FKBP5, termed FKBP51s, is a PD-L1 foldase, assisting the immune checkpoint molecule in maturation and expression on the plasma membrane. The concept that PD-L1 supports tumor-intrinsic properties is increasingly emerging. The aim of the present work was to confirm the pro-tumoral effect of PD-L1 on human glioma cell survival, stemness capacity and resistance, and to address the issue of whether, by targeting its foldase either chemically or by silencing, the aggressive tumor features could be attenuated. PD-L1-depleted glioma cells have a reduced threshold for apoptosis, while PD-L1 forced expression increases resistance. Similar results were obtained with FKBP51s modulation. The ability of PD-L1 to counteract cell death was hampered by FKBP51s silencing. PD-L1 expression was particularly high in glioma cells with a cancer-stem-cell profile. Moreover, PD-L1 sustained the spheroid formation capability of glioma cells. Targeting of FKBP51s by small-interfering RNA (siRNA) or the specific inhibitor SAFit2, reduced the number of formed spheroids, along with PD-L1 expression. Finally, in an orthotopic mouse model of glioblastoma, daily treatment with SAFit2 significantly reduced tumor PD-L1 expression, and tumor growth. In treated mice, caspase-3 activation and reduced vimentin expression were observed in excised tumors. In conclusion, targeting of FKBP51s hampers PD-L1 and its pro-tumoral properties, thereby affecting the self-renewal and growth capacities of glioblastoma cells in vitro and in vivo.Gliomas aberrantly express programmed cell death ligand-1 (PD-L1), which has a pivotal role in immunoevasion. The splicing isoform of  The interaction of PD-L1 with its counter-receptor programmed cell death-1 (PD-1) expressed by activated T lymphocytes, inhibits T-cell activation and proliferation, thus contributing to cancer immune evasion5. So far, anti-PD-1 treatment has shown a therapeutic efficacy limited to selected subsets of glioma patients6. Several studies are in progress, especially exploring the combination of anti-PD-1 antibodies with chemotherapies or targeted therapies6. It is worth noting that PD-L1 not only binds to PD-1 but also competes with CD28-to-CD80 binding7, suggesting that a PD-1 blockade may sometimes be insufficient to prevent signals emanating from PD-L1 receptors.Glioblastoma multiforme (GBM) is the most common, but also the most dangerous and aggressive form of primary brain cancer, for which no contemporary treatments are curative. Gliomas, indeed, express several co-inhibitory molecules that negatively regulate immune system functions6. Azuma et al.8 found that PD-L1, upon ligand stimulation, acts as a receptor that transduces pro-survival signals that enhance the threshold for apoptosis induced by both immune effectors and proapoptotic drugs. Zheng et al.9 found that PD-L1 sustains the self-renewal capability of malignant melanoma-initiating cells. Most recently, Qiu et al.10 demonstrated that PD-L1 binds to Ras and activates the Ras/Erk pathway. Via this mechanism, PD-L1 promotes the epithelial-to-mesenchymal transition (EMT) and GBM cell migration.Recent studies show that, in addition to its immunomodulatory function, PD-L1 conveys a survival signal to the cancer cell and sustains tumor aggressiveness11. PD-L1 glycosylation is an essential element influencing protein stability12. Most recently, in glioma, the splicing isoform of the FK506-binding protein-51 (FKBP51s)13 was found to serve as a PD-L1 co-chaperone, assisting in protein glycosylation14. PD-L1 post-translational modifications have been thoroughly reviewed by Hsu et al.15. According to Jiang et al.16 and the Oncomine database (www.oncomine.org), FKBP51 is among the top 10% of the most highly expressed genes in GBM. The FKBP51 protein structure includes a C-terminal TPR three-tandem-repeat domain responsible for protein-protein interaction, and two N-terminal FK domains, of which the one with most N terminals exhibits peptidyl-prolyl cis-trans isomerase (PPIase) activity18. FKBP51s is generated by alternative splicing of FKBP5 pre-mRNA13, which causes a frameshift with a premature stop codon, leading to a distinct C-terminus, compared to the canonical isoform. FKBP51s retains the PPIase activity but loses the TPR domain. An IHC study on 29 GBM specimens showed that FKBP51s is broadly expressed in this tumor, albeit with different proportion/intensity scores, with nuclear and/or cytoplasmic localization14. Biochemical evaluation of glioma cell lines showed that naive PD-L1 is complexed with FKBP51s in the endoplasmic reticulum, whereas the glycosylated form was detected in the Golgi apparatus14. FKBP51s knockdown severely reduced the level of glycosylated PD-L1, whether constitutively expressed or induced by ionizing radiation14. The essential role of the PPIase activity in PD-L1 protein maturation was confirmed by use of a selective inhibitor of this catalytic function, i.e., SAFit2, which produced similar results to those found in FKBP51s silencing14.Akt supports aberrant PD-L1 expression in glioma cells, through a translational mechanism favouring the assembly of a polyribosome complex that allows better entrance of the PD-L1 transcript into polysomesThe present study confirms that PD-L1 exerts important tumor-intrinsic properties in GBM. In particular, we show that PD-L1 sustains cell survival, resistance and stemness capability. PD-L1 expression was highest in GBM cancer-initiating cells, due to a post-transcriptional regulatory effect involving FKBP51s. Targeting of FKBP51s by gene silencing or via the selective inhibitor SAFit2, downmodulated PD-L1 expression and inhibited spheroid formation when GBM cancer-initiating cells were cultured under non-adherent conditions. In an orthotopic GBM mouse model, SAFit2 showed an anti-tumor effect, as assessed by reductions in tumor volumes, caspase activation and attenuated expression levels of PD-L1 and the mesenchymal marker vimentin.14. For PD-L1 downmodulation, cells were treated with specific siRNAs targeting PD-L1 or its co-chaperone FKBP51s. Twenty-four hours after transfection, some of the cells were harvested for lysate preparation. After a further 24 h, the remaining cells were collected for cell-death measurements via PI incorporation. Figure UTR1 and siFKBP51sUTR2) and another (siFKBP51) on the coding region. The PD-L1 signals at ≈50 kDa are those of mature (glycosylated) forms and those under 37 kDa correspond to the naive protein14 . The procaspase-7 level was decreased by the same siRNAs, with a cleaved fragment at ≈20 kDa also observable, consistent with apoptosis activation    . To confirm the role of FKBP51s in the resistance of GBM cells, we overexpressed FKBP51s and analyzed cell death using EV cells for comparison. In line with the FKBP51s knockdown sensitizing effect, we registered a significant decrease in etoposide-induced cell death in D54MG and U251MG cells transfected with FKBP51s vector, compared with EV cells   Fig. . Similar01) Fig. . FKBP51s03) Fig. . In etop03) Fig.  (p = 0.0ly) Fig. . To invely) Fig. , and mealy) Fig. . The silly) Fig. . The celly) Fig. , as wellly) Fig. .Fig. 2FK10, supports the concept that this immune checkpoint is a hallmark of GBM malignancy. To address whether PD-L1 expression increased with the aggressiveness of GBM cells, we used two GBM cell lines previously established by Kroonen et al.19 from the tumor mass (TM-GBM) and the subventricular zone (SVZ-GBM) of an orthotopic GBM model. TM-GBM and SVZ-GBM cell lines, characterized by an increasing level of aggressiveness from TM to SVZ, were derived from the human U87MG cell line. SVZ-GBM cells are particularly rich in GBM cancer stem cells and have a high tumorigenic potential, as assessed by mouse reinjection19 or by their capacity to grow in spheres when cultured under non-adherent conditions20. We confirmed the high tumorigenicity of SVZ-GBM cells showing increased levels of p-Akt, along with its substrate p-S6K1  were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% FBS, 200 mM glutamine, and 100 U/mL penicillin–streptomycin. TM-GBM cells and SVZ-GBM cells were U87MG cells obtained by microdissection of xenografted mice brain19, from, respectively, the tumor mass and the subventricular zone, and seeded in cultures19. For FKBP51s and PD-L1 knockin and knockdown, cells were transfected using the K2 Transfection System , as previously described14 and in accordance with the manufacturer’s recommendations. For overexpressing FKBP51s and PD-L1 a True-ORF-Myc-DDK-tagged and a True-ORF-GFP-tagged expression vectors were used , which carried the complementary DNA (cDNA) of the human FKBP5 transcript variant 4 and CD274, respectively. Control cells were transfected with the related empty vector (EV). FKBP51s silencing was performed using short-interfering oligoribonucleotides. The siFKBP51 was a mix of two siRNAs designed on the coding region; siFKBP51sUTR1and FKBP51sUTR2 were designed on 3′-UTR14. The non-silencing RNA (NSRNA) and PD-L1 siRNA were from Qiagen (siPD-L1)  or Novus Biologicals (siPD-L1.2) . U87MG cells used for xenograft implantation were previously transduced with lentiviral vectors allowing the dual expression of eGFP and luciferase as previously described20. In experiments with etoposide, the drug was added 24 h after transfection at a concentration of 20 μM. For experiments with SAFit222, which was provided by Prof. Felix Hausch laboratory, a stock solution (60 μM in DMSO) was used at 1:1000 dilution in DMEM-F12 media; for control cells, DMSO has diluted accordingly.Human glioma cell lines D54MG, U251MG, SF767MG were obtained and cultured as described14 and assayed by immunoblot as previously described14. The primary antibodies against FKBP51 , FKBP51s13 and CD274/PD-L1  were used diluted 1:2500. CD133  was used diluted 1:1000. A further antibody Pdcd-1L1  was used for PD-L1 detection at the 1:1000 dilution. Antibody against M2-Flag, caspase-7 and γ-Tubulin  were used diluted 1:5000. Anti β-Actin-Peroxidase  was used diluted 1:10000. Anti-phospho-Akt (Ser473), Akt1/2/3, phosphor-S6 kinase, G3PDH and Sox-2  were used diluted 1:1000. Anti-p70S6 kinase  was used diluted 1:500.Whole-cell lysates were homogenized in modified RIPA buffer23. Apoptosis was assessed by two methods: propidium iodide (PI) incorporation to analyze the DNA content in permeabilized cells24, and the annexin V-binding assay in double staining with PI, according to Vermes et al.25. Cells were analyzed with a BD Accuri™ C6 Cytometer .Expression of PD-L1 was assessed using anti-B7H1-phycoerythrin (PE)  at a concentration of 0.05 μg/ml. A PE-conjugated control Ig isotype was used to assess non-specific binding. Briefly, cells were harvested and incubated with the antibodies mentioned above for 30 min in the dark at 4 °C, washed and then analyzed with a BD Accuri™ C6 Cytometer . Cleaved caspase-3 immunostaining was performed as previously described26. Quantification of vimentin expression in-brain slices was performed as followed: mean fluorescence intensity (MFI) was calculated for each GFP+ cell stained with anti-vimentin along with GFP+ cell stained with control antibody. Positivity was assigned when vimentin