directed at the content of the image. We introduce VQA-RAD, the first manually constructed dataset where clinicians asked naturally occurring questions about radiology images and provided reference answers. Manual categorization of images and questions provides insight into clinically relevant tasks and the natural language to phrase them. Evaluating with well-known algorithms, we demonstrate the rich quality of this dataset over other automatically constructed ones. We propose VQA-RAD to encourage the community to design VQA tools with the goals of improving patient care. Deep learning models require vast amounts of data to approach abilities of humans and as a result, the most popular training datasets are crowdsourced with general public knowledge4–8 or synthetically generated10. The images in most of these datasets were taken from Microsoft Common Objects in Context (MSCOCO)11. While researchers have tried balancing these large datasets12, bias in selection of images and questions makes it difficult to transfer models trained on these datasets to specific applications. For example, the creators of VizWiz13, the first VQA dataset designed from images taken by blind users and visual questions from those use cases, demonstrated challenges in using the state-of-the-art models trained on the larger VQA datasets to predict answers from their VizWiz blind users generated data.Visual question answering (VQA) is a computer vision and artificial intelligence (AI) problem that aims to answer questions about images. As more of medicine is digitized and medical data continues to grow, there is enormous opportunity for multimodal tools such as VQA to benefit patients, clinicians, and researchers. Radiology, with its wealth of images and textual reports, is a prime area where VQA could assist radiologists in reporting findings for a complicated patient or benefit trainees who have questions about the size of a mass or presence of a fracture. Many different techniques are applied to build VQA systems including computer vision, natural language processing, and deep learning. These systems need to be trained for the task and evaluated on large data collections consisting of images and pairs of questions asked about the images with corresponding answers. Although there has been great progress in image recognition in radiologyWhile visual questions are prevalent in medicine, most communications between clinicians are not usually documented in the patient’s record or available for researchers. Medical education is one area where collections of visual questions exist. Through their training and continued medical education, clinicians will take exams testing their knowledge of medical images. However, these test questions often go beyond the scope of the image and require extra knowledge about epidemiology or next step treatments. Most exam questions may not be suitable for teaching machines to answer descriptive questions about images, but they still have a role for more abstract machine assisted question answering in the future.14 and coordinated the first community-wide VQA challenge in medicine. While the dataset is an excellent starting point for the medical domain, several design issues prevent useful clinical applications. To overcome the lack of readily available natural visual questions, questions and answers were automatically generated from corresponding captions. Unfortunately, this resulted in many artificial questions that do not always make sense, to the point where a human could not reason what the questions were trying to ask. Another issue with the dataset is that images were automatically captured from PubMed Central articles. This automation may not help VQA support clinical tasks as the dataset included composites of a series of images and 3D reconstructions that are rarely useful for direct patient care. If VQA tools are to assist in clinical processes, the datasets need to be designed with aligned goals.In 2018, ImageCLEF-Med released a radiology datasetWe introduce VQA-RAD, a manually constructed VQA dataset in radiology where questions and answers about images are naturally created and validated by clinicians. Trading off quantity of automatic generation, VQA-RAD is a high-quality design with only 60 hours of specialist contributions. Structured like other existing VQA datasets, each question can be answered with a single image alone. The images are a balanced sample from MedPix®, an open-access radiology archive of case reports and teaching cases. These images were presented to clinicians who wrote unguided questions they would ask a colleague or radiologist. To further explore the natural clinical language, clinicians also paraphrased questions in both free-form and template structures. All question-answer pairs are manually validated and categorized, helping to characterize clinically important areas of focus. The flow of building the VQA-RAD dataset is shown in https://medpix.nlm.nih.gov/, an open-access database of radiology images and teaching cases. Our sampling criteria were as follows: (1) Only one image for each teaching case, so that all images represented unique patients. (2) All images are sharp enough to identify individual structures. (3) Images are clean of radiology markings, such as arrows or circles. (4) Images have captions that correspond to the image and are detailed enough to describe at least one structure.We sampled images from teaching cases in MedPix, Today, diagnostic imaging often contains stacks of images, which is a challenge that VQA tools will need to incorporate to be useful for assisting radiology. For a dataset just emerging into medicine, we wanted to start with simple one image and one question pairs, enabling comparisons with current existing VQA dataset structures and algorithms. To reduce bias of having multiple images of the same pathology, we chose to limit one image per case, since each case is a unique patient. In addition, MedPix images are stored in JPG format, which needs to be taken into consideration when using the images for teaching and for training AI approaches.Captions include plane, modality, and image findings that were generated and reviewed by expert radiologists. In total, we selected 104 head axial single-slice CTs or MRIs, 107 chest x-rays, and 104 abdominal axial CTs. The balanced distribution from head, chest, and abdomen should help determine if visual questions differ for each organ system and if the algorithms perform differently on different regions.12, annotating radiology reports from MedPix, and categorizing CME questions from open-access domains such as MedPix, AuntMinnie (https://www.auntminnie.com/), and Radiopaedia.org (https://radiopaedia.org/). Question types were further refined after analyzing an initial sample of visual questions from annotators. The final taxonomy of medical VQA in our dataset is shown in In addition to categorizing images, we define several question and answer types. Broad question types were initially developed from a combination of using Task Directed Image Understanding Challenge (TDIUC) categoriesThe tasks of assisting clinicians with radiology are different and range from support for those just learning the basics of reading a plain film x-ray, to those who are senior radiologists investigating rare presentations of diseases. We selected medical students and fellows who had at least their core clinical rotations completed because they will better understand how radiology is incorporated into every day clinical decision making and patient care. These trainees’ questions and tasks are closer to those of attending trainees, compared to students just starting medical school. However, we do expect that there will be additional needs from resident trainees as their personal skill in reading radiology images improves and they have more nuanced questions. For example, a junior trainee may ask if there is presence of a pneumothorax, while a senior, who can already recognize air in the lungs, may ask more questions about the size of the pneumothorax since this has implications for invasive treatment.rd year of school and exposes students to major fields of medicine such as surgery, internal medicine, neurology, etc. This ensures that all participants have basic clinical radiology reading skills and were exposed to a variety of settings where radiology was vital to the management of patients. Our participants had training from different regions of the U.S. and there was one board certified pathologist, three applying into ophthalmology, four applying into family medicine, two applying into internal medicine, two applying into emergency medicine, one applying into radiology, one applying into orthopedics, and one dermatology.Questions and answers were generated by 15 volunteer clinical trainees using a web-interface developed for collecting the questions and the answers. We asked for volunteers from the clinical fellows from the NIH and students from class of 2018/2019 University of Massachusetts, classmates of one of the authors, Jason Lau. All participants had completed the core rotations of medical school, which typically occurs during the 3Participants enerated questions and answers in a two-part evaluation shown in  from DecFor the next 20 images, participants were randomly paired and given another participant’s images and questions. They were asked to generate “rephrased” and “framed” questions based off the given “free-form” questions with corresponding image and caption. We asked the participants to paraphrase the question in a natural way and generate an answer that agreed with both the original and the paraphrased questions.Participants generated “framed” questions by finding the closest question structure from a list of templates and filling in the blank spaces to retain the answer to the original questions.After completion of the evaluations, we used several methods to validate questions answer pairs and question types. During the paraphrasing part of the evaluation, participants answered another person’s questions. The answers could have strict or loose agreement. We defined strict agreement when the question and answer format and topic were the same. In loose agreement, the topic of the questions is the same or similar even though the answers may