TAR DNA-binding protein-43 KDa (TDP-43) and fused in sarcoma (FUS) as the defining pathological hallmarks for amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), coupled with ALS-FTD-causing mutations in both genes, indicate that their dysfunctions damage the motor system and cognition. On the molecular level, TDP-43 and FUS participate in the biogenesis and metabolism of coding and noncoding RNAs as well as in the transport and translation of mRNAs as part of cytoplasmic mRNA-ribonucleoprotein (mRNP) granules. Intriguingly, many of the RNA targets of TDP-43 and FUS are involved in synaptic transmission and plasticity, indicating that synaptic dysfunction could be an early event contributing to motor and cognitive deficits in ALS and FTD. Furthermore, the ability of the low-complexity prion-like domains of TDP-43 and FUS to form liquid droplets suggests a potential mechanism for mRNP assembly and conversion. This review will discuss the role of TDP-43 and FUS in RNA metabolism, with an emphasis on the involvement of this process in synaptic function and neuroprotection. This will be followed by a discussion of the potential phase separation mechanism for forming RNP granules and pathological inclusions. TARDBP [FUS [UBQLN2 [VCP [TBK1 [CHMP2B [C9ORF72 gene [The first descriptions of ALS and FTD were provided in the late 19th century by Jean-Martin Charcot 1874) and Arnold Pick 1892), respectively, highlighting the defining features of both diseases. In the classical form of ALS, upper and lower motor neurons degenerate, leading to muscle wasting, paralysis, and eventual death typically within 5 years from disease onset. By contrast, FTD is a progressive neuronal atrophy with loss in the frontal and temporal cortices, characterized by personality and behavioral changes, as well as a gradual impairment of language skills. It is the second most common dementia after Alzheimer's disease (AD). Although seemingly different, symptoms unique to FTD and ALS can occur in the same patient, and either ALS or FTD or both can present in the same families, suggesting genetic and clinical overlaps of the two disease entities [, respectTARDBP , 4, FUS  and ArnoDBP [FUS , UBQLN2 CP [TBK1 , CHMP2B  [CHMP2B , 13, andF72 gene , 15, conTARDBP gene) and FUS (encoded by the FUS gene) are the major components of pathological inclusions in over 90% of all ALS and 55% of FTD cases regardless of the cause [TARDBP and FUS genes are causal for ALS and FTD; (2) regardless of the causes, the pathogenic process converges on TDP-43 as pathological TDP-43 inclusions are present in the majority of ALS and FTD patients (to a much lesser extent for FUS); and (3) the pathogenic mechanisms for TDP-43 and FUS are likely to be a combination of both loss-of-function and gain-of-function properties. Thus, it is critical to first understand the physiological and pathophysiological roles of TDP-43 and FUS in ALS and FTD.Remarkably, TDP-43  is an ubiquitously expressed 414 amino-acid protein containing two RNA recognition motifs (RRMs) followed by a glycine-rich, low-sequence-complexity prion-like domain , 36 Fig. TDP-43 ogenesis . Howeverin vivo UV-cross-linked immunoprecipitation and high-throughput sequencing (CLIP-seq or HITS-CLIP) and other more conventional methodologies such as combining immunoprecipitation and deep sequencing (ribonucleoprotein immunoprecipitation (RIP-seq)), were used to identify the in vivo RNA targets for TDP-43 in mice, the human brain, and cell lines [In contrast, overwhelming evidence demonstrates that TDP-43 is involved in many aspects of RNA metabolism, including splicing, miRNA biogenesis, RNA transport and translation, and stress granule formation , 33, 34.ll lines , 41, 42.ll lines . Importall lines , 25. Simll lines  possiblyll lines . Furtherll lines , 45. TakIntriguingly, a novel mechanism by which TDP-43 binds to cryptic exons to repress the splicing of these exons has been recently discovered , 47. Delin vivo and in vitro models [SLC1A2), or glutamate transporter 1 (GLT-1)), amyloid beta precursor protein (APP), presenilins, huntingtin, ataxins, synuclein, progranulin, and tau [Gene expression profiling using postmortem FTD human samples with TDP-43 pathology showed downregulation of genes enriched with gene ontology (GO) terms of synapse formation, long-term potentiation, and long-term depression , 54. Theo models , 25, 26, and tau . Curious and tau , such