MFI was at least four-fold higher than control MFI. When required, the brightness, contrast, and color balance of the images were adjusted in Photoshop CS2 . This adjustment was applied to every pixel in each image.Before the immunostaining, cells were seeded for 3 h on coverslips coated with polyornithine (0.1 mg/ml), then, after washing with PBS the cells were fixed in 4% paraformaldehyde (PFA) for 15 min. Brain coronal sections or cells on coverslips were permeabilized and unspecific binding sites were blocked for an hour at room temperature using a 10% donkey serum and 0.2% Triton X-100 PBS solution. Tissue sections or coverslipped cells were then incubated overnight at 4 °C with primary antibodies diluted in PBS containing 0.1% of donkey serum and 0.1% of Triton X-100, followed by tetramethylrhodamine (TRITC)-, fluorescein isothiocyanate (FITC)-conjugated, or cyanine 5 (Cy5) secondary antibodies  diluted 1:500. Anti Ki-67  was used diluted 1:500. Anti-Vimentin  was used diluted 1:400. Anti-PD-L1  was used diluted 1:200. Anti-Active Caspase-3  was diluted 1:250. Anti-GFP  was used diluted 1:500. Counterstaining with DAPI  was performed and slides coverslipped with Fluoromount-G . Images were acquired with OLYMPUS FV1000 confocal microscopy. For PD-L1 and active caspase-3 expression, fluorescence intensity was quantified according to “corrected total cryosection fluorescence” (CTCF)27, using the SsoAdvancedTM SYBR Green Supermix (Bio-Rad) and specific qPCR primers. Amplification of FKBP51s was performed as previously described13. Oligo sequences used for PD-L1 mRNA quantitation along with co-amplified housekeeping genes are:Total RNA was extracted from cells by Trizol . Each RNA was used for cDNA synthesis with iScript Reverse Transcription . Relative gene expression was quantified by qPCR with 2–ΔCt comparative methodFw-PD-L1 5′-GCTTTTCAATGTGACCAGCA-3′Rev-PD-L1 5′-TGGCTCCCAGCCTTACCAAG-3′Fw-18S 5′-CGATGCGGCGGCGTTATTC-3′Rev-18S 5′-TCTGTCAATCCTGTCCGTGTCC-3′Fw-GAPDH 5′-GGACTCATGACCACAGTCCAT-3′Rev-GAPDH 5′-GTTCAGCTCAGGGATGACCTT-3′Fw-β-Actin 5′-CGAGGCCCAGAGCAAGAGAG-3′Rev-β-Actin 5′-CGGTTGGCCTTAGGGTTCAG-3′TM- and SVZ-GBM cells were cultured in DMEM/F12 serum-free medium containing B-27-Supplement without vitamin A  and supplemented with recombinant epidermal growth factor and fibroblast growth factor 2 . After 4 days in culture, the number of spheroids was counted using an optical microscope.TM- and SVZ-GBM cells were daily treated with 60 nM SAFit2 or opportunely diluted DMSO. Every 2 days, cells were harvested, and cell number was estimated by the Countess II Automated Cell Counter (Life Technologies). After a 4-day treatment, cells were seeded on a coverslip and processed for Ki67 immunostaining as previously described.22. Before obtaining brains, mice were anaesthetized with an injection of Nembutal  followed by an intracardiac perfusion with a NaCl 0.9% solution  plus 4% PFA at 4 °C . Then after, brains were collected, postfixed in 4% PFA and cryoprotected overnight in a 20% PBS/sucrose solution. Brains were frozen at −20 °C in a 2-methylbutane solution (Sigma-Adrich) and cut into 14 μm thick coronal sections using a cryostat. The tumor volume was calculated on GFP+ areas using ImageJ and, according to Kim et al.28, the formula (Volume = 0.5 × length × width × height). For bioluminescence imaging: immunodeficient nude mice bearing intracranial xenografts were injected intraperitoneally with d-luciferin . After anesthesia using 2.5% isoflurane, mice were imaged with a charge-coupled device camera-based bioluminescence imaging system .P40 female immune-deficient mice (Crl:NU-Foxn1nu) were obtained from Charles River Laboratories  and handled according to the Animal Ethical Committee of the University of Liège. All animals were cared for in accordance with the Declaration of Helsinki and the guidelines of the Belgium Ministry of Agriculture, in agreement with the European Commission Laboratory Animal Care and Use Regulation . For intracranial transplantation, mice were anesthetized with an intraperitoneal injection of ketamine  and xylazine  solution (V/V). The cranium was exposed and a small hole was drilled 2.5 mm lateral and 0.5 mm anterior to the bregma with a size 34 Dremel Inverted Cone Burr. Mice were positioned in a stereotactic frame and 50000 GFP+ luc+ U87MG cells in 2 µl PBS were injected into the right striatum through a 27-gauge needle over 1 min at 3 mm below the dura mater. The incision was closed with 3 M Vetbond Tissue Adhesive . For mice treatment, with SAFit2, this was performed as previously describedt-test was used to analyze differences between means of values. A p-value of ≤0.05 was considered statistically significant.Student’s Supplementary figures to the manuscript


While doctors should analyze a large amount of electronic medical record (EMR) data to conduct clinical research, the analyzing process requires information technology (IT) skills, which is difficult for most doctors in China.In this paper, we build a novel tool QAnalysis, where doctors enter their analytic requirements in their natural language and then the tool returns charts and tables to the doctors. For a given question from a user, we first segment the sentence, and then we use grammar parser to analyze the structure of the sentence. After linking the segmentations to concepts and predicates in knowledge graphs, we convert the question into a set of triples connected with different kinds of operators. These triples are converted to queries in Cypher, the query language for Neo4j. Finally, the query is executed on Neo4j, and the results shown in terms of tables and charts are returned to the user.https://github.com/NLP-BigDataLab/QAnalysis-project.The tool supports top 50 questions we gathered from two hospital departments with the Delphi method. We also gathered 161 questions from clinical research papers with statistical requirements on EMR data. Experimental results show that our tool can directly cover 78.20% of these statistical questions and the precision is as high as 96.36%. Such extension is easy to achieve with the help of knowledge-graph technology we have adopted. The recorded demo can be accessed from Our tool shows great flexibility in processing different kinds of statistic questions, which provides a convenient way for doctors to get statistical results directly in natural language. More hospitals have integrated data from different information tools into clinical data repository (CDR), and regional platforms have also gathered EMR from dozens of hospitals in an area. The data can be utilized in many different types of clinical research. For example, the CALIBER  themselves. In this paper, we design a novel tool QAnalysis. The tool allows doctors to enter their analytic requirements with questions in their natural language, and it returns the query results with charts and tables.While there are many studies on knowledge-based QA , 2, our 1. Questions must be analytics instead of fact-oriented. Existing work focuses on fact-oriented questions. The question always like “Who is Yao Ming’s wife” which can be constructed as a corresponding triple, and the answer is an entity in the knowledge base. Our questions are analytic, and they often include statistical operators, such as ratio, maximum, and average. Besides, a question usually contains logic operators, such as NOT, AND, and OR. Negations and combinations are important concepts in clinical medicine. In general, the answer of a fact-oriented question can be directly extracted from data in the knowledge base. For analytic questions, the answers are derived statistically from data in the knowledge base. In particular, analytic questions may include vocabularies referring to logic operator, and statistic operators such as average and summarization.2. Questions contain medical terms and require domain knowledge. The questions inevitably have medical terms, such as names of diseases, tests and drugs. These named entities lead to difficulties in parsing questions. Specially, the word-segmentation step may split the word incorrectly. Furthermore, doctors may use types of drugs instead of drug names in the questions. For example, they may ask “The number of patients who use ACEI (angiotension converting enzyme inhibitors) drugs”. However, the EMRs only contain common drug names; therefore, drug knowledge bases may be used to answer these kinds of questions.3. High accuracy is required. For QA on the common domain, the precision can be fairly low. For example, the best precision score of question answering over linked data (QALD) in 2016  to describe the clinical research questions. VT is the terminator set; the elements in the set are concept types listed in Table As shown in Table Property-condition chunks can be composed of class/instance, attribute names, and attribute values, including numeric, enumerated, and date attributes. For example, “葡萄糖化验结果偏低” (“glucose test results are low”), in which the “葡萄糖”(“glucose”) is an instance, “化验结果” (“test results”) belongs to attributes, and “偏低” (“low”) belongs to enumeration values. The subject can also be omitted, for example, “住院天数超过10天” , where “住院天数”  is an attribute and “超过10天” (“exceeds 10 days”) indicates numeric values of attributes in a range form.The query-item chunks represent the statistical operations on the records that satisfy the combination condition. For example, the question about etiology “What is the ratio of heart failure in patients with hypertension, diabetes, and coronary heart disease?” uses the ratio operation. The question about effectiveness “How many female patients with hyperglycemia and no hypertension have low levels of glucose tests after they took hypoglycemic drugs?” contains several logic and time operators to filter the patient’s records and uses the count operation.All the previous examples show the expressiveness of our grammar, and these kinds of questions are complex and are hard to interpret in current fact-oriented knowledge-based QA tools.Although context-free grammar covers most questions used in clinical research, grammars still fail to parse in some special cases. For example, “patients who suffer from hyperglycemia and hypertension”, our grammar cannot capture this relationship because of the remote dependency of “suffer from” and “hypertension.” So, we use Stanford parser to do a dependency analysis of the sentence, which can obtain the parallel relationship “conj” between “hyperglycemia” and “hypertension.” Through the parallel relationship, we get the verb “suffer from” of “hypertension.” The concept-type sequence is extended to “Relation Instance LogicalOperator Relation Instance” for normal parsing. Currently, we use “cc” and “conj” relations, to solve the above problem. In the future, we will use more parsing results of Stanford parser to make the input query more flexible.We convert the sequence of concept types and the results of parse tree into an internal knowledge representation. As mentioned in Concept linking, the representation is basically triples connected with logical and time operators, coupled with distribution operators.The relation condition chunk typically consists