differ. Three subcategories of loose agreement are defined: inversion, conversion, and subsuming.Examples of each as follows:Inversion: Q1: “Are there abnormal findings in the lower lung fields?” is a negation of Q2: “Are the lower lung fields normal?”Conversion: Q1: “How would you describe the abnormalities?” is open-ended while Q2: “Are the lesions ring-enhancing?” is closed-endedSubsumption: Q1: “Is the heart seen in the image?” subsumes Q2: “is the heart seen on the left?”We calculate F1 scores of user agreement for questions considered ‘evaluated’, meaning they were reviewed by two annotators. Disagreements amongst question category types were reviewed at weekly team meetings to determine general rules for all content. Disagreements in objective findings of images were reviewed by using MedPix metadata and advice from expert radiologist to find ground truth. This in-depth review was only required for 280 question-answer pairs. Questions are labeled as ‘not evaluated’ if they are not reviewed by a second participant or the paraphrased question is not similar enough to be used as validation. Both the evaluated and not evaluated questions are used as part of the test and training set.We validated question types assigned by the participants. Final categorization was determined through consensus with the research team to resolve disagreements.15.We anticipate that a VQA-RAD pipeline is extensible to produce more test data and training data for traditional machine learning approaches. We believe that more training data sufficient for training deep learning approaches could be produced automatically, bootstrapping from the VQA-RAD collection and using recent approaches to image synthesis and data augmentationThe full dataset is archived with Open Science Framework (Data Citation 1). All question-answer pairs referencing images are stored in a single dataset that we provide in three common formats: JSON, XML, and Excel. The dataset contains 14 variables of unique identifiers and categorization with a total of 2,248 elements. The 315 corresponding radiological images are contained in a separate folder. Naming conventions and descriptions of all files can be found in a provided Readme file.The final VQA-RAD dataset contains 3,515 total visual questions. Of these, 1,515 (43.1%) are free-form. 733 questions are rephrased which means 48.3% of the free-form questions have corresponding paraphrasing. The remaining 1,267 questions are framed and have corresponding free-form and rephrased questions. On average, there are 10 questions per image.Of the free-form and rephrased questions, 75%  are evaluated using seven pairs of annotators. User agreement F1-scores range from 0.78 to 0.95 with a mean of 0.85.All questions are short, with medians, mean, and modes ranging from 5 to 7 words per question. After converting all words to lowercase and comparing sentences for unique structures, the free-form and rephrased questions have more distinct questions (87% and 93% questions are distinct) compared to framed questions which had 49% uniquely structured questions. Answers are also short, with median and modes of 1 word per question (mean of 1.6). Comparing all lowercase answers for unique structures, we observed 486 distinct answers. This represents 32% distinct answers of the total 1,515, which we expect because about half of all answers are either ‘yes’ or ‘no’.Subgroup analysis of free-form questions demonstrates the relationship between question types and answer types. Of the 11 question types, Presence questions dominate, with 36% (483) of free-form questions. The least frequent question type is Counting, with only 1% (15) questions. The QA pairs are split into 42% (637) open-ended answer types and 58% (878) close-ended. Yes/no questions represent 92% of the close-ended QA pairs, and ‘yes’ (405) is as frequent as ‘no’ (406.)Subgrouping question and answer types, we observe that people think about certain categories differently See . AbnormaWe next analyzed the types of words for each question type . For each question category, we tokenized the words and listed the top 10 most common distinct words. Many of these words are unique to the question category and can be used as keywords to help guide question categorization. The frequencies of words give insight into the context in which certain questions are likely to be asked. For example, ‘heart’, ‘cardiac’, ‘cardiomegaly’, and ‘dilated’ are frequent words in the size questions, which suggests that participants thought about size of the heart more commonly than other organs.Examining the question categories in relation to image type, we observed several differences in what clinicians ask when they see images of the head, chest, or abdomen. Presence questions are asked evenly about abdomen and chest, with fewer head images, although remaining the most commonly asked question type. Positional and Color questions were more prevalent with images of the head. Contrastingly, positional questions were less frequent with abdominal images. Organs in the head, in particular structures of the brain, are much more fixed in location compared to organs in the abdomen. Therefore, positional questions are more relevant in the head where one can reference locations. In the abdomen, asking if something is seen in the image is more clinically relevant or easier to determine than knowing the location. Another trend is that size questions are asked most frequently in chest images, agreeing with the distinct words we described earlier. The relationship between question type and image type gives some insight into the patterns of clinical relevancy that clinicians may have. Not all questions are useful and maybe shouldn’t be asked for every image.We validated the dataset by evaluating the performance of well-known VQA algorithms and their accuracy at answering visual questions from the VQA-RAD dataset.16 and Stacked Attention Network (SAN)17. The MCB model, winner of the 2016 VQA challenge, uses a multimodal compact bilinear pooling method that first predicts attention visually and textually then combines these features with question representation. MCB includes three components: a CNN image model, an LSTM question model, and MCB pooling that first predicts the spatial attention and then combines the attention representation with the textual representation to predict the answers. For the image model, we used ResNet-152 pre-trained on imageNet. For the question model, a 2-layer LSTM model was used.We include two well-known VQA methods: Multimodal Compact Bilinear pooling (MCB)The SAN model is a stacked attention model that queries images multiple times to progressively narrow an attention. Similarly to MCB, SAN includes three components: the image model based on a CNN to extract high level image representations, the question model using an LSTM to extract a semantic vector of the question and the stacked attention model which locates the image regions that are relevant to answer the question. We used the last pooling layer of VGG-16 pre-trained on imageNet as image features, and the last LSTM layer as question features. The image features and the question vector were used to generate the attention distribution over the regions of the image.18 and VGGNet19 respectively to extract image features and then both trained on VQA V1.0 dataset training and validation4. We refer to these baseline models as MCB-VQA1.0 and SAN-VQA1.0, when trained on VQA V1.0. We then trained the models on the ImageCLEF-VQA-Med14 training set to see how an existing medical VQA dataset influences the models. We refer to the ImageCLEF trained models as MCB-CLEF and SAN-CLEF. We then train the two networks with VQA-RAD training set creating MCB-RAD and SAN-RAD. Since VQA-RAD is a small dataset, we combined ImageCLEF-VQA-Med with VQA-RAD and trained the networks to explore any synergistic effects. As part of a baseline reference for closed-ended questions, we include predicted answer sets of all ‘yes’ and ‘no’.The MCB and SAN were pre-trained on ResNetFrom VQA-RAD we separated out a training set and test set. VQA-RAD test set is composed of 300 randomly chosen free-form questions and 151 corresponding paraphrased questions. The VQA-RAD training set is the remainder of the dataset. We conducted a manual review to calculate simple accuracies for each question type and use several accepted metrics to calculate overall performance for each model. For the manual review, we judge the predicted answers of the models to the gold standard VQA-RAD test. In addition to exact match, a full point is given for predicted answers that appropriately answer the original question. For example, “right lung” and “right upper lobe” are correct answers for the question “where is the lesion?” since the question does not indicate a degree of specification. Half a point is awarded for answers that incorporate part of the answer. For example, “right lung” would be given half a point for “which lobe is the lesion in?” where the correct answer is “right upper lobe”.12. The BLEU20 method, a commonly used metric for evaluating machine translation, measures the difference based on number of different words used. Following the ImageCLEF-Med methods, we pre-process the human and machine answers by converting answers to lower-case, removing punctuation, tokenizing individual words, and removing stop words using an English list of 172 words.We employ multiple overall scoring methods to demonstrate limitations of current metrics and need for newer metrics to evaluate VQA in medicine. Simple accuracy is calculated as the number of correct answers over total answers. Mean accuracy is the average of accuracy of the question types, similar to TDIUC dataset16 and 58.9%17 accuracies for open and closed ended public domain questions. Predicting open-ended VQA-RAD questions, the models performed much lower with the MCB_RAD scoring the best at 25.4% simple accuracy and 19.3% mean accuracy. The contrast between open and closed-ended questions suggests that these models are currently still guessing and may require more data. Potential improvements can come from learning medical terminology and jargon, greater quantity and diversity of questions, and extracting image features from radiological images as opposed to public domain.As