asNRXN1 and NRXN3, resp.) and neuroligin 1 (NLGN1), whose mutations are found to associate with various neurological diseases, including autism spectrum disorders [One of the unique RNA classes bound by TDP-43 includes RNA transcripts with exceptionally long introns (average size > 100 kb), which are enriched in the brain and are often involved in synaptic activity and functions . Transcrisorders . Furtherisorders . Furtherisorders . Thus, dTDP-43 has been shown to colocalize with fragile X mental retardation protein (FMRP) and Staufen, two proteins that mark transporting RNP granules and P-bodies , in dendin vitro indicates that TDP-43 could regulate neuronal morphogenesis and spinogenesis, although the underlying mechanisms remain controversial in the literatures [Varying TDP-43 levels in primary neurons cultured eratures –62. Overeratures . Howevereratures , which ieratures .A315T showed age-dependent reduction of dendritic spines that correlates with lower synaptic transmission within the motor cortex [Q331K mice correlating with increased excitatory synaptic transmission [Drosophila sensory neurons, where overexpression of fly and human TDP-43 increases and knockdown of TDP-43 decreases dendritic branching [in vitro [in vivo [The majority of studies in the transgenic animals expressing disease-linked mutations in TDP-43 have been focused on the motor phenotype and ALS-like pathology . Althougr cortex , the spismission . Neverthranching . The conin vitro , 68 and [in vivo , 70. FurTDP-43 RNP granules showed bidirectional microtubule-dependent transport in axons of fly motor neurons, primary rodent cortical and motor neurons, and iPSC-derived human motor neurons –73. In c(Nefl) [(Map1b), the latter of which is the homologue of Drosophila Futsch [NEFL and synaptic vesicle proteins are among the most downregulated genes in the postmortem ALS and FTD human samples [Similar to the observation in dendritic transport, RNP granules containing disease-linked mutations  showed reduced mobility . Recent (Nefl)  and micra Futsch , 76. Fura Futsch , 26, 77. samples , 54. Takin vivo axonal damage either by axotomy or axon ligation triggered a transient accumulation of TDP-43 at the injury sites with concomitant loss of nuclear TDP-43 [Two independent studies showed that r TDP-43 , 79, sugr TDP-43 . As the r TDP-43 , 82, it r TDP-43 .Recent work from the Shen group provides evidence that TDP-43 and FMRP coregulate dendritic translation of ~160 common targets, including Rac1, GluA1, and Map1b . The resSharing the structural and functional properties with TDP-43, FUS is a 526-amino acid protein containing a prion-like, low-complexity domain that is enriched with glutamine, glycine, serine, and tyrosine (Q/G/S/Y) residues , 88, folSimilar to TDP-43, FUS is involved in the transport of RNA granules in dendrites  using coFUS clearly is required for proper neuronal functions, as primary cortical neurons cultured from FUS-knockout mice showed altered neuronal dendritic morphology, that is, more dendritic branches, with more immature filopodia-like spines . The spiGria1 mRNA, which in turn reduced the protein expression of GluA1, a subunit of the α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) type of ionotropic glutamate receptor. AMPA receptors are typically heteromeric ion channels with a combination of 4 different subunits, GluA1–4 (encoded by Gria1–4) [Gria1 mRNA [α2 isoform via binding and stabilizing the 3′-UTR of SynGAP mRNA. Depletion of FUS leads to reduction of SynGAP, which caused the internalization of PSD-95 into the dendritic shaft and reduced spine density. Reintroducion of SynGAP α2 mRNA was able to rescue the spine and behavioral abnormalities caused by FUS depletion [More recently, the Sobue group showed that depletion of FUS led to destabilization of poly(A) tails of Gria1–4) . HeterodGria1–4) . Among tGria1–4) , 121. Ania1 mRNA . In addiepletion . Taken tUsing a super-resolution microscope, Schoen et al. reveal that FUS could also localize to the presynaptic terminal . InteresIn NIH/3T3 cells, FUS appears to facilitate translation for RNAs within the adenomatous polyposis coli- (APC-) containing RNP granules independent of stress-granule-like morphology . Given tDrosophila and zebrafish support the notion that TDP-43 and FUS function in common RNA maturation pathways [Biochemically, a small proportion of TDP-43 and FUS is bound in common protein