of two tokens, such as <Relation, Instance>. To convert the chunk into a triple, we may use the definitions of domain and range in the patient-schema graph the nearest class node to find the subject of the triple. For instance, in “eat Coldrine”, “eat” is binded with the predicate “take” since the schema graph contains a synonym relation < eat, sameAs, take >. In the concept linking step, “Coldrine” can be linked to “cold” and “Coldrine capsule”. Based on the patient-schema graph, < patient, suffer from, cold >、 < patient, take, Coldrine capsule >, the latter is chosen since the predicate is “take”. The property-conditional chunks are similar to relation-condition chunks. Our query combines statistic operators with classes and property values. With the help of query-item chunks, we can find out the object that the statistic operator works on.Among the Not, LogicalOperator and TimeOperator that appear in the grammar tree, Not has the highest priority. TimeOperator is least prioritized among the three. We use “” to indicate priority. Besides, the chronology of the relations may not explicitly appear in the sentence. We use some heuristic rules to detect them, which can be a part of the patient-schema graph.About the example in Overview of the method, the ultimate expression is ) after <patient, take, hypoglycemic drugs> after  < patient, sex, female> < patient, count,? > .This step is to translate the semantic representation of the query into Cypher, which is a query language of graph database Neo4j. The translation process consists of three parts: the translation of match clause, where clause, and return clause. Similar to the tree-like semantic representation, the match clause is also made up of nodes and edges, in which “” denotes node and “-- >” denotes edge. The attribute-value constraint in semantic representation corresponded to the conditions in the where clause. The target attribute values or classes in the semantic representation corresponds to the return clause in the query statement. For example, the patient node directly connects with the disease node, the test node, and the drug node; and the structure corresponds to the match clause in Fig. We execute the Cypher query in Fig. Since no standard question sets are applicable in our contexts for evaluation, we collect our questions from both related literature and clinical doctors. We search literature on Wanfang Data, a literature-resource library with major research journals in Chinese, using different combinations of three groups of key words: electronic medical records, electronic health records, statistical, data analysis and epidemiological investigation, clinical research. Out of the 50 returned papers, 16 are with statistical problems in clinical research. For English questions, we also select published papers from 2016 to 2017 in the CALIBER research project. About 12 papers with statistic requirements  from EMR are selected. There are altogether 28 papers in Chinese and English, and we gathered 161 questions from them. Since a part of the question set is taken from CALIBER, and is translated by the doctor. While translating these questions into Chinese, many entities do not exist in the current system knowledge base, such as “HDL cholesterol ratio,” “England,” and “depression.” We replace them with similar entities in our knowledge base. For example, “How many female patients in England” will be replaced with “How many female patients in Shanghai?”. We merge these questions with 50 others collected from the doctors and get 211 questions altogether. The question lists can be accessed from our GitHub URL.Besides, we organize a joint team with physicians and IT professionals to generate the gold standard (benchmark results) for the 211 questions. The team are divided into three groups. Each group contain two persons, one is a physician whose task is to verify whether the query is correctly interpreted, the other is an IT professional who execute the query against the dataset, then both of them verify whether the result is correct. Each question is assigned to two groups. If the two groups get different answers, then the third groups decide the ultimate results.The EMR data used to test the performance and accuracy of our tool in this paper is from Shanghai Shuguang Hospital, which is a large hospital for integrated Chinese and Western Medicine. It has used EMR systems for more than a decade. Patient health-care encounters are well-documented and time-stamped. The hospital has built their CDR for three years. We construct a sub-repository with congestive heart failure for clinical research. There are 486,525 edges and 21,728 vertices in our graph, and the average degree is 44.78. As shown in Table We define coverage and precision as the quality indicators of the tool. Here, coverage means the percentage of natural language questions that our tool can recognize and return results. Precision means for all the results returned from our tool, the percentage of correct results is returned. Coverage is more suitable to use than other similar ones, such as recall, since we can extend our tool with more rules on grammar or translation, and the tool can cover more types of questions.The evaluation result is shown in Table There is one question with a statistical vocabulary “annual growth rate” that is not supported by the tool. We can solve this problem by adding this word to the statistical vocabulary and setting up its related functionThere are 17 questions whose semantics cannot be interpreted directly, for example, “What is the comparison of the prevalence of hypertension between 1991 and 2014?” Currently, the tool’s grammar cannot parse and interpret the term “comparison” correctly. However, we can extend the vocabulary and parse them into two tree-like structures later.Two questions contain operations not supported in Neo4j, for example “What is the sex ratio of the top 10 diseases among inpatients in 2012?” we need to sort the group by operation for each disease first, but it is not supported in Neo4j.Twenty-six questions contain properties that do not appear in our patient graph, such as “current smoker.” as mentioned before, adding new nodes and edges to extend our patient graph is easyResults for six questions are wrong, and the precision is as high as 96.36%. The reasons are the following:Questions, which collected from doctor, contain vocabularies that do not exist in our knowledge base. For example, “How many patients are suffering from sepsis, heart failure, and coronary heart disease?”, “sepsis” is not in our knowledge base. Our tool automatically ignores the entities, so the result is the same as “How many patients are suffering from heart failure and coronary heart disease?” To solve the problem, we plan to add a named entity recognition step. If the entity cannot link to any entity in the knowledge base, we will prompt the user.Entities in the question are linked to wrong entities in the knowledge base. Entities in the question may not exist in the knowledge base, so they are linked to the wrong entities, for example, “Who are the patients with coronary heart disease and rheumatic heart disease?” The tool will link rheumatic heart disease to heart disease. In this case, a larger knowledge base is required.Our tool can only recognize the time relation between two entities where temporal preposition exists. In “High blood pressure patients with coronary heart disease after heart failure”, our tool can only know that heart failure occurs before coronary heart disease, but the hidden semantics that hypertension happens earlier than the previous two cannot be captured. For this case, we intend to define a set of heuristic rules to recognize such domain-specific implications.The remaining 46 questions whose results are not returned can be divided into four categories:If none of the above three situations occur in the question, the accuracy will be 100%. At the same time, we are trying to prevent errors that may be caused by the above situations. So, we output the results of the parser to the doctor for review.The number of nodes in questions is large and mostly more than three, and the query is generally completed within two seconds. These questions accounted for 10% of the question set.Not only is the number of nodes more than three, but it also contains several logic operators, especially OR and TimeOperator. These kinds of questions require two to six seconds, and account 2% of the question set.The start node of the Cypher query is a large collection, which makes it time-consuming to filter and match time operations later. The query time may take as long as 25.88 s. We encounter five questions with the problem. In general, the performance is basically acceptable in current phase.The test environment is a PC with Centos 6.8, 16G memory, and 4 CPU core. The JVM maximum heap size of Neo4j is set to 4G. Fifty questions are used; the result is shown in Table Our work is closely related to two important research topics: question answering using a knowledge base (KB-based QA) and question answering on statistical linked data.The traditional methods for KB-based QA is based on semantic parsing –13. It rThe overall performance of the semantic-parsing method is not promising since errors may occur in each step from converting original questions to logic forms. Another way for QA on a large knowledge base is to convert both questions and answers to similar representations and check similarities between the two representations. Also,  transforBoth QALD and TREC have special for medical field. Recently, the TREC-CDS  track requires to retrieve relevant scientific articles that contain the answers to medical questions. Goodwin et al.  propose QALD 2016 has a special task about question answering on statistical linked data . The appThe cube-based approach requires the original graph-based dataset to be converted into multidimensional data. It not only needs a large amount of skills and hard work but also limits the possible ways questions are raised. Faceted-search approach, in general, does not support complex logical operations, such as negative, or relations, between events, which is important in the medical field.We designed and implemented a statistical analysis tool in the natural language, which aims to help doctors who lack IT skills and ease their clinical research. Our tool shows great flexibility in processing different kinds of statistic questions, which is important in domain-oriented applications and is also the key difference between our tool and the current fact-oriented knowledge-based QA tool. Besides, our designed clinical knowledge graph is easy to extend and to connect with other knowledge graphs.In the future, we will use more dependency relation in Stanford parser to allow questions to be expressed more flexibly. Once enough user logs are accumulated, we plan use machine learning techniques to handle these question-translation challenges. Since, the schema of our patient graph and our questions representation do not limit our questions to statistic ones, we plan to combine statistical requirements with normal KB-based QA requirements in our tool.