might be expected, the models that best predicted VQA-RAD test set are trained on VQA-RAD training alone or partially. For closed-ended questions, MCB_RAD had slightly better simple accuracy of 60.6%, while SAN_RAD had a slightly better mean accuracy at 54.6%. This performance is comparable to the MCB and SAN models trained and tested on VQA1.0 dataset which had reported 64.2%Manual evaluation of the accuracy of automatic answers to naturally occurring questions in the VQA-RAD test set is shown in In medicine, seemingly small changes in wording can result in very different questions and answers. The opposite also holds true, the same idea can be written in simple or complicated phrasing. The VQA-RAD test set contains 151 matched pairs of free-form and paraphrased questions. We analyzed the MCB_RAD model to better understand the variance in predicted answers for similarly written questions. We observed 48 pairs (31.8%) had differing behavior, Overall scores may be useful for picking a winner of a challenge, but they may also bias towards algorithms that can provide answers that are more common. In medicine, the best answer is the most clinically relevant, not necessarily the most common. Calculating accuracies for different question types can highlight unique features of algorithm designs. For example, the SAN_RAD model scored high for open and closed ended size questions suggesting for this type of task, the SAN design might have certain advantages over the MCB design. However, further refinement is needed to understand these differences, as it is not clear if the SAN model is able to better recognize image regions of interest or is better at answering questions.VQA-RAD demonstrates the value of natural questions for the specialized medical domain. Models without VQA-RAD training scored the same or lower than the all ‘yes’ or ‘no’ baselines, suggesting they were no better than guessing yes/no as answers. While the models may have been able to recognize yes/no structured questions, they lacked image features and vocabulary. For example, we observed that the MCB model trained on VQA1.0 dataset predicted many open-ended questions with “face”, “clock”, or “banana” as answers.Analysis using BLEU scoring shows the need for improvement to metrics measuring visual tasks in medicine. We observe an opposite pattern between BLEU and manually judged accuracies where all ‘no’ resulted in better scores. BLEU penalizes answers with varying lengths, which favors the short one-word VQA-RAD answers and does not consider semantic similarity. While BLEU may be appropriate for certain machine learning techniques, it is not useful for medical VQA where there are many ways to phrase an answer and semantics are more valuable than exact wording. Using VQA-RAD, improved metrics can be developed that are aligned with the task of clinical care.To ensure that all researchers understand how to use the data, we provided instructions in the Readme file in the Open Science Framework (Data Citation 1).How to cite this article: Lau, J. J. et al. A dataset of clinically generated visual questions and answers about radiology images. Sci. Data. 5:180251 doi: 10.1038/sdata.2018.251 (2018).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Despite the ubiquity of single-best answer multiple-choice questions (MCQ) in assessments throughout medical education, question writers often receive little to no formal training, potentially decreasing the validity of assessments. While lengthy training opportunities in item writing exist, the availability of brief interventions is limited.We developed and performed an initial validation of an item-quality assessment tool and measured the impact of a brief educational intervention on the quality of single-best answer MCQs.x2(1) =38, p <0.001.The item-quality assessment tool demonstrated moderate internal structure evidence when applied to the 20 practice questions  and excellent internal structure when applied to the true dataset . Quality scale scores for pre-intervention questions ranged from 2–6 with a mean ± standard deviation (SD) of 3.79 ± 1.23, while post-intervention scores ranged from 4–6 with a mean ± SD of 5.42 ± 0.69. The post-intervention scores were significantly higher than the pre-intervention scores, Our study demonstrated short-term improvement in single-best answer MCQ writing quality after a brief, open-access lecture, as measured by a simple, novel, grading rubric with reasonable validity evidence. The use of single-best answer multiple-choice questions (MCQ) in examinations is ubiquitous in medical education. Although guidelines for writing MCQs exist, item writers often receive little to no formal training, potentially reducing the validity of examinations by introducing construct-irrelevant variance.10The National Board of Medical Examiners (NBME) provides both a detailed, open-access guide for exam- question writing and an online training module.We sought student and resident volunteers from the American Academy of Emergency Medicine Resident and Student Association, and conducted the educational intervention in September 2017. The study was granted exemption status by the University of Pennsylvania Institutional Review Board.We created a MCQ quality assessment tool based on expert opinions  of the most important components contained in the question-writing lecture; it is based on multiple, well-accepted sources, supporting content evidence.The training module was created by an item-writing expert (JJ) using PowerPoint  with recorded voice-over , allowing for independent completion by learners. The training module itself has been previously published in an open- access curriculum database and was based on principles of item writing as described by the NBME.Pre- and post-intervention MCQ quality scores were determined by two item-writing experts  via the item quality assessment tool. Discrepancies were decided by a third item-writing expert (LC).We first performed descriptive summaries including mean and standard deviation (SD), frequencies, and total responses. Internal reliability was assessed using Cohen’s kappa. We decided a priori to compare pre- and post-lecture scores using the non-parametric Friedman’s analysis of variance (ANOVA), given the expected range to be relatively small and low likelihood of having an even distribution of the standard error of the mean. Friedman’s ANOVA is essentially a non-parametric, repeated measures one-way ANOVA. A p-value less than 0.05 was considered statistically significant. We performed all analyses using SPSS version 24 .The internal structure evidence was moderate when the tool was applied to the 20 practice questions . The tool demonstrated excellent internal structure when applied to the true dataset of questions created by the students and residents  with only eight discrepancies in 264 cases , evaluated by two different researchers. Evidence of consequence was demonstrated as part of the other primary objective of this study, in which pre- and post-lecture scores were different. As this was a stand-alone study, we were unable to evaluate for relationships with other variables.x2(1) =38, p <0.001.A total of eight residents and students consented and participated in the lesson, of whom seven provided both pre- and post-lecture MCQs. One participant provided two pre-lecture questions rather than three, and another provided no post-lecture questions, thus totaling four total missing questions of the 48 possible total questions (8 × 3 × 2). Missing questions were excluded pairwise since the post questions were edits of the original questions; therefore, four missing questions led to elimination of eight total questions. We analyzed a total of 40 questions (20 pre- and 20 post-lecture). The MCQ quality scale scores for pre-intervention questions written by the learners ranged from 2 - 6 with a mean ± SD of 3.79 ± 1.23, while post-intervention scores ranged from 4 – 6. The post-intervention scores were significantly higher than the pre-intervention scores, The current study supports the efficacy of a short, high-yield lecture to teach best evidence in developing single-best answer MCQs. The study also provides strong validity evidence for a novel tool by which to evaluate the structure of single-best answer MCQs.Journal of Education and Teaching in Emergency Medicine (JETem) and its brief duration provide a practical advantage to this educational intervention as well.Although multiple prior studies have evaluated outcomes from an educational intervention to improve MCQ writing, the current study is the first available remotely, free to the public, and at approximately 30 minutes in length is the shortest.Another important contrast to prior studies is the target group. Much focus has been placed on faculty development, yet educators are seeing the benefits of learners writing questions.This study lastly provides a checklist with reasonable validity evidence and strong inter-rater reliability when applied to the real-world questions. This is in contrast to other checklists that exist but are limited to content validity by experts.Our study must be interpreted in the context of several limitations. Most importantly, we studied a short-term outcome. This variable must be a precursor to follow-up, long-term learning outcomes to fully elucidate the efficacy of the intervention. It is also important to highlight that the intervention and assessment tool are intended to improve the structure of MCQs. Such proper practices are associated with good question quality as ascertained through psychometric analysis, but they are beyond the scope of our initial study. Additionally, our study recruited volunteers who may have been more motivated to improve their MCQ writing skills than students and residents in the general population. Finally, although the MCQ quality tool was applied against a test group of questions and a real-life group of questions, it was nonetheless a small sample of questions with a small number of participants, and the tool should be tested against more questions and more raters.Our study demonstrated short-term improvement in single-best answer MCQ writing quality after a brief, open-access lecture, as measured by a simple, novel, grading rubric with reasonable validity evidence.