complexes , 128. A pathways –131, whepathways . Recent pathways , furtherpathways  and >25%pathways , supportOne of the key questions is why and how TDP-43 and FUS are prone to form pathological inclusions in ALS and FTD. Recent work has shown that full-length TDP-43 forms amyloid oligomers that are toxic to neurons , possiblThe discovery that the low-complexity prion-like domains within TDP-43 and FUS self-assemble is rather serendipitous , 135. Unin vivo time-lapse imaging in the germline P granules in Caenorhabditis elegans [in vitro and in vivo [In other words, RNA granules are essentially hydrogel-like liquid droplets enriched with RNA and RNA-associated proteins, which may explain how RNA granules could readily switch from between different identities . In fact elegans . In soma elegans , are sor elegans ; stress  elegans ; and tra elegans . Many of elegans , suggest in vivo , 141–148 in vivo –145, 147 in vivo . Thus, iThe concept that membraneless organelles, such as RNA granules, are formed by intrinsically disordered proteins, such as LC-domain-containing RNA-binding proteins, via multivalent weak interactions has now been recognized as an underlying principle for how intracellular compartments can be generated , 151. ThOn the molecular level, TDP-43 and FUS regulate diverse functions affecting RNA metabolism, in particular, splicing and polyadenylation site selection, which take place in the nucleus. The enrichment of TDP-43 and FUS binding to 3′-UTR of cytosolic mRNAs further suggests that TDP-43 and FUS could influence the fates of RNA beyond their nuclear actions. Together, these molecular regulations influence the half-lives, localization, and translation of their target mRNA . As many


We aimed to investigate sarcoidosis mortality in a large, population-based cohort, taking into account disease heterogeneity.Individuals with incident sarcoidosis (n=8207) were identified from the Swedish National Patient Register using International Classification of Disease codes (2003‒2013). In a subset, cases receiving treatment ±3 months from diagnosis were identified from the Prescribed Drug Register. Nonsarcoidosis comparators from the general population were matched to cases 10:1 on birth year, sex and county. Individuals were followed for all-cause death in the Cause of Death Register. Adjusted mortality rates, rate differences and hazard ratios (HRs) were estimated, stratifying by age, sex and treatment status.versus 6.7 in comparators (rate difference 2.7 per 1000 person-years). The HR for death was 1.61 (95% CI 1.47‒1.76), with no large variation by age or sex. For cases not receiving treatment within the first 3 months, the HR was 1.13 (95% CI 0.94‒1.35). The HR was 2.34 (95% CI 1.99‒2.75) for those receiving treatment.The mortality rate was 11.0 per 1000 person-years in sarcoidosis Individuals with sarcoidosis are at a higher risk of death compared to the general population. For the majority, the increased risk is small. However, patients whose disease leads to treatment around diagnosis have a two-fold increased risk of death. Future interventions should focus on this vulnerable group. Sarcoidosis is associated with a higher risk of mortality; the risk varies greatly by severity at diagnosishttp://ow.ly/60Ll30h6my8 Sarcoidosis is a granulomatous disease of unknown aetiology affecting primarily the pulmonary and lymphatic systems. The disease is characterised by heterogeneity in terms of presentation and disease course . In someMany estimates of sarcoidosis mortality are derived from death certificate data –6. InterWe used nationwide Swedish registers to conduct a large, population-based cohort study. The objective of our study was to compare the mortality experience of individuals recently diagnosed with sarcoidosis to that of the general population, overall, by age, sex and by use of treatment around the time point of diagnosis as a marker for more severe sarcoidosis.In Sweden, residents are entitled to universal healthcare coverage that is provided by a tax-funded system of hospitals. The National Patient Register (NPR) captures information on hospitalisations and outpatient (non-primary care) visits. The inpatient component has national coverage since 1987 and the outpatient component since 2001. The Cause of Death Register holds information on the date and causes of death (main and contributory) for almost all residents since 1961. Dispensed