Frontotemporal dementia (FTD) and Amyotrophic Lateral Sclerosis (ALS) are incurable, progressive and fatal neurodegenerative diseases with patients variably affected clinically by motor, behavior, and cognitive deficits. The accumulation of an RNA-binding protein, TDP-43, is the most significant pathological finding in approximately 95% of ALS cases and 50% of FTD cases, and discovery of this common pathological signature, together with an increasing understanding of the shared genetic basis of these disorders, has led to FTD and ALS being considered as part of a single disease continuum. Given the widespread aggregation and accumulation of TDP-43 in FTD-ALS spectrum disorder, TDP-43 may have potential as a biomarker in these diseases.We therefore conducted a systematic review and meta-analysis to evaluate the diagnostic utility of TDP-43 detected in the cerebrospinal fluid (CSF) of patients with FTD-ALS spectrum disorder.p = 0.02).From seven studies, our results demonstrate that patients with ALS have a statistically significantly higher level of TDP-43 in CSF (effect size 0.64, 95% CI: 0.1–1.19, These data suggest promise for the use of CSF TDP-43 as a biomarker for ALS. Frontotemporal dementia (FTD) is an umbrella term for a spectrum of neurodegenerative disorders. These disorders primarily affect behaviour, personality, executive function and language (and may include motor impairment). It is the second most common form of dementia after Alzheimer’s disease (AD) in people under 65 years old, with the average age of onset being between 45 and 65 years old, and the peak prevalence being around age 65 to 69 . PrognosFTD also affects the motor system and is now increasingly thought to be on a syndromic spectrum with amyotrophic lateral sclerosis . ALS isC9orf72, found to be present in the majority of cases of familial cases of FTD-ALS spectrum disorders The title and abstract of each paper were screened for relevance with respect to inclusion and exclusion criteria. For studies meeting our predefined criteria the full text was retrieved, imported to Excel and duplicate records were discarded. The quality of each paper was then evaluated by the QUADAS-2 quality score and relevant data were extracted.All studies that measure TDP-43 in the CSF of living patients with ALS or FTD, compared to controls.Inclusion criteriaNo control groupAnimal studiesPost mortem studiesReviewsLetters and commentsAbstractsNon-parametric results Exclusion criteriaStudy ID: (i) Author and (ii) YearIntervention: FTD , ALS or both (FTD-ALS)Medical diagnosis of comparison/controlsIntervention quantification method (western blot or ELISA)Age: mean age of patients and controlsSex: of patients and controlsDiagnosis method of patientsGenetic factorsSample size: of patients and controlsStudy characteristics to be extracted:Two similar searches were done, with the similar objectives. These were to look at the effect size of lymphocytic bound TDP-43 in FTD, ALS and FTD-ALS patients compared to controls, and the effect size of plasma TDP-43 in FTD, ALS and FTD-ALS compared to controls. However, neither of these searches produced enough papers in order to do a systematic review. AND (Lymphocyte [MeSH] OR White Cell [MeSH] or White Blood Cell [MeSH]) AND TDP [MeSH]. AND (Plasma [MeSH] OR Serum [MeSH] OR Blood [MeSH]) AND TDP [MeSH]2-values, and a funnel plot and Egger’s regression test was used to assess publication bias.An individual meta-analysis was carried out for each group of patients (i) FTD alone, (ii) ALS alone and (iii) FTD and ALS patients combined as an overall evaluation of FTD-ALS spectrum disorders. Additional subgroup analyses included: an assessment of heterogeneity and a quality score. Outcome measures were calculated for each of the studies identified and included on a forest plot. Given the variability of intervention quantification included in the analysis, outcome measures were recorded in standardised mean differences (SMD), to allow for meaningful comparisons between studies. SMD was compared using Hedges g-statistic, to account for bias from small sample sizes, using a random effects model. SMDs were reported as odds ratios with 95% confidence intervals. Heterogeneity was assessed for all outcome measures using Ip = 0.8029) or funnel plot. Systematic review, risk of bias and quality assessment revealed that only 3 of the 7 identified papers reported on measures to reduce bias, such as blinding  demonstrating a significant increase in detectable TDP-43 in the CSF of patients with FTD or ALS compared to controls. However, Chi2 was 20.57 and I2 was 76% showing significant heterogeneity in this dataset . There was a similarly significant heterogeneity with a Chi2 of 10.75 and an I2 of 81%. Meta-analysis of studies assessing TDP-43 in the CSF of patients with ALS alone showed an effect estimate of 0.64 (95% CI: 0.10–1.19). A Z-score of 2.3 (p = 0.02) showing a significant increase in detectable TDP-43 in the CSF of ALS patients. Chi2 was 11.80 and I2 was 66% showing moderate heterogeneity  FTD-ALS spectrum disorders (ALS and FTD combined), (ii) FTD alone, and (iii) ALS alone. Meta-analysis of FTD-ALS spectrum disorders versus controls using standard mean difference found an effect estimate of 0.55 (95% CI: 0.01–1.09) and a Z-score of 2.0  showed a significantly increased CSF TDP-43. We also detected substantial heterogeneity and relative risk of bias in these studies. This heterogeneity is likely due to the wide variety of methods used to analyse CSF TDP-43 concentrations, but could also reflect the disease heterogeneity  seen in these spectrum disorders and the heterogeneity of controls used for comparison. Two of the studies used normal healthy controls , 20 whilC9orf72 hexanucleotide expansion might provide an indication as to how early TDP-43 aggregation occurs, and potentially would allow us to diagnose FTD-ALS spectrum disorders pre-symptomatically in these patients [C9orf72) that might skew data. The study should be carried out longitudinally in order to map the progression of TDP-43 in both controls and patients. Diagnosis would be done using currently approved diagnostic tools, with post-mortem pathological verification of diagnosis. Both the sampler and the pathologist should be blinded to whether the CSF is from a control or a patient. Equally, analysis should be done with an ELISA and presented in empirical concentrations to improve transparency. As of yet, there are not enough studies into the TDP-43 in the blood, either in lymphocytes or in plasma. However, these are avenues for future meta-analyses (given more studies), as they provide less invasive diagnostic tests than lumbar puncture.Given the significant effect size shown in this meta-analysis, demonstrating an increase in the levels of detectable TDP-43 in patients with ALS, further research to refine the use of CSF TDP-43 as a diagnostic tool is warranted. Furthermore, a longitudinal study of CSF TDP-43 concentrations in patients with FTD-ALS spectrum disorders might show if CSF TDP-43 increases with disease progression. Similarly, a longitudinal study investigating carriers of the patients . This woOur data demonstrate a significant difference in TDP-43 levels in ALS patients compared to controls, implying that CSF TDP-43 levels show promise as a potential biomarker in ALS. It is probable that the statistically significant result observed in FTD-ALS spectrum disorder cases Fig. , could bOur systematic review and meta-analysis reveals early supportive data indicating that TDP-43 detected in the CSF of patients with FTD-ALS spectrum disorders in particular ALS patients could be a promising biomarker in these diseases. Given the current paucity of diagnostic and prognostic biomarkers in these disorders, this result clearly indicates that further studies in to its utility are warranted.


The synthesis of a graphene derivative with exclusively azide groups (graphene azide) is however still a challenge. In comparison also hydrogenated graphene, called graphene or halogenated graphene remain challenging to synthesize. A route to graphene azide would be the desoxygenation of azide functionalized oxo-G1. Here we show how treatment of azide functionalized oxo-G1 with HCl enlarges the π-system and removes strongly adsorbed water and some oxo-functional groups. This development reflects one step towards graphene azide.In the last decades, organic azides haven proven to be very useful precursors in organic chemistry, for example in 1,3-dipolar cycloaddition reactions (click-chemistry). Likewise, azides can be introduced into graphene oxide with an almost intact carbon framework, namely oxo-functionalized graphene (oxo-G Intriguing properties like a high flexibility, transparency of 97.7% (visible regime) [3 up to 106 cm2/Vs [1) bears defined amounts of lattice defects and they play therefore a minor role and surface chemistry is enabled. A typical density of lattice defects of about 0.3%–0.8% is achieved [1, but recently we succeeded in converting C-O bonds to C-N bonds by introducing the azide group [1 with sodium azide and the major chemical functional groups of that derivative are hydroxyl and azide groups. Consequently, hydroxyl and azide functionalized graphene was produced with hydroxyl groups in majority. While graphene oxide is known for a long time, graphene decorated with exclusively azide groups is completely unknown. Also the synthesis of other “pure” derivatives, like graphane (fully hydrogenated graphene) [2-carbon lattice with only a single type of functional group attached to a sp3-carbon atom. It can be expected that graphene azide could act as a precursor for nitrogen doped graphene or functional graphene derivatives by introducing further addends.Graphene is a 2D nanomaterial consisting out of exclusively sp regime) ,2,3, cha6 cm2/Vs ,5 and a 6 cm2/Vs ,9. In ad6 cm2/Vs ,13,14,15raphene) ,17 and graphene) ,19. Such1 with azide groups (oxo-G1-N3) followed by HCl treatment. We observe an increased amount of aromatic C=C bonds and a significant removal of adsorbed water leading to a hydrophobic derivative of oxo-G1-N3. The product is analyzed by thermogravimetric analysis coupled with mass spectrometry (TGA-MS), UV-Vis, FTIR, Raman and elemental analysis (EA). Although hydroxyl groups are still present in HCl treated oxo-G1-N3, the expanded π-system is one further step towards the synthesis of graphene azide . Oxo-G1 was subsequently treated with sodium azide to obtain azide-functionalized oxo-G1. A nitrogen content of 3.3% was determined by EA and due to the carbon content of about 45% it can be calculated that one azide group is bound to around every 50th carbon atom 28.8(O)5.7N3(OH2)2.1(OSO3)0.5. Labelling of azide by 15N enables the detection of 15N14N by TGA-MS at m/z 29  to oxo-G1-N3 leads to a decreased solubility depending on the reaction time and acid concentration . Thus, structural changes must be responsible for that observation. The product was therefore isolated and thoroughly washed with water by centrifugation and redispersion in a 1/1 mixture of water and 2-propanol. Subsequent analyses via FTIR spectroscopy, EA, TGA-MS, UV-Vis and Raman reveal that graphene azide is not yet fully formed and hydroxyl groups are still present, but π-conjugated regions increased.The addition of HCl1-N3 is depicted in 1 FTIR features have been investigated since many years [1 and oxo-G1-N3 can be related to the following functional groups: 3600–3000 cm−1 (C-OH); 2360* cm−1 (CO2); 2123/2112 cm−1 (14N3/15N3) 1730 cm−1 (C=O); 1630 cm−1 (adsorbed H2O); 1575 cm−1 (C=C); 1433 cm−1 (C-OH or carboxylate); 1254 cm−1 (C-O-C), 1060 cm−1 (C-O or C-C) [1-N3 dispersion began to precipitate within minutes. A precipitate was immediately formed upon treatment with concentrated HCl. FTIR spectra were recorded on drop casted samples on ZnSe-windows , respectively. Moreover the absorption at 1215 cm−1 of reduced oxo-G1 is different compared to 1254 cm−1 for HCl treated oxo-G1-N3 (oxo-G1-N3-HCl). Importantly, the azide-peak  is preserved upon treatment with diluted HCl (diluted 0.5 M–2 M for 60 h) and concentrated HCl treatment for very short reaction times (minutes to few hours). Long reaction times and higher acid concentration result both in cleavage of the azide group, e.g., 7 days treatment with 1 M HCl. Moreover the azide group is not stable upon treatment with a reducing agent, such as NaBH4, as well as temperature annealing [Interestingly, the peak at 1575 cmsappears . Since t1-N3-(HCl) by EA (1-N3-(HCl) (2M HCl treatment) of 54.8%, a decreased sulfur content to 0.4% and 4.5% nitrogen. Interestingly, the hydrogen content remained constant with 2.6%. TGA-MS reveals that the m/z 18 peak up to a temperature of 120 °C is smaller compared to oxo-G1 or oxo-G1-N3. This detection is in line with the observation that oxo-G1-N3 is almost not dispersible in water but in a mixture 1/1 mixture of 2-propanol. Also for purification, by means of centrifugation and redispersion, 2-propanol was necessary as an additional solvent. Consequently, the powder of isolated oxo-G1-N3-(HCl) (freeze-fried) still contains some 2-propanol, as indicated by the m/z 45 signal, that relates to the H3COH+ mass fragment of 2-propanol, with cleaved methyl radical (1-N3-(HCl) is quite high, it seems likely that some 2-propanol is still present that consist of 1 oxygen and 8 hydrogen atoms. With that in mind we can calculate a molecular formula of C42(OH)20.4N3(OSO3)0.2(2-propanol)0.5. Within the experimental error, one can conclude that at least epoxy- and some hydroxyl groups were removed from the carbon lattice of oxo-G1-N3, leaving azide bound to the graphene lattice. A growing π-system upon HCl treatment of oxo-G1-N3 is also apparent by comparing UV-Vis spectra. As depicted in To clear up a potential loss of moieties, we further analyzed washed and freeze-dried oxo-Gl) by EA  and and l) by EA D. EA ind radical D, green.