We present a p-n-p monolayer graphene photodetector doped with titanium dioxide nanotubes for detecting light from visible to near-infrared (405 to 1310 nm) region. The built-in electric field separates the photo-induced electrons and holes to generate photocurrent without bias voltage, which allows the device to have meager power consumption. Moreover, the detector is very sensitive to the illumination area, and we analyze the reason using the energy band theory. The response time of the detector is about 30 ms. The horizontal p-n-p device is a suitable candidate in zero-bias optoelectronic applications. Integrating the graphene with TiO2 nanotubes improved the photoresponse intensity relative to the monolayer graphene. Moreover, we use the energy band theory to analyze detectors that are very sensitive to the illuminated area. It was found that the graphene/TiO2 nanotubes photodetector works without external bias and it is expected to be a promising zero-bias broadband photodetector. In addition, the p-n-p junction makes the photodetector sensitive to the irradiation area, which means this kind of device is a candidate for dynamic object imaging device.As the most representative two-dimensional material, graphene has attracted a large amount of attention from researchers all over the world. Due to its high mobility, unique optical properties, being gapless at the Dirac point, and its excellent conductivity, graphene has become a potentially useful material for optoelectronic devices, such as light modulators , photodeparation ,23. Zhanetection ,24. We h2 nanotubes arrays were prepared on 0.1 mm–thick Ti sheets of 99.8% purity, . We reported in a previous article that TiO2 nanotubes were prepared by anodic oxidation method [4F/glycol organic electrolyte aqueous solution at room temperature. A total of 180 min thermal annealing in the air atmosphere at 450 °C was followed by anodization to form high ordered anatase TiO2 nanotubes arrays. After annealing, the titanium oxide nanotubes need to be carefully treated because the titanium oxide nanotubes are easily detached from the titanium sheet and are drawn into the body through the air [2 nanotubes after depositing gold electrodes.The TiOn method . The thi the air ,27,28. M the air  and was 2 nanotubes is necessary. After the graphene has been transferred to the substrate, it is brought into contact with the substrate by Van der Waals force. This force is much weaker than the force of the molecular bond. Despite the single-atom-thick nature, graphene is very compact—water and gas molecules do not readily penetrate monolayer graphene. Tan et al. used the monolayer graphene to keep their perovskite film away from the water [2 substrate, the surface of the TiO2 nanotubes manufactured by the anodic oxidation method is rugged, and the nanotubes have a hollow structure, as shown in 2 nanotubes as the graphene can fall off the TiO2 nanotubes after the dying and annealing treatment. To solve this problem, we first deposited gold on the TiO2 nanotubes to create a relatively flat surface, and then we transferred the monolayer graphene onto the substrate. The 60-nm gold layer, deposited with using electron beam evaporation can fill the holes of the hollow TiO2 nanotubes, as shown in 2 nanotubes. With the assistance of a flat gold surface, graphene can fit smoothly over the surface of the substrate.We used monolayer graphene synthesized on a copper foil with the CVD method to fabricate the photodetector. Transferring the graphene to the preset TiOhe water . Therefo2 nanotubes. Raman spectroscopy  was used to identify the number of layers of graphene. Atomic force microscopy  characterized the surface morphologies of the TiO2 nanotubes. The current–time curves were measured with the Keithley 2636B sourcemeter.A scanning electron microscope  was used to investigate the morphology of the graphene and the TiO2 grown on the titanium sheet provides many unique characteristics, such as a highly ordered one-dimensional structure, high chemical stability, low cost, and tunable morphology [2 nanotubes can enhance the interaction with light. 2 nanotubes with gold electrodes. 2 nanotubes and Si/SiO2 substrate with 514 nm excitation laser. The 2D peak at 2697.53 cm−1 is significantly higher than the G peak at 1579.8 cm−1,which means the graphene on the nanotubes is a single layer. Due to the unevenness of the TiO2 nanotubes, the graphene is still wrinkled on a small scale after being transferred to the substrate, as shown in 2D/IG < 2. the 2D peak of graphene on the Si/SiO2 substrate appears at 2685.3 cm−1. The varieties fit well with previous research results [2 nanotubes arrange from 90 to 160 nm.TiOrphology ,32,33. T results , as the 2 nanotubes/graphene device. The device can work under low bias voltages and even no bias. The laser frequency is 405 nm, and the power density of the laser is 3.63 mW/cm2. The current responsivity R(I) = Iphoto/P, where R(I) is the current responsivity, Iphoto is the photocurrent, P is the effective illumination power [0, where A is the active illumination area and P0 is the optical power density. The optical power density of the 405 nm laser is P0 = 3.63 mW/cm2, and the active illumination area is about A = 0.34 mm2. The detector generates a 55 nA photocurrent with different bias voltages, and the computational current responsivity is about 4.5 mA/W. To compare with a pure monolayer graphene and TiO2 nanotubes, we tested the photoresponse of pure graphene and TiO2 nanotubes without graphene as shown in 2 nanotubes without graphene has no photoresponse at 405 nm laser illuminating as shown in 2 nanotubes device. Although the carrier mobility of graphene is very high (∼1810 ± 710 cm2 /(V∙s) with isopropanol treatment at 300 K [r ≈ 30 ms and fall time Tf ≈ 37 ms are shown in on power ,36. Moreat 300 K ), the liat 300 K . With th2 nanotubes provide photo-induced carriers for the monolayer graphene. The characteristic of the zero bandgaps of graphene makes the detection frequency of the photodetector range from visible to near-infrared, as shown in 2 nanotubes photodetector is a promising candidate for the broadband photodetector. With the same laser power, the photocurrent of the 405 nm laser is more significant than other frequencies, which means that the TiO2 nanotubes, the nanotubes acted as a donor and doped the middle region of the graphene with electrons. The monolayer graphene was divided into three states, and a p-n-p junction formed, as shown in 2 nanotubes are separated by gold. As the light illuminates the junction, light-induced electronhole pairs are separated by the built-in electric field and then captured by the external circuit to form a photocurrent. Because the graphene on the TiO2 nanotubes is wrinkled randomly, the character of the graphene on the opposite side will not be the same. The direction of the built-in electric field at both sides are opposite. If the light illuminates only one side of the graphene (p-n or n-p junction), a positive response I1 is generated when the built-in electric field is consistent with the direction of the external electric field, as shown in 2 occurs, as shown in When the light was illuminating on the different side of the graphene, the photocurrent would be positive or negative. Since graphene usually adsorbs some moisture, oxygen, etc., on the surface, it usually shows p-type as shown in 1 + I2| if the light illuminated the whole device. However, the photocurrent I1 and I2 are in the opposite direction. The photocurrent weakens each other in the two different areas. Therefore, if the light spot illuminating on the whole device, the photoresponse is much weaker than the light illuminating on only one side of the device, as shown in The band structure of the horizontal p-n-p junction is shown in 2 nanotubes doped the horizontal p-n-p monolayer graphene photodetector. The photodetector is sensitive to visible light to near-infrared. The reason why the device is sensitive to the illumination area is analyzed from the energy band structure of the device. We get a broad spectrum zero-bias photodetector from 405 nm to 1310 nm. The built-in electric field makes the device work without bias, which is friendly to the environment and to sustainable development. This kind of horizontal p-n-p device will be a candidate in low power consumption optoelectronic applications.In conclusion, we demonstrated TiO