prescribed medications in pharmacies across Sweden have been recorded in the Prescribed Drug Register since July 2005. Records in population-based registers can be linked using a unique personal identification number.We conducted a matched cohort study based on data from 2003 to 2013. Information on visits for sarcoidosis was obtained from the NPR using International Classification of Diseases (ICD) codes . We identified individuals who had their first ever sarcoidosis visit on January 1, 2003 or later, 2 years after the NPR's outpatient component became available to capture incident sarcoidosis. We required patients to have at least two visits for sarcoidosis >14 days apart and at least one visit at which sarcoidosis was the primary discharge diagnosis. Start of follow-up began on the date they fulfilled the inclusion criteria (index date). To compare sarcoidosis mortality to that of the general population, sarcoidosis cases were individually matched on birth year, sex and county of residence 1:10 to nonsarcoidosis comparators identified from the Total Population Register, which includes all residents. They were required to be resident in Sweden at the time of the case's index date.To minimise misclassification, the study population was restricted to individuals aged 18‒85 years at start of follow-up. Subjects with a lung cancer or lymphoma diagnosis listed in the Swedish Cancer Register  6 months before or after the first visit for sarcoidosis (or the matched case's first visit for comparators) were excluded. Ethical permission for this study was granted by the regional ethics review board in Stockholm (DNR 2014/230–31).dx.doi.org/10.1787/193a2829-en), deaths that occurred in individuals aged <70 years were considered premature. During the study period, the average life expectancy at birth in Sweden was 83 years for females and 80 years for males (www.statistikdatabasen.scb.se).We obtained information on the date of death from the Cause of Death Register. Individuals were followed for all-cause death, first emigration  or the end of the study , whichever occurred first. Following the definition of premature mortality by the Organisation for Economic Co-operation and Development (online supplementary table E1 for ICD codes and weights). By restricting the time window for identifying comorbidity, we attempted to capture the load of multimorbidity that was relevant to mortality at baseline and to avoid detection bias affecting the sarcoidosis group.The date of birth , sex, county of residence , and the country of birth  were collected from the Total Population Register. Attained education was obtained from the Education Register . Using information from the NPR, we calculated the Charlson comorbidity index using the original Charlson weights . We seare.g. incapacitating symptoms or involvement of vital organs) [We hypothesised that individuals in need of treatment at the time of diagnosis represent a group with more severe disease and probably have a different mortality rate. According to national and international guidelines, individuals in need of treatment are those with severe clinical disease ( organs) , 12. Sys organs) . We usedIn addition, using information obtained from the Cause of Death Register, we identified the 10 most common underlying and contributory causes of death in the sarcoidosis group, and calculated their corresponding proportion in the comparator group.We used Poisson models to estimate age- and sex-adjusted mortality rates and their corresponding 95% confidence intervals in the sarcoidosis and comparator groups. Rate differences were estimated using an additive Poisson model  and hazawww.folkhalsomyndigheten.se) and previously published literature: smoking prevalence in sarcoidosis (range 9‒16%) and comparators (21‒27%), and a relative risk of death due to smoking of 2.6 (see online supplementary table E2 for detailed definitions) [We tested the robustness of the HR against the potential confounding of current smoking (data for which we did not have) using probabilistic bias analysis methods . Smokingnitions) –19. We ssd age at inclusion was 49±14.4 years and 56% were male. Individuals with sarcoidosis had more comorbid conditions than comparators . 12% of individuals with sarcoidosis and 7% of general population comparators had at least one comorbidity. 