−1 to the G peak at about 1580 cm−1 can be used to determine the size of intact graphene patches [1 and GO in general. For comparison, oxo-G1 was chemically reduced by vapor of hydriodic acid and trifluoroacetic acid and the spectrum indicated graphene with a density of defects between roughly 0.3%–0.8% (ID/IG ratio of 3.0) [1-N3-(HCl), treated with 12M HCl, indicate the growth of graphene like patches by the increased ID/IG from 1.1 to 1.4. By measuring unreduced and so highly functionalized oxo-G1-N3-(HCl) an increase of ID/IG ratio indicates a reduction of defect sites as we are in the high defect regime [Raman spectroscopy is a tool that developed to the major characterization technique for graphene materials and the ratio of the intensity of the defect induced D peak at about 1340 cm patches ,30,31. A of 3.0) ,30,32. Rt regime ,32.®  and double distilled water from Carl Roth® . A Sigma 4K15 centrifuge was used with 200 mL plastic beakers. Transmission FTIR spectra were recorded on a Bruker Tensor27  and ZnSe-windows had been used. TGA-MS was performed on a Netzsch Skimmer STA 409 CD coupled with mass spectroscopy . Raman spectra were recorded with the confocal Raman spectrometer LabRAM Aramis  equipped with a second harmonic 532 nm Nd-YAG laser for excitation. The acquisition was set to 0.2 s. The absorbance of our samples was recorded using quartz cuvettes and a Lambda 1050  in the wavelength from 200 to 600 nm.Graphite grade 3061 was purchased from Asbury carbon mills . Potassium permanganate, sulfuric acid, hydrogen peroxide, sodium azide and hydrogen chloride solution were purchased from Sigma Aldrich1 and oxo-G1-N3 powder were synthesized as described in the literature [1 dispersion with main flake sizes of 1 μm to 20 μm. The concentration of 0.488 mg/mL was calculated by freeze-drying a small amount of the dispersion.Dry oxo-Gterature . Graphit1-N3, the dispersion containing 100 mg oxo-G1 was stirred with 80 mg of Na14N3 for 1 h followed by freeze-drying. Purification was accomplished by repetitive centrifugation . The yield after lyophilization was 77.6 mg [1-15N3 was produced similarly, but on half scale (yield: 38.8 mg).To obtain oxo-G(aq). To this, 10 mL of differently concentrated HCl solutions  were added. The samples were centrifuged for purification  and redispersed with water or 50:50 water/isopropanol.All dried samples had been dispersed in pure water to obtain dispersions with a concentration of 1 mg/mL  before the treatment with HCl1-N3 samples, few drops of the dispersion had been dropped onto a ZnSe window and put onto a heated magnetic stirrer to obtain a temperature annealed sample . For a chemical reduced reference, a few mL of the dispersion (10 mg oxo-G1-N3) were treated with a small excess of sodium borhydride (16 mg), purified by repetitive centrifugation and dropped on ZnSe windows after redispersion.To obtain a temperature annealed oxo-G1-N3. FTIR spectroscopy confirmed an enlarging of aromatic areas due to assumed rearrangement and reduction. A reduction is plausible if chloride can be oxidized by protonated oxo-G1-N3. Regular reduction protocols of oxo-G1-N3 samples as for examples NaBH4 or HI/TFA lead to an unselective cleavage of all surface moieties resulting in graphene [1-N3 does not lead to a complete cleavage of oxygen containing moieties, but at least the π-system enlarged as evidenced by TGA-MS, EA, Raman, FTIR and UV/Vis. The regiochemistry of hydroxyl groups and azide groups remains, however, speculative. Thus, the work presented here represents one step towards the synthesis of graphene azide, a derivative of graphene that stands in line with graphene or halogenated graphene.We observed changes of the physical properties of HCl treated oxo-Ggraphene ,33. Ther


The invisibility cloak, a long-standing fantastic dream for humans, has become more tangible with the development of metamaterials. Recently, metasurface-based invisibility cloaks have been proposed and realized with significantly reduced thickness and complexity of the cloaking shell. However, the previous scheme is based on reflection-type metasurfaces and is thus limited to reflection geometry. In this work, by integrating the wavefront tailoring functionality of transparent metasurfaces and the wave tunneling functionality of zero-index materials, we have realized a unique type of hybrid invisibility cloak that functions in transmission geometry. The principle is general and applicable to arbitrary shapes. For experimental demonstration, we constructed a rhombic double-layer cloaking shell composed of a highly transparent metasurface and a double-zero medium consisting of dielectric photonic crystals with Dirac cone dispersions. The cloaking effect is verified by both full-wave simulations and microwave experimental results. The principle also reveals exciting possibilities for realizing skin-thick ultrathin cloaking shells in transmission geometry, which can eliminate the need for spatially varying extreme parameters. Our work paves a path for novel optical and electromagnetic devices based on the integration of metasurfaces and metamaterials. Chinese scientists have developed an innovative technique for making an invisibility cloak that could pave the way for new optical and electromagnetic devices. Although invisibility cloaks by bending light around an object have been around for some time, they are bulky and complex devices with spatially gradient parameters, limiting their uses. Now, Yun Lai and his colleagues from Nanjing University and Soochow University, in collaboration with researchers from Fudan University, have developed a new technique that combines the properties of metamaterials and metasurfaces to produce an invisibility cloak that is significantly thinner and less complex than currently available. The hybrid device integrates the wavefront tailoring functionality of transparent metasurfaces with the wave tunneling functionality of materials with near-zero-refractive index, and enables a new approach of cloaking by creating the illusion of free space. However, invisibility cloaks designed by transformation optics2 are normally bulky devices with complex spatial distributions of material parameters. Recently, metasurfaces, which are ultrathin metamaterials composed of tailored planar microstructures, have demonstrated extraordinary abilities to control the phase, amplitude, and polarization of electromagnetic waves38. With the use of metasurfaces, the thickness and complexity of invisibility cloaks have been significantly reduced38. By covering a corrugated surface with a layer of carefully designed metasurface of skin-like thickness, the wavefront of the reflected waves can be properly modified so as to imitate plane waves reflected from a flat surface37, creating the illusion17 of a plane mirror5. However, this method only works in reflection geometry. For an invisibility cloak that functions in transmission geometry, in which the illusion of free space is created instead of that of a plane mirror, the requirements are completely different. First, the cloak itself should be non-reflecting. Second, the wave energy should be either directed around a hidden space or compensated by using lossy and gain media38. These requirements are beyond the capabilities of reflection-type metasurfaces.The intriguing concept of invisibility cloaks has aroused great enthusiasm55, that is, metamaterials with a near-zero-refractive index, offer extraordinary wave tunneling functionality to direct the electromagnetic wave energy around a hidden space. Such ZIM-assisted wave tunneling phenomena have been demonstrated in channels and bends using various ZIMs, such as metallic metamaterials42, waveguides at cut-off frequencies43, double-zero media54 (with simultaneous zero permittivity and zero permeability) composed of dielectric photonic crystals with Dirac cone dispersions53, and doped epsilon-near-zero materials54. Nevertheless, in free space, oblique incident waves are reflected due to the zero-refractive index of ZIMs, which disables the wave tunneling functionality. Therefore, an efficient coupling method is required to exploit the full functionalities of ZIMs in free space.Zero-index materials (ZIMs)In this work, for the first time, we propose a general principle to integrate transparent metasurfaces and ZIMs into a unique hybrid invisibility cloak that works in transmission geometry. The key functionalities of wavefront tailoring with metasurfaces and wave energy tunneling with ZIMs are combined together to achieve invisibility through an illusion of “free space.” Numerical simulations not only prove the validity of the cloaking effect for almost any shape of cloak but also demonstrate possibilities in realizing skin-thick cloaking shells in transmission geometry, avoiding the need for spatially varying extreme parameters. By constructing a rhombic double-layer cloaking shell that is composed of a highly transparent metasurface and a double-zero medium consisting of dielectric photonic crystals with Dirac cone dispersions, we have experimentally verified the cloaking effect. Our work not only demonstrates a new principle of cloaking, that is, hybrid invisibility cloaks, but also unveils a novel strategy of integrating metamaterials and metasurfaces together for more sophisticated and advanced functionalities that are beyond each of them alone.17, which are composed of volumetric gradient structures  within the double-layer cloaking shell. The simulated electric field distributions under a plane wave incident from the left is calculated with COMSOL Multiphysics and shown in Fig. We have performed both simulations and experiments to verify this new principle of cloaking based on the integration of metasurfaces and ZIMs. Without loss of generality, we consider a two-dimensional demonstration with transverse electric (TE) polarization. The fundamental idea is proven through finite element simulations using the COMSOL Multiphysics software. The metasurface is represented by an ultrathin layer of an anisotropic effective medium, which exhibits perfect transmission as well as a full range of phase-tuning abilities see . For theOwing to the generality of this principle, the shape of the cloaking device can be almost arbitrary, although its smallest feature size is limited by the unit structure of the metasurface and ZIM. In the second and third examples, a rhombic cloak and an irregular cloak with a complex polygon shape are designed using the same effective medium models for metasurfaces and ZIMs. The corresponding simulated electric field distributions under a plane wave incident from the left are displayed in Fig. 21. Six ABA metasurface units with a linear phase shift covering the entire range of [0, 2π) and high transmittance are configured near 10 GHz. The ABA structures as well as the calculated phase shift and transmittance through each unit are shown in Fig. Γ point) in the photonic band diagram. This unique type of photonic crystal effectively functions as a double-zero medium at the frequencies near the Dirac point, which has been previously experimentally verified in both microwave and optical frequencies51. Here, the designed photonic crystal consists of a square array (with lattice constant of 17.46 mm) of alumina cylinders (with relative permittivity εr = 8.1 and radius 3.95 mm). The frequency of the Dirac point, that is, the