Reactions applying amidation- or esterification-type processes and diazonium salts chemistry constitute the most commonly applied synthetic approaches for the modification of graphene-family materials. This work presents a critical assessment of the amidation and esterification methodologies reported in the recent literature, as well as a discussion of the reactions that apply diazonium salts. Common misunderstandings from the reported covalent functionalization methods are discussed, and a direct link between the reaction mechanisms and the basic principles of organic chemistry is taken into special consideration. In 2004, Geim and Novoselov reported the first experimental isolation of the graphene sheet and the measurement of its properties . Since t2 carbons from the graphene sheet. The carboxyl groups contained in GO and RGO constitute important targets for structural expansion, as COOH moieties can be transformed into, e.g., amide- or ester-type linkages. On the other hand, the functionalization of sp2 carbons is most commonly performed utilizing a diazotization approach; in other words, the diazonium salt is generated from the corresponding aromatic amine (preparation of the reagent), and then the aryl radical is added to the graphene sheet.To trigger some desired effects and to open new avenues for the application of GO and RGO, a chemical functionalization was conducted –12. TherThe crucial parts of a significant number of the articles on the application of GO and RGO focus on the chemical functionalization of the graphene-family material. In fact, the structural modification of GO and RGO constitutes a key starting point of such research. Even though many studies on chemical functionalization have presented very interesting and novel applications of GO and RGO derivatives they also present yet common misunderstandings and inaccuracies. This work deals with common issues in the field of GO and RGO functionalization; it discusses the carboxyl-based approach and includes remarks regarding reactions that utilize diazonium salts. Direct links are provided to basic principles of organic synthesis, reaction mechanisms, and state of the art of organic chemistry. Point-by-point recommendations are also given for the proper application of organic chemistry principles in covalent functionalization of graphene-family materials. The chemistry of the reactive groups of graphene-family materials covers many areas of research, including colloid chemistry and interface science; nevertheless, the basic rules of organic chemistry should be regarded as playing a leading role in covalent functionalization.O-acylisourea intermediate, which is highly electrophilic and which bears the urea-based good leaving group. Such activation of carboxyl groups onto a graphene material makes these functionalities very reactive toward the nucleophilic reagents.A primary amine or a primary alcohol constitutes a nucleophilic partner in a reaction with a carboxyl group (COOH). As a result of derivatization, an amide or ester bond is formed between a graphene-family material and a given chemical. The nature of the derivatization of carboxyl functionalities onto the graphene sheet is directly associated with improvements in the reactivity of carboxyl moieties. Carboxyl groups are not as reactive as the corresponding acyl chlorides or anhydrides. Activating a carboxylic group is therefore a crucial step in improving its reactivity toward nucleophilic reagents. A common approach is to employ carbodiimide-promoted reactions –14. As pO-acylisourea intermediate is also reactive toward water molecules and in some cases may react slowly with the desired nucleophile. To increase the stability of the active O-acylisourea intermediate and to promote the creation of amide- or ester-type linkages, an additional coupling reagent is included in the process pyridine (DMAP) is as an additive in the carbodiimide-coupling protocol. In the second step, DMAP forms active amide intermediates via a reaction with an O-acylisourea individual  react with epoxides more rapidly than do weak nucleophiles . This functionalization approach based on the epoxides’ opening enables the introduction of the reactive groups to the surfaces of graphene-family materials. The properties of these materials can thus be tuned, and the introduced functional groups can be utilized for further reactions [The structure of GO includes a number of epoxide moieties, which are also reactive toward the nucleophilic reagents. The epoxides’ opening by nucleophiles can therefore act as a competing side reaction in many coupling processes with the inclusion of graphene-family materials ,16. In oeactions ,17–19.The literature features many examples of amide- or ester-bond formation with the inclusion of graphene-family materials –21. Howep-phenylenediamine and graphene oxide with no additive (heating at 80 °C for 24 h), should be directly observed using IR spectroscopy. In this case, benzene rings were claimed to be introduced to GO via the presence of a strong absorption band located at ca. 1500 cm−1 (C=C stretching vibrations) in the product’s IR spectrum. Therefore, it is not clear why a similar, prominent adsorption band corresponding to the amide moiety was not observed in the spectrum, and this issue was not discussed. It is noteworthy that both a lower intensity of the absorption band coming from the C=O of GO (above 1700 cm−1) and a new absorption band in the wavenumber range of 1550 cm−1 to 1620 cm−1 can be directly inferred from the formation of either a hydrogen bond between primary amine and the carboxyl groups of GO or an inner salt formation . The presence of the amide bond in the functionalized GO was implied by the deconvolution of the C1s (287.8 eV for NH–C=O vs 289.1 eV for O–CO) and N1s (399.4 eV for N–C=O vs 400.3 eV for O–CO) peaks in the X-ray photoelectron spectroscopy (XPS), only. However, the C=O stretching vibrations in the amide groups can indeed be easily observed in the infrared spectrum of the functionalized graphene-family materials; thus, XPS should be regarded as a supporting analysis, as many researchers have demonstrated . Moreover, the most prominent absorption band (located at 1587 cm−1) was surprisingly neither discussed nor assigned [−1 (N–H stretching vibrations) and 1385 cm−1 (C–N stretching vibrations) were assigned. However, these features cannot be considered direct evidence for the formation of amide-type linkages. Importantly, no mechanism for generating the amide bond (if any such bond formed) using L-cysteine has been proposed or discussed. Most plausibly, the attack of cysteine’s highly nucleophilic sulfur on GO’s epoxides did occur in this case [−1 (asymmetric vibrations of C=O of –COO− and N–H of NH3+) and 1475–1385 cm−1 (symmetric vibrations of C=O of –COO− and N–H of NH3+) ranges indeed implies. The absorption band located at 3016 cm−1, interestingly, was assigned to the stretching vibrations of the alkyl chain (commonly observed at up to 2980 cm−1); this absorption band corresponds to the N–H stretching vibrations of NH3+. The further reaction with amphotericin B, which is a compound containing unsaturated bonds, was most plausibly a result of π–π stacking.The same conclusion about the structure of the obtained material applies for the derivatization of GO and RGO based on heating or mixing a carbon nanostructure with an amine or hydroxy-containing component –26. For e, e.g., ,28–29). e, e.g., , the apphis case –31, base−1 to 1735 cm−1). Another consideration is that this process further influences the obtained material’s morphology, properties and thermal stability [−1, C=O coming from ester bond stated for functionalized GO: 1639 cm−1). This unexpected statement was supported, not by further spectroscopic analyses, but by morphological studies (transmission electron microscopy (TEM) and atomic force microscopy (AFM)) only. Once again, the possible electrostatic adsorption or hydrogen bonding-dependent interactions are not considered in