frequency at which the photonic crystal behaves as a ZIM, is tuned to be 10.2 GHz . Optimization through a more meticulous design or utilization of the concept of Huygens’ metasurface may further reduce the total scattering of the cloak.By combining the designed metasurface and ZIM, a rhombic cloaking shell with a side length of 234.3 mm is realized, as shown in Fig. ε and μ parameters of the metasurfaces in Fig. d/R→0, where d is the thickness of the cloak and R is the radius of the cloaked area. When d/R→0, traditional transformation optics cloaks require divergent parameters in the cloaking shell  polarization is applied instead of TE polarization. First, we note that for a ZIM layer composed of a double-zero medium, there is almost no limit on its shape except that the smallest feature size is determined by the unit structure. For instance, as shown in Fig.  see the , which mdia Fig. , single-ε→0 Fig. , and ani⊥→0 Fig. . Here, ε43. Moreover, there are stringent requirements in the shape and thickness of ZIMs to match the shape of the wavefront of the incident waves and the phase accumulation in free space, respectively. Interestingly, all the above-mentioned restrictions can be removed via the introduction of metasurfaces, which enables cloaks to have arbitrary shapes, as shown in Fig. In the design of hybrid cloaks, the metasurface plays the crucial role of bridging the propagating waves in free space and the tunneling waves in ZIMs. Without such metasurface, the wave tunneling-based cloaking effect of ZIMs is limited in the waveguide configuration, as has been previously experimentally demonstratedAlthough the hybrid invisibility cloak designed in this study can have arbitrary shapes and possess an ultrathin thickness, it still has limitations set by the metasurfaces, ZIMs, and general physical laws that limit the full realization of cloaking. Therefore, we provide the possible solutions to solve these limitations. For instance, all types of invisibility cloaks are dependent on the morphology and shape of the cloak, including the hybrid invisibility cloaks designed in this study. Nevertheless, we note that an advantage of the hybrid invisibility cloak is that the parameters of the ZIM layer are almost independent of the morphology or shape of the cloak, which greatly reduces the complexity of the cloak design.38, the metasurface of the hybrid invisibility cloak is designed for a fixed incident angle, and therefore, the cloaking effect is limited to a certain angle with small tolerance. Interestingly, such a limit in angle may be relieved by employing the recently developed coding/programmable metasurfaces32 or nonlocal metasurfaces33. Coding/programmable metasurfaces in principle can change the metasurface parameters based on the direction of incident waves. Nonlocal metasurfaces inherently exhibit different material parameters for different incident angles. In the Another limitation is the incident angle. Similar to previously reported metasurface cloaks in reflection geometry16. To overcome this limitation, one feasible solution is to ignore the requirement of phase continuity and design the cloak in the ray-optics limit, for example, ray-optics cloaking16. Such a principle can also be applied to hybrid cloaks. The hybrid invisibility cloaks demonstrate, for the first time, that the functionality of cloaking can be divided into two parts: the import and reconstruction of the light field, and the guiding of light. Here, the metasurface is for the import and reconstruction of the light field, while the ZIM is for the guiding of light. But when phase continuity is no longer required, both the metasurfaces and ZIMs can be replaced by other optical devices with larger bandwidths to enhance the total bandwidth of the cloaking device.As for the limitation on bandwidth, when phase continuity is required (as for transformation optics cloaks and hybrid invisibility cloaks), the cloaking effect is limited to a narrow frequency regime due to the speed limit of light53, or the design of 3D ZIMs, which can be realized by combining the Dirac cones in the in-plane and out-of-plane directions in a single photonic crystal. We note that such dielectric ZIMs can be attained at optical frequencies51. The other requirement is the design of a metasurface for both polarizations, which has been experimentally realized by adopting symmetrical units in the reflection-type metasurface cloak37 or other metasurface designs. In the Although in this paper we mainly discuss the scheme of hybrid cloaks in two dimensions for single polarization (TE polarization for Figs. 37 that create the illusion of a “plane mirror,” this hybrid invisibility cloak creates new possibilities for the realization of various-shaped and ultrathin cloaking shells in transmission geometry, which leads to the ideal illusion of “free space.” Hybrid invisibility cloaks, a union between metasurfaces and metamaterials, unveil a novel strategy for realizing advanced optical and electromagnetic devices beyond the capabilities of metasurfaces or metamaterials alone.In conclusion, we have numerically and experimentally demonstrated a unique type of hybrid invisibility cloak based on the integration of metasurfaces and ZIMs as a new principle of cloaking. Unlike previous reflection-type metasurface invisibility cloaksSupplementary informationSupplementary Movie S1


Assay of Transposase Accessible Chromatin sequencing (ATAC-seq) is widely used in studying chromatin biology, but a comprehensive review of the analysis tools has not been completed yet. Here, we discuss the major steps in ATAC-seq data analysis, including pre-analysis , core analysis , and advanced analysis . We also review the reconstruction of transcriptional regulatory networks with multiomics data and highlight the current challenges of each step. Finally, we describe the potential of single-cell ATAC-seq and highlight the necessity of developing ATAC-seq specific analysis tools to obtain biologically meaningful insights.Supplementary information accompanies this paper at 10.1186/s13059-020-1929-3. Mammalian DNA is highly condensed through three major hierarchical scales; the first is the nucleosome which then wraps into chromatin leading to the third hierarchy, the chromosome –6. ChromRecent gene regulation studies have focused on epigenetics, and through the advances of high-throughput sequencing technologies, various assays have been developed to decipher the epigenetic landscape. These include Assay of Transposase Accessible Chromatin sequencing (ATAC-seq) , 10, DNaAmong assays designed for detecting chromatin accessibility, ATAC-seq has gained particular popularity since first described in 2013. An exponential increase of curated ATAC-seq datasets and publications indicates its value in a wide spectrum of biological questions  < 100 bp) and mono-, di-, and tri-nucleosomes   and is the basis for advanced analysis. A similar process has been comprehensively reviewed for ChIP-seq , 59 and Unlike in ChIP-seq, input controls for ATAC-seq, in which Tn5 transposase randomly cleaves protein-free DNA, are often unavailable due to high sequencing costs to obtain comparable coverage. Thus, peak callers which require input controls are impractical for ATAC-seq. Moreover, the direct pile-up of paired-end fragments from ATAC-seq represents both nucleosome-free and nucleosome-bound regions Fig. a. Open cPopular peak callers for ATAC-seq can be divided into two major categories: count-based or shaped-based. The count-based peak callers employ different statistical methods to compare read distribution shape in a candidate region to a random background. MACS2 , HOMER , and SICShape-based peak callers are not currently used in ATAC-seq, but they utilize read density profile information directly or indirectly and are believed to improve peak calling in ChIP-seq . PICS 7 models fCurrently, HMMRATAC is the only peak caller that is exclusive for ATAC-seq . It emplOther considerations should include whether the peak caller accounts for Tn5 cleavage bias and how it deals with biological replicates. Similar to DNase-seq, the enzymatic cut by Tn5 will introduce bias due to binding preference , 31, 77,Peak tracks generated by these tools can be visualized in Fig. Because by its nature ATAC-seq reveals multiple aspects of transcriptional regulation, the third major step involves interpretation at four different levels: peak, motif, nucleosome, and TF footprint. However, only a few tools are designed specifically for ATAC-seq.Currently, no differential peak analysis tools have been specifically developed for ATAC-seq data analysis. A straightforward approach would be to find the candidate regions (consensus peaks or binned genome), normalize, and count the fragments in these regions and compare with other conditions statistically . This coP values and more false positives [Among the consensus peak-based tools, HOMER, DBChIP , and Difositives .Sliding window approaches do not require pre-generated peak sets. Instead, they evaluate all windows along the binned genome and tend to yield more false positives and require stringent filtering and false discovery rate (FDR) control. PePr  and DiffCurrently, most studies assume that ATAC-seq reads in peak regions follow a NB distribution, as is the case for RNA-seq data. However, no shape-based differential analysis tools exist for ATAC-seq data. The peaks contain not only read count information, but also the distribution shape profile. It is especially important for broad peaks, as broad peaks can contain multiple local maxima, and those shifts can indicate biologically relevant perturbations, which could be detected in sliding window or shape-based methods. Although not systematically studied, we believe incorporating shape information will improve differential peak analysis. Nevertheless, considering replicate handling, external peak caller dependency and backend statistical methods, csaw is worth a first try due to its easily explainable edgeR framework.cis-regulatory elements including enhancers and promoters [After obtaining peak sets, annotation of peaks can associate chromatin accessibility with gene regulation. Normally, peaks are annotated by the nearest genes or regulatory elements. HOMER, ChIPseeker , and ChIromoters . After oromoters , KEGG [9romoters , and Rearomoters . In geneAlthough peak annotation provides functional interpretation, it does not directly explain the underlying mechanism. Open chromatin can affect transcription through TFs, which facilitate transcription by recognizing and binding to specific sequences on DNA. This sequence is known as a motif and the binding positions are called TF binding sites (TFBS). There are approximately 1600 TFs in human, and more than half have motifs obtained experimentally or computationally . Most TFE. coli. However, there is no central database, which contains comprehensive and consistent motif information, and the discrepancies can arise from differences of original ChIP-seq experiments and the software used to perform de novo motif discovery.In order to exploit motif information, great efforts have been made to compile databases of motif sequences from either experimental methods or computational predictions. Popular databases such as JASPAR  contain The motif information is mainly stored in text format, for example, as a position weight matrix (PWM). HOMER and Bioconductor packages TFBSTools  and motiZ-test. These methods employ different statistical tests to compare the motif frequency in peaks and the background regions.Based on aforementioned motif search tools, the position and frequency of motifs in each peak region can be obtained and compared to a random background or another condition. HOMER uses the hypergeometric test, while MEME-AME  uses theZ-score and is adjusted for known technical bias . It is specifically designed for scATAC-seq data with a large number of cells that could be considered as replicates. However, its performance in bulk ATAC-seq has not been evaluated yet. DiffTF generates a distribution of accessibility fold changes for all TFBSs, adjusted for GC content for each motif and is then compared to a permutated null background to evaluate significance [Apart from an overrepresentation test, accessibility at each putative TFBS is assumed to associate with TF activity and can be measured by fragment counts. ChromVAR  calculatificance , 124. InAll tools mentioned so far predict putative TFBSs indirectly from sequences found within peak regions. Such TFBSs can contain a significant fraction of false positives and are likely to be incomplete and confounded. This is because not all TFs have identified motifs and TFs from the same family can share very similar motifs . MoreoveAnother way to decipher the TF regulation is to use footprints. A footprint in ATAC-seq refers to a pattern where an active TF binds to DNA and prevents Tn5 cleavage within the binding site. This leaves a relative depletion within the open chromatin region Fig. a 47, 12, 127. ThHowever, there are hurdles for ATAC-seq footprinting analysis. First, it is important to shift the raw reads in the pre-processing step to account for the 9-bp duplication for accurate footprint detection . Second,Footprinting analysis tools mainly fall into two categories: de novo and motif-centric methods. De novo methods predict all footprint sites across peaks, according to features of a typical footprint pattern (peak-dip-peak). Then these putative footprint sites are used to match known motifs or identify novel motifs. Instead, motif-centric methods require the input of a priori TFBSs and discriminate these sites as bound or unbound using supervised or unsupervised methods Table .Table 1For de novo methods, it is important to mathematically define what is a footprint and denoise the footprint pattern from Tn5 cleavage bias , 134. BoMotif-centric methods focus on a priori TFBSs and consider TF-specific footprint profiles compared to de novo methods. The challenge is to avoid ascertainment bias where TFs with high-quality motifs are enriched.The unsupervised motif-centric methods classify putative TFBSs as bound or unbound, based on features extracted from genomic regions, e.g., distance to TSS, PWM match score, and sequence conservation score , 146, asIn contrast, supervised motif-centric tools require high-quality ChIP-seq to annotate true TFBSs as training data. MILLIPEDE and BinDNase both use logistic regression , 143, whIf global TF footprint pattern changes between conditions is of interest, BaGFoot  can be eThere are several caveats for footprinting analysis. First, supervised motif-centric footprinting tools generally outperform unsupervised counterparts and de novo methods, with the trade-off of less generalizability , 131. ThThe nucleosome consists of a histone octamer complexed with approximately 147 bp of DNA Fig. a and affSoftware developed for MNase-seq, such as DANPOS2, PuFFIN, iNPS, and NucTools, can be applied to ATAC-seq data after filtering for nucleosome-associated fragments –153, whiHowever, all these tools suffer from the same underlying drawbacks of a typical ATAC-seq experiment, which is low coverage beyond open chromatin. In the future, new experimental protocols coupled with bioinformatic methods for ATAC-seq will be required to more efficiently and precisely capture nucleosome occupancy. Here, we believe HMMRATAC and NucleoATAC are two useful and specific tools for ATAC-seq nucleosome detection.Along with the specified requirements for ATAC-seq data analysis we have articulated so far, the integration of ATAC-seq with other high-throughput sequencing technologies such as RNA-seq and ChIP-seq is gaining increasing interest to understand gene regulation.Because open chromatin is the pre-requisite for most TFs to bind, ATAC-seq peaks generally overlap with TF ChIP-seq peaks but are often broader. Thus, TF ChIP-seq and ATAC-seq can mutually validate the quality and reliability of each other within the same experimental system . Unique Researchers are also interested in qualitatively or quantitatively associating changes in chromatin accessibility with changes in gene expression by RNA-seq. Intuitively, researchers can discover whether DE genes also have significantly differential chromatin accessibility surrounding the respective TSS . MoreoveWhile ATAC-seq can simultaneously detect hundreds of TF motif occurrences or footprints, it is possible to reconstruct cell-specific regulatory networks by linking footprints/motifs with downstream genes. Similar approaches have been demonstrated in DNase-seq Fig. c 104, 1. HoweverAlthough it is possible to reconstruct undirected TF-gene regulatory networks with ATAC-seq alone, the directional regulation can be further inferred as activation or repression when RNA-seq is integrated. Duren et al. proposed a model with paired gene expression and chromatin accessibility (PECA) data to predict the target gene expression as a function of TF expression, chromatin remodeller expression and chromatin accessibility . MiraldiIn order to further improve network reconstruction, publicly available ChIP-seq datasets can be integrated to improve the accuracy of footprinting. Incorporating known enhancer-promoter interactions from chromatin conformation data would also be helpful. With the surge of deep learning, it would require more work on feature construction and selection in order to build effective algorithms to predict transcriptional regulatory networks. In summary, integrating ATAC-seq with multiomics data yields biologically meaningful results, which can uncover underlying mechanisms of gene regulation.There is growing need for integrated pipelines to process ATAC-seq data. Several have been developed but have different focus for downstream analysis by stitching together previously discussed tools.To name a few, esATAC  and CIPHThese pipelines will provide a helpful and convenient entry for researchers with minimal programming skills to explore ATAC-seq data. However, a general problem for these pipelines is the lack of flexibility for parameter tuning. Most parameters are hard coded empirically because the combination of them increases exponentially with the number of tools, which makes pipelines difficult to modify for any given context. Overall, a pipeline with visualization and user interface will be more appropriate for nonprogrammers to explore the data.Chen et al. about clustering methods in scATAC-seq showed that SnapATAC, Cusanovich2018 and cisTopic outperformed other methods [Enabled by microfluidic, nano-well, and combinatorial indexing technologies, scATAC-seq is now able to measure the chromatin accessibility for thousands of cells with easy protocol at a low cost –35. The  methods , 173–175New techniques such as scNMT-seq, sci-CAR, and Pi-ATAC were recently developed to measure chromatin accessibility, transcriptome, and proteome simultaneously from exactly the same cell , 177. DaATAC-seq has developed rapidly over recent years and has become a method of choice to investigate chromatin accessibility. There are now optimized protocols that work with single cells, blood samples, and frozen tissue with improved signal-to-noise ratio –35, 178.In this review, we have systematically discussed all major steps in an ATAC-seq analysis pipeline for the reader to consider, starting with raw sequencing reads to the endpoint of biological meaningful interpretation. Here, we offer a guide of available tools and suggested steps of analysis to consider to facilitate proper biological interpretation of ATAC-seq data. The alignment and QC steps are similar to RNA-seq and ChIP-seq. As for the peak calling, most ChIP-seq derived tools are compatible with ATAC-seq data. However, a comprehensive benchmarking would help to select appropriate tools and to guide future development of ATAC-seq-specific peak callers. There is growing evidence that improvement or parametrization of current tools can be applied to fit ATAC-seq data.For downstream interpretation, differential peak analysis can give an overview of the changes of chromatin accessibility. Nevertheless, these changes can arise from both read numbers and the shapes of peaks and can be detected by count-based or sliding window approaches. The performance of these two approaches still requires further evaluation in ATAC-seq and could be specific to particular contexts. In order to infer biological function and related TFs, peak annotation and motif enrichment analysis is a good first pass analysis for initial insight.Motifs and footprints are direct and indirect indicators of regulatory events respectively. The difficulty in detecting footprints comes from both enzymatic cutting bias and weak signals from transient TFs. Instead of defining footprints with a mathematical formula, recent publications made a good first attempt to embrace the fast development of machine learning algorithms with supervised learning , 144. MoAnother consideration for analysis is on reconstructing gene regulatory networks from ATAC-seq data alone or integrating with multiomics data. This is particularly tempting because ATAC-seq can work with as low as 500 cells and allow the study of well-defined subpopulations especially in developmental biology and clinical samples. ScATAC-seq provides another option to study chromatin biology in heterogeneous cell populations.In summary, ATAC-seq, an information rich assay, is in great demand for specific bioinformatic analysis tools for further exploitation in analyzing chromatin state, TF footprint, nucleosome position, and regulatory network reconstruction. As a starting point, we suggest researchers can build an effective workflow, by combining FastQC, trimmomatic, and BWA-MEM for pre-analysis, and MACS2 for peak calling. For advanced analysis, we suggest csaw for differential peak analysis, MEME suite for motif detection and enrichment, ChIPseeker for annotation and visualization, HMMRATAC for nucleosome detection, and HINT-ATAC for footprint analysis. If RNA-seq data is available, regulatory networks can be reconstructed using PECA method. However, researchers can always refer to this review for alternative tools for each step and we recommend selecting the tool based on the context of the experimental system and the data collected.We envisage that this review will encourage researchers to appreciate the complexity and current major hurdles in ATAC-seq data analysis. New ATAC-seq-specific tools and comprehensive benchmarking studies would enable the answering of more biological questions with ATAC-seq in the near future.Additional file 1.Review history. (DOCX 21 kb)


Graphene and its derivatives such as graphene oxide (GO) and reduced GO (rGO) offer excellent electrical, mechanical and electrochemical properties. Further, due to the presence of high surface area, and a rich oxygen and defect framework, they are able to form nanocomposites with metal/semiconductor nanoparticles, metal oxides, quantum dots and polymers. Such nanocomposites are becoming increasingly useful as electrochemical biosensing platforms. In this review, we present a brief introduction on the aforementioned graphene derivatives, and discuss their synthetic strategies and structure–property relationships important for biosensing. We then highlight different nanocomposite platforms that have been developed for electrochemical biosensing, introducing enzymatic biosensors, followed by non-enzymatic biosensors and immunosensors. Additionally, we briefly discuss their role in the emerging field of biomedical cell capture. Finally, a brief outlook on these topics is presented. Graphene is a one-atom thick form of carbon, where the carbon atoms are arranged regularly in a hexagonal lattice A 1]. Be. Be1]. BTo produce high-quality graphene monolayers that exhibit excellent sheet properties, the mechanical exfoliation and the chemical vapor deposition (CVD) methods have remained popular ,10,11. Igraphene . In contmaterial .In 2006, Ruoff and co-workers employed a solution-based approach to produce graphene monolayers and thin-films on a large scale, which is termed as the chemical exfoliation method F 16,17],1716,17]Traditionally, since the key interest is to obtain large-area, single sheets of high electrical quality graphene with no defects or oxygens, large efforts have been put into studying the process of reduction of GO, i.e., removal of oxygen atoms from the basal plane. Generally, researchers have used two methods to achieve this goal: (1) Thermal reduction is where high temperatures are utilized to break C-O bonds and thus remove oxygen groups ,26,27,28en atoms . (2) Cheic acids ,30. In aic acids  and micric acids  have alsSince the scalability and solution-processability allows for facile fabrication of GO- and rGO-based electrodes, they have been widely employed to design electrochemical sensors . FurtherspspGO is known to contain several oxygen functional groups (epoxy and hydroxyl primarily) that are bonded to the underlying graphene plane. In addition, the sheet edges are known to be functionalized with carboxyls and lactols . GO is tne plane ,37,38. Tne plane . It is ane plane .spspspspspThe reduction of GO leads to many changes in the sheet’s chemical structure, which in turn provides opportunities for tailoring its properties. Freshly prepared GO is an insulator with a sheet resistance of about 9 Gom sites ,39. Uponon atoms A 23,40],40Ω/sq. in films . These pin films .Experiments and theory have shown the formation of CO and CO process ,25,26. T process ,34. Thes process ,42.Another important property of 2D graphene films is their large surface area, which can help load more biomolecules onto the surface ,44. FurtDetection and determination of biomolecules are clinically highly significant for diagnosis and treatment of various diseases. Enzymatic biosensor is a very well accepted system for sensing biomolecules based on their electrochemical reaction (oxidation or reduction) with the enzyme, which is immobilized on the electrode surface ,49,50,51π–π interactions between AP and rGO sheets resulted in improved electron transfer across the nanocomposite, enabling suitable detection of HHications . Althougfer rate . Recentl of H2O2 . Other n of H2O2 ,60, for In addition to rGO, other graphene derivatives have also been employed. Zhou et al. used graphene electrodes to immobilize sarcosineoxidase for the detection of H samples . Muthuraetection . Shao etpure one . Wang etGraphene derivatives offer suitable matrix for immobilization of oxidase enzymes and hence are ideal for glucose detection. There are several reports on glucose oxidase (GOx) immobilized graphene electrodes for glucose detection ,64,65,66Other than rGO, Kang et al. reported a graphene–chitosan nanocomposite matrix to immobilize GOx for glucose detection with wider linear range of detection, lower detection limit and much higher sensitivity . Shan etvs. Ag/AgCl whereas CrGO modified glassy carbon (solid line) electrode reduced the potential to 0.42 V which is about 300 mV less for NADH oxidation . . 