such cases. Researchers have previously discussed this and directly shown that non-covalent adsorption should also be taken into account, even though that the carbon material is functionalized using the covalent crosslinker-based approach [As mentioned above in the discussion of reaction mechanisms, water molecules significantly lower the reaction rates for the desired nucleophiles. Importantly, water molecules also influence the hydrolysis of the activated carboxyl groups , step f.tability –42. Additability  or grindtability  for the tability , it was approach –45. Indeapproach  have pre2 carbon in the graphene sheet [The diazotization reaction is a widely applied protocol for the functionalization of spne sheet ,46–48. Tne sheet .o-Dichlorobenzene is the most commonly used solvent in radical processes because it is not reactive toward radical species. Several researchers have reported protocols for functionalizing graphene-family materials using an organic solvent as the reaction medium and amyl nitrites as the additive [This reaction’s mechanism is not fully understood, but several researchers have discussed the reaction pathway –50. Mostadditive –52. Theradditive –57. It io-dichlorobenzene) can therefore (i) enable an efficient electron-transfer process (2 functionalization that use the diazonium salts’ chemistry.Importantly, it has been well documented that carbon materials are some of the best known adsorbents of phenolic compounds –61. Wate process , step b, process , step c,With the growing number of articles on the application of graphene-family materials, a proper and rational design of a functionalization route is of the highest importance. Many scholars have employed the basic laws of organic chemistry to the covalent functionalization of graphene-family materials. These research works showcase that the chemistry of graphene, which includes many areas of science, both fulfils and heavily relies on the principles of organic synthesis. However, some of the works that are focused on graphene’s organic chemistry still contain major misunderstanding and inaccuracies. This synopsis includes a major discussion on the crucial role that water molecules and coupling reagents play in covalent modification processes. Special consideration was also given to the applied reactants, and in-depth analysis of obtained material’s structure is also of the highest importance. This work includes recommendations for the proper application of the basic organic synthesis principles for the functionalization of graphene-family materials.


This study carried out a novel duplex surface treatment on aluminum alloy base to explore the potential improvement of wear and corrosion resistance. Regular arrayed dimple surface texture (DST) and groove surface texture (GST) were fabricated by using laser processing on 6065 aluminum alloy matrix (6065Al). Electrochemical deposition of Ni and Graphene/Ni coatings on textured surface was then performed in electrolytes with concentrations of 0, 0.5, 1 and 1.5 mg graphene. Surface morphology such as diameter of dimple and width of groove measured by C-PSCN stereo microscope presents addition of graphene helps to refine and homogenize the coating. Corrosion resistant properties of the duplex surface treatment were examined by electrochemical corrosion tests and wear resistant properties were tested by UMT-Tribo Lab friction and wear tester in a dry sliding condition at room temperature. Electrochemical corrosion tests results show that the corrosion resistance of samples is related to the specific surface texture and the dimple texture can improve the electrical corrosion parameters, such as the electrode potential, greatly. Friction and wear tests show that the textured Gr/Ni electroplating coating with the 1.5 mg graphene content has best wear properties under vertical friction and each index, such as the coefficient of friction and wear trace width, are superior to other conditions of samples. However, difficulty still arises with the electroplating process is the formation of natural oxide film on the surface of the aluminum alloy, hindering the direct reduction of metal ions on the surface of the aluminum alloy to obtain a plating layer with ideal adhesion situation [Aluminum alloy has been considered a good choice as a material in the aviation, aerospace, automotive, marine and chemical industries, due to its excellent specific strength and plasticity, high electrical conductivity, thermal conductivity and remarkable corrosion resistance . Yet, liituation . TherefoGraphene, with excellent electrical, thermal, chemical and mechanical properties, has gained extensive attention since its advent. Single-layer graphene is a planar thin film material which composed of carbon atoms in sp2 hybrid orbitals ,5. This Previous studies have demonstrated that graphene can be used as an additive for electroplated composite. Szeptycka  finishedWith the development of textured surface science, people now begin to re-recognize the wide application of non-smooth surfaces in various fields. Among of them, surface texture (ST) has been confirmed as an effective and economical surface treatment technique that can be applied to a great range of materials and it has been attached greater importance in various engineering fields. Ta  utilized2O3 composite cladding on AISI 1040 substrates by the cost-effective process of gas tungsten arc welding. The microscopic images showed coaxial fine grains in the weld composite surface, there are no cracks in the surface layer created and Al2O3 particles are distributed uniformly in the surface layer. The purpose of the research could achieve optimal parameters of claddings in an inexpensive method. Liu [However, neither the surface texture nor the surface coating can solve the corrosion resistance and wear resistance of aluminum alloy well . A combihod. Liu  preparedThus far, however, there has been little discussion about duplex treatments of surface modification-surface texturing for 6065 aluminum alloy matrix (6065Al). Inspired by bionic studies and taking the advantages of surface texturing and surface modification, in the present work, the surface texturing/composite electroplating (ST/CE) duplex treatments were applied on 6065Al. Regular arrayed dimple surface texture (DST) and groove surface texture (GST) were adopted and fabricated by using laser processing on 6065Al matrix respectively and electrochemical deposition of Ni and Gr/Ni coatings on textured surface was then performed in electrolytes with concentrations of 0, 0.5, 1 and 1.5 mg graphene. The influences of different texture and content of graphene on friction behavior and corrosion resistance were investigated by micrographs, electrochemical experiments and friction and wear experiments.This section presented procedures of the duplex treatments of surface modification-surface texturing on 6065Al matrix. The scheme of this processing is shown in The material selected for the present research work is Aluminum 6065 alloy, which is commercially available with dimension of 70 mm × 20 mm × 2 mm under cold-rolled condition and its composition is given in A P50Q pulsed fiber laser  with 1060 nm wavelength was employed here to fabricate the needed surface texture. Referring to other researchers, dimple and groove surface texture were adopted, which are based on the two typical wear resistant and corrosion resistant surfaces of lotus leaf and shark skin ,22. Deta2 saturated solution and 37% HCl mixed solution for 50–60 min at room temperature to remove surface rust. Electrolyte [In this experiment, an Ni plate  with a surface of 20 mm × 20 mm was used as the anode for electrodeposition. The cathode, with distance of 60 mm towards the anode, was a textured 6065Al plate with size of 20 mm × 20 mm. To improve the adhesion of coating, both electrodes were firstly immersed in 75% anhydrous ethanol and deoiled by ultrasonic heating with a PS-20A Digital Ultrasonic Cleaner  for 10 min. Then, the nickel plate was pickled in 3% hydrochloric acid for 15 min, and the textured sample plate was soaked in NiClctrolyte , as can 2 was applied. The electroplating time is 5 min at 40 °C and the stirring speed is 300 rpm. The