150]. HIn 2015, a device which integrated GO nanosheets with single domain antibodies was developed for selective capture of Class II MHC-positive (MHC+) and CD11b-positive (CD11b+) cells from small volumes (≈30 geometry . These sng of GO B 35]. T. TμL) ofng of GO C. By simng of GO D,E. In aAnother publication has illustrated the ability of GO in multiplex detection by using aptamer-conjugated GO membranes for capturing and identifying multiple types of CTCs, including SKBR3 breast cancer cells, LNCaP prostate cancer cells, and SW-948 colon cancer cells . MultiplThe above studies have illustrated that GO can serve as a foundational nano-bio interface in rare cell isolation, detection and characterization devices. By integrating physical and chemical properties of this biocompatible nanomaterial into cellular-level research, not only can the sensitivity and specificity be enhanced, but also the human-involved sample pre-processing or complex system architecture can be reduced.spThe ultrahigh surface area  applications in which graphene can absorb and transfer UV laser energy to the molecules efficiently for their detection. Using this technique, aromatic pollutant molecules, ssDNA and proteins have been detected using graphene as an affinity probe ,164,165.Excellent electrochemical properties of graphene and GO-based nanomaterials and nanocomposites offer great opportunities to develop electrochemical biosensors and immunosensors for the detection of clinically important biomarkers. Moreover, these nanomaterials are promising for drug and gene delivery applications, especially for the treatment of cancer.Graphene has also started making its way toward commercial applications. Nokia has a patent on a graphene-based photodetector in which graphene is used as a photon-collecting layer. These photodetectors are claimed to be cheaper than commercial photodetectors . Nokia hEven though graphene-based nanomaterials are vastly studied for biomedical applications, certain challenges still exist in this field. For instance, controlling the biomolecule orientation and function on the graphene surface are still challenges and they limit the performance of the biomedical devices. To overcome this limitation, synthesis of uniform graphene is highly essential to improve the repeatability and accuracy of the biomolecule detection. It opens future directions to develop new fabrication methods. Similarly, mass production of graphene quantum dots with high yield is required for bioimaging applications. Moreover, it is also very important to study the long-term toxicity of graphene nanomaterials and the cellular uptake mechanism.Although GO has an abundance of oxygen functional groups on its surface, effective utilization of these functional groups has remained difficult owing to their amorphous nature. Effective synthesis protocols to prepare GO structures with a particular oxygen functional group or developing ways to control the chemical structure of GO would be an interesting direction to pursue in the future. Selectively functionalizing a specific functional group over another in a controllable manner would enable multi-functionalization of GO. To further tailor graphene-based biosensing systems for clinical use and, to obtain consistent results, researchers have to develop scalable methods for immobilizing different biomolecules to achieve one-step, no-wash multiplex detection. Overall, graphene-based nanomaterials emerge as promising platform for biomedical applications with a continuing need for effective collaboration among different scientific communities such as chemistry, physics, biology and medicine to enable its applicability.


P = 0.02) and numerically better PFS  than atezolizumab, while they had similar ORR, all cause AE and grade 3–5 AE. For PD-L1 high patients, pembrolizumab and atezolizumab showed similar OS and PFS. However, for PD-L1 low/negative patients, pembrolizumab had superior OS  and better PFS  than atezolizumab. Our analysis raises the hypothesis that anti-PD-1 antibody therapy in combination with chemotherapy may have superior efficacy compared to anti-PD-L1 antibody combination for patients with PD-L1 low/negative advanced squamous NSCLC.Recent randomized phase III trials (KEYNOTE-407 and IMpower131) reported that adding anti-programmed death (ligand) 1 (anti-PD-(L)1) antibodies in combination with taxane-platinum improve the therapeutic efficacy for advanced squamous non-small-cell lung cancer (NSCLC). However, there is no head-to-head comparison of pembrolizumab (anti-PD-1) plus chemotherapy vs. atezolizumab (anti-PD-L1) plus chemotherapy. Therefore, we performed an indirect comparison to explore the optimal choice of anti-PD-(L)1 treatment for advanced squamous NSCLC in combination with chemotherapy. The clinical outcomes were overall survival (OS), progression-free survival (PFS), objective response rate (ORR) and adverse event (AE). For overall patients, pembrolizumab had significantly superior OS (hazard ratio (HR) with 95% confidence interval, 0.67, 0.47–0.94;  Adenocarcinoma and squamous carcinoma are two most common histological subtype of advanced non-small-cell lung cancer (NSCLC). Patients with lung adenocarcinoma whose tumor harbor specific gene mutations, such as epidermal growth factor receptor (EGFR) mutation or anaplastic lymphoma kinase (ALK) fusion, derive significant benefit from targeted agents, tyrosine kinase inhibitors (TKIs), and have better prognosis . Howeverlog HRAB =  log HRAC −  log HRBC, and its standard error (SE) for the log HR was P-value≤0.05 was considered as significant.The clinical outcomes for our study were overall survival (OS), progression-free survival (PFS), objective response rate (ORR) and adverse event (AE). Data of OS and PFS were extracted as hazard ratio (HR) and its 95% confidence interval (CI), while data of ORR and AE were extracted as risk ratio (RR) and its 95% CI. All of above data were derived from KEYNOTE-407 and IMpower131. HR and RR represented pembrolizumab vs. atezolizumab. Based on the assumption that there is no significant treatment efficacy of carboplatin plus paclitaxel in comparison to carboplatin plus nab-paclitaxel for advanced squamous NSCLC, . The logN = 278) and atezolizumab (N = 343) in combination with chemotherapy as the first-line treatment of advanced squamous NSCLC  and numerically better PFS  than atezolizumab, while they had similar ORR, all cause AE and grade 3–5 AE (Table P <0.01) and numerically better PFS  than atezolizumab for PD-L1 low patients. Furthermore, pembrolizumab showed significantly longer PFS  compared with atezolizumab for PD-L1 negative patients  instead of absolute value  for analyses to lower the bias. Besides, the proportion of PD-L1 high patients was slightly higher in KEYNOTE-407, while the proportion of PD-L1 negative patients was slightly higher in IMpower131, both in experimental group and control group. It might cause imbalance of the patient population which affected the comparability of this indirect comparison. Moreover, PD-L1 expression was scored by SP142 assay in IMpower131, while it was scored by 22C3 assay in KEYNOTE-407, thus might have influence on PD-L1 level evaluation. Recent studies demonstrated the percentage of PD-L1-stained tumor cells was highly comparable among 22C3, 28–8 and SP263 PD-L1 assays, while SP142 assay exhibited fewer stained tumor cells,  which waThese limitations aside, our study firstly compared pembrolizumab plus chemotherapy and atezolizumab plus chemotherapy for advanced squamous NSCLC and found the former seemed to be superior in terms of OS and PFS than the latter, especially in PD-L1 low/negative patients. Our analysis provides a hint that anti-PD-1 antibody might have superior efficacy compared to anti-PD-L1 antibody in combination with chemotherapy for patients with PD-L1 low/negative advanced squamous NSCLC. Additional studies are warranted to confirm this.


Hepatocellular carcinoma (HCC) is a prime public health concern that accounts for most of the primary liver malignancies in humans. The most common etiological factor of HCC is hepatitis B virus (HBV). Despite recent advances in treatment strategies, there has been little success in improving the survival of HCC patients. To develop a novel therapeutic approach, evaluation of a working hypothesis based on different viewpoints might be important. Long interspersed element 1 (L1) retrotransposons have been suggested to play a role in HCC. However, the molecular machineries that can modulate L1 biology in HBV-related HCC have not been well-evaluated. Here, we summarize the profiles of expression and/or activation status of L1-related genes in HBV-related HCC, and HBV- and HCC-related genes that may impact L1-mediated tumorigenesis. L1 restriction factors appear to be suppressed by HBV infection. Since some of the L1 restriction factors also limit HBV, these factors may be exhausted in HBV-infected cells, which causes de-suppression of L1. Several HBV- and HCC-related genes that interact with L1 can affect oncogenic processes. Thus, L1 may be a novel prime therapeutic target for HBV-related HCC. Studies in this area will provide insights into HCC and other types of cancers. Hepatocellular carcinoma (HCC) is a prime public health concern that causes almost 90% of the primary liver malignancies in humans. HCC is the sixth most common cancer and is the fifth leading cancer in males and ninth most common cancer in females ,2. The mpre-core/HBc, Pol, HBs and HBx . HB. HBHBx-Lgression  [38,132,In HBV-related HCC, the expression and/or activation status of L1-related genes is altered, which may contribute to L1 activation and HCC tumorigenesis. To investigate this hypothesis, it is important to evaluate the L1 activation status in each cancer cell and surrounding non-cancer cells because HCC is highly heterogenous and L1 activity in each cell might be different ,36,37. SAlthough L1 is likely to be involved in the oncogenic processes of HBV-related HCC, it has not yet been demonstrated whether HBV indeed modulates L1 retrotransposition. HBV genes often contribute to the development of HCC. Among them, HBx is the best-studied viral protein in HBV-related HCC. HBx associates with various host factors in multiple cancer-related biological pathways. Therefore, HBx may be a candidate that modulates L1 expression and/or retrotransposition, by which oncogenic processes are potentiated. At present, we cannot exclude the possibility that other HBV proteins contribute to the regulation of L1 activity. Further investigation will be required for clarifying this point. Intriguingly, we have demonstrated that another oncogenic virus, Kaposi’s sarcoma-associated herpesvirus, can enhance L1 retrotransposition , which mRecently, we have also reported that capsaicin, a compound with anti-tumor activity, can suppress L1 retrotransposition . This re