samples were then washed with deionized water after electroplating process and finally put into the SX2-4-10 electric furnace  at 200 °C for 0.5 min for drying. Detailed electroplating parameters are shown in Here, multi-layer graphene aqueous solution was used supplied by Nanjing Ji nano company produced by using physical method . The composite plating solution was obtained through adding 1 ml with different concentration graphene aqueous solution  for comparing into nickel electrolyte and stirred sufficiently by ultrasonic vibration. Composite graphene electroplating were then started. During electro-deposition process, both the anode and the cathode were connected to the MS-155D electroplating power supply  and a constant current power with 13.75 A/dmSurface morphology such as diameter of dimple and width of groove were measured by C-PSCN stereo microscope (Nikon Corporation. Japan). Scanning electron microscopy (SEM) images of the samples were analyzed using an Su 8000 FE-SEM  and the amount of electrodeposited graphene was estimated by using an energy dispersive spectroscopy (EDS-550I) which was attached to the FE-SEM. The sectional images of the textured electroplating coatings were captured by MA100N-optical microscope .Electrochemical corrosion tests were carried out on CS2350 double channel electrochemical workstation  equipped with three-electrode system. Reference electrode and auxiliary electrode are saturated calomel electrode and platinum electrode, respectively. Sample obtained above was used as the working electrode. Results of the electrochemical corrosion test were obtained through the corresponding software CS Studio 5. Tafel lines were recorded at a scanning rate of 1 mV/s, which is low enough to have a good response for the system, from 0.8–0.2 V versus open circuit potential. Electrochemical impedances were obtained in the frequency range of 0.01 Hz–10 kHz and the amplitude of sinusoidal signal was 10 mV . These t3N4 ball with the diameter of 6 mm was selected as a counterpart. Test parameters are as follows: a normal load of 10N, sliding velocity of 5 mm/min, reciprocatory displacement of 10 mm and test time of 10 min. The worn surface was observed and examined for analyzing.UMT-TriboLab friction and wear tester  was employed to investigate the tribology behavior of coatings in a dry sliding condition at room temperature. A SiHere, to explore different texture surface influence on the friction result, two friction directions  for dimple texture surface were tested, referred to as D0 and D45 in the following 2+s would be preferably electrodeposited onto the graphene and lead to the formation of microbulges on the electroplating coatings. In the process of composite electrodeposition, Ni2+s would be preferentially electrodeposited onto graphene and encapsulated graphene which finally lead to form micro-convex bulges on the electroplating surface. This phenomenon can be found in 2+ to reduce, resulting in a fine grained and intact arrangement of Ni crystals [The average diameter of the original dimple shown in crystals . The chlFurthermore, by comparing 2, respectively. For DST/CE(Ni) samples, the corrosion potential increases to −618.63 mV, which means the corrosion resistance of 6065Al sheet is improved to a certain extent. Furthermore, there was a significant positive correlation between the corrosion resistance and graphene content from test result. The DST/CE(Ni+1.5Gr) get the most notable improvement of corrosion resistance. The corrosion potential of the composite coating moves forward to −601.69mV, and the corrosion current also decreases by 13.90% to 404.58 μA/cm2. Meanwhile, the corrosion test parameters of stripe textured composite electroplating coatings also appeared a similar trend. This result is consistent with another study [As can be seen from er study , in whicQuite possibly, these results are due to the excellent electrical conductivity of graphene that make the nickel have more nucleation points on the surface of the coating. The metal particles crystallized at these nucleation points will deposit at a faster rate, thus, the nucleation rate of the composite electroplating coating is higher than that of the pure nickel electroplating coating. During the process, graphene can effectively prevent the growth of metal grains, thereby refining the grain. Therefore, the addition of graphene composite electroplating coating can obtain a much more compact coating reflecting a better anti-corrosion effect.- ions to reach the matrix of dimple surface texture [Further analysis with data in  texture . On the The corrosion resistant properties of textured graphene/nickel composite electroplating coatings were evaluated by the electrochemical impedance tests. The Nyquist plots of all the surfaces with different treatments were shown by Friction coefficients of textured surfaces coated with different electroplate composites under stable friction and the variation trend of COF on the surface for all the samples are shown in From By comparing the friction coefficients of dimple and groove surface texture coatings, it can be seen from Besides this, by comparing the frictional coefficients of the groove texture surfaces under different frictional directions, it can be found that the lower the coincidence degree between frictional direction and stripped direction is, the more obvious the decrease of frictional coefficients is. When the frictional direction is perpendicular to the groove direction, the frictional coefficient is the lowest, which is 0.735. This is due to the spacing of the groove isolates the propagation of cracks and limits the increase of friction coefficient on the textured surface of the fringes .All the micrographs of those worn samples surface after the test are shown in Comparing with the friction scratches of all the specimens in It is also possible to verify that graphene reduces friction by comparing the trend of worn trace width on composite coatings with different graphene additions in (1)Graphene/Ni composite electroplating coating on groove and dimple textured 6065Al surface were prepared in this paper. Sectional images of the samples show the electroplating coatings and alloy matrix are connected uniformly and tightly. Electrochemical corrosion test results show that the corrosion resistance of the dimple textured graphene/Ni composite electroplating coating is better than that of the groove textured one due to the effect of those small air pockets created by dimple textured surface.(2)Friction and wear experiments revealed that the surface friction properties are related to the texture, the amount of graphene added and the direction of friction. Anti-friction performance of the groove textured graphene/Ni composite electroplating coating is higher than that of the dimple textured one.The following conclusions can be drawn from the present study:


The plasmon-induced transparency (PIT), which is destructive interference between the superradiation mode and the subradiation mode, is studied in patterned graphene-based terahertz metasurface composed of graphene ribbons and graphene strips. As the results of finite-difference time-domain (FDTD) simulation and coupled-mode theory (CMT) fitting, the PIT can be dynamically modulated by the dual-mode. The left (right) transmission dip is mainly tailored by the gate voltage applied to graphene ribbons (stripes), respectively, meaning a dual-mode on-to-off modulator is realized. Surprisingly, an absorbance of 50% and slow-light property of 0.7 ps are also achieved, demonstrating the proposed PIT metasurface has important applications in absorption and slow-light. In addition, coupling effects between the graphene ribbons and the graphene strips in PIT metasurface with different structural parameters also are studied in detail. Thus, the proposed structure provides a new basis for the dual-mode on-to-off multi-function modulators. At present, surface plasmon polaritons (SPPs), as a carrier for transmitting information and energy, have become a research hotspot in the sub-wavelength optics. Generally, they are produced by the interaction between the photons in the incident light field and the electrons on the metal or insulator surface , 2. The 2/(Vs), the group delay can be as high as 0.7 ps, representing the proposed metasurface also has distinguished slow-light functions. Moreover, coupling effects between the graphene ribbons and the graphene strips in PIT metasurface with different structural parameters also are studied in detail. Therefore, this research lays a solid foundation for the dual-mode on-to-off multi-function modulator.In this study, the proposed PIT metasurface, which consists of the periodic graphene ribbons and graphene strips, is easier to implement and fabricate. Through chemical vapor deposition (CVD) , the graVg1. Moreover, the graphene strips are connected with the right electrode using thin metal wires, and a gate voltage Vg2 is applied to modulate their Fermi levels , where ξ = ε/kBT. Moreover, ω, kB, σg, σinter, and σintra are the angular frequency of incident light, the Boltzmann constant, the conductivity of single-layer graphene, the inter-band, and intra-band contributions, respectively. In this work, the room temperature is T = 300 K; the thickness of the graphene is Δ = 0.34 nm. σinter can be ignored owing to kBT ≪ 2Ef in the terahertz band. Thus, σg can be expressed asHere, G(τ = μ0Ef/(evF2) [μ0 = 1 m2/(Vs) being the graphene mobility. Besides, the propagation constant β of the incident light on the graphene surface can be expressed as [Here, the electron relaxation time can be expressed as f/(evF2) , with μ0essed as 7\documeε1, ε2, and k0 are relative permittivity of silica and air, and the wave vector of the plane wave, respectively.Here, 1 and A2 serve as two antennas to describe the coupling effect between the graphene ribbon and the graphene strip. When the incident light is illuminated from A and exited from B, the relation can be obtained byIn Fig. γ1(2)= (iω – iω1(2) – γi1(2)– γo1(2)), in which the inter-loss coefficient is γi1(2)= ω1(2)/(2Qi1(2)) and the extra-loss coefficient is γo1(2)= ω1(2) /(2Qo1(2)). Additionally, Qi1(2) = Re(neff)/Im(neff) [neff= β/k0. The intra-loss quality factor can be obtained by 1/Qt1(2) = 1/Qi1(2) + 1/Qo1(2), with Qt1(2) = f/Δf being the quality factor of the whole system (Δf is 3 dB bandwidth). Following the conservation of energy, coupling relationship between two antennas is as follows:Here, Im(neff)  is the iμnm  and φ are the coupling coefficients and the phase difference between two antennas, respectively. Thus, we can obtain the transmission coefficient and the reflection coefficient of the proposed PIT metasurface.Here, the subscripts “+” and “–” represent that the antennas are illuminated in the same and opposite directions; the superscripts “in” and “out” represent the sign of the incident light entering and exiting the antennas. In addition, χ1(2) = iμ12(21) +(γo1(2)γo2(1))1/2eiφ. Then, the transmission and absorption of the proposed PIT metasurface can be obtained byWhere Very recently, the graphene ribbons, as one of the most promising candidates in the graphene series owing to the fact that they are greatly easy to achieve experimentally and can support localized plasmons (mainly based on Fabry-Perot-like standing wave resonance) –50 and pSo as to discuss the physical origin of the PIT effect, simulated transmission spectra of three graphene metasurfaces and electric field distributions of the entire structure and graphene strip at the resonance frequency are illustrated in Fig. Ef2 of the graphene strip is fixed at 1.0 eV, the Fermi level Ef1 of the graphene ribbon is changed to explore the PIT effect. In Fig. Ef1 increases from 0.6 eV to 1.2 eV, there is a significant change in dip1. For one thing, the transmittance of the dip1 is remarkably reduced indicating that an on-to-off modulation can be obtained. For another, the dip1 has an obvious blue-shift demonstrating that it is sensitive to the change of Fermi level Ef1 and can realize frequency modulation. Besides, when the Fermi level Ef1 of the graphene ribbon is fixed at 1.0 eV, a similar phenomenon occurs in Dip2 with the increase of the Fermi level Ef2. However, the blue-shift is more significantly observed in the left dip in both cases. When Fermi levels of the graphene strip and the graphene ribbon are both 1.0 eV, the resonance frequency of the superradiation mode and the monopole resonance frequency of the subradiation mode are basically 6.2 THz. Thus, the coupling between them forms a symmetrical PIT. When the Fermi level Ef1 of the graphene ribbon is increased from 0.6 eV to 1.0 eV, the monopole resonance frequency of the subradiation mode shifted from the left side to the 6.2 THz due to the change of the graphene ribbon conductivity. In the case, the coupling between the subradiation mode and the superradiation mode is weak owing to different resonance frequencies, generating a highly asymmetric PIT. The obvious blue-shift of the dip1 in Fig. Vg1 mainly regulates the left transmission dip, yet the right transmission dip is chiefly tailored by the gate voltage Vg2. Therefore, a dual-mode on-to-off modulator is realized. Meanwhile, the dual-mode modulation of plasmon-induced absorption (PIA) is also obtained in Fig. The dual-mode on-to-off modulation of PIT can be achieved by two gate voltages applied to the graphene ribbons and the graphene strips, as illustrated in Figs. Ef1 = Ef2 = 1.0 eV. On this basis, the graphene mobility is increased from 1.0 m2/(Vs) to 3.0 m2/(Vs) in a 1.0 m2/(Vs) step. As the graphene mobility increases, not only the transmission spectra show apparent red-shift, but also the 3 dB bandwidth of the transmission dips becomes narrower, meaning graphene mobility can also be used to dynamically modulate the PIT and quality factors of transmission dips. Here, transmission spectra of FDTD simulation and CMT fitting are still perfectly matched. It is known that the performances of the slow-light effect are better with the higher quality factor of the transmission dip. Therefore, the transmission phase shift and the group delay with different graphene mobilities are plotted in Fig. In addition, transmission spectra with different graphene mobilities are also studied, as illustrated in Fig. ϕ(ω) is the phase shift calculated by ϕ(ω) =  arg (t). The results show that both the group delay and the phase shift are 0 when the transmittance of the system is close to 1. Moreover, the large group delay occurs at the transmission peak and its surroundings owing to the fact that the graphene ribbon and the graphene strip have a strong coupling effect at the resonance frequency. When the graphene mobility reaches 3μ0, the group delay of the system can be as high as 0.7 ps. However, the group delay at the transmission dips reaches a large negative value, meaning fast light propagation in the system. Meanwhile, the phase shift has also changed dramatically at the transmission dips. Zhang et al. recently has proposed an absorption efficiency of 50% and slow-light performance with a patterned graphene structure [where tructure . Howeverl4 results in stepped red-shift in the left transmission dip and its quality factor is getting smaller, indicating the dependence of the graphene strip length on left transmission spectra. Fig. Finally, coupling effects between the graphene ribbons and the graphene strips in PIT metasurface with different structural parameters are studied in detail, as illustrated in Fig. In short, we have numerically simulated and theoretically calculated the PIT in the patterned metasurface composed of the graphene ribbons and the graphene strips, which is caused by destructive interference between the superradiant mode and the subradiant mode. Interestingly, the dual-mode on-to-off modulation of PIT can be achieved by two gate voltages applied to the graphene ribbons and the graphene strips. Moreover, an absorption rate of 50% and slow-light property of 0.7 ps are achieved, demonstrating the proposed PIT metasurface has important applications in absorption and slow-light. Furthermore, coupling effects between the graphene ribbons and the graphene strips in PIT metasurface with different structural parameters are studied in detail. Thus, this work provides potential applications for the implementation of dual-mode on-to-off multi-function modulators.