6191 (42%) out of 2599 individuals with sarcoidosis entering the cohort starting October 1, 2005 received treatment at the time of diagnosis. Baseline characteristics of the two subgroups of individuals with sarcoidosis and of matched comparators are presented in online supplementary table E3.We identified 8207 individuals with sarcoidosis and 81 119 matched general population comparators between 2003 and 2013. online supplementary table E4). On average, individuals with sarcoidosis died 2 years younger than their general population comparators .During a similar median follow-up of 5.9 years (interquartile range 3.4‒8.7 years), 528 deaths occurred in the sarcoidosis group and 3204 in the comparator group . SarcoidThe age- and sex-adjusted mortality rate was 11.0 per 1000 person-years in sarcoidosis (95% CI 10.1‒12.0) and 6.7 per 1000 person-years in comparators  for those who did not receive treatment at diagnosis (online supplementary table E5). Survival functions stratified by age and treatment status at diagnosis are depicted in The fully adjusted HR for all-cause death was 1.61  was relatively robust even under extreme bias assumptions . The HR In this study, individuals with sarcoidosis had a higher mortality rate compared to matched comparators from the general population. After adjusting for relevant confounders, individuals with sarcoidosis had a 62% higher risk for all-cause death compared to the general population and an excess of three deaths per 1000 person-years were related to sarcoidosis. In addition, sarcoidosis was associated with a greater risk of premature death (death at <70 years of age). Despite the overall differences in mortality, our results suggest that individuals with sarcoidosis who did not receive treatment at diagnosis had only a marginally greater risk of death. However, the risk was increased more than two-fold for those treated, representing more severe disease at diagnosis.The results of this study are in line with most, but not all previous investigations. A small study from the USA of mainly Caucasian incident cases reported no association between sarcoidosis and mortality  . Howeverukeyet al. [ukeyet al. included cases at various disease stages in their cohort , whereas we limited inclusion to newly diagnosed cases. Another explanation could be that individuals of African American descent have worse prognosis, with previous studies suggesting that sarcoidosis is diagnosed earlier, more extrapulmonary organs are involved and the rate of sarcoidosis-related hospitalisations is higher in black individuals compared to white individuals [In the report by Tyet al. , sarcoidividuals , 21.Mortality estimates in this study, both absolute and relative, are generally lower than those reported in previous investigations for sarcoidosis . This miTo address our concern that any potential greater risk for death in sarcoidosis compared to the general population was possibly driven by a smaller group of patients with more severe disease, we defined a proxy for severe disease based on dispensation of sarcoidosis-related treatments around the time of disease diagnosis. We showed that the risk of death in individuals treated was more than twice that of the general population, whereas the risk was only slightly increased for patients who did not require such treatment. This finding, while in line with a single-centre study from the 1990s in pulmonary sarcoidosis patients , 25. In e.g. cardiac sarcoidosis), time since diagnosis, reporting practices or sarcoidosis misclassification in this study might influence this observation. Therefore, it should be noted that while studies utilising death certificate datasets provide an interesting snapshot of disease burden at death, they are methodologically limited to account for the whole picture of disease course, as they capture only a subset of sarcoidosis patients [Our analysis of death certificate data of individuals with sarcoidosis in our cohort showed that the disease is the most commonly mentioned underlying and/or contributing cause of death in these individuals. However, sarcoidosis was listed in only 30% of the deaths in the sarcoidosis cohort. Factors such as organ engagement  were available for all individuals. Moreover, two potential sources of bias were quantitatively addressed using advanced analysis techniques, indicating the robustness of our results.In conclusion, we have demonstrated that the overall risk of death in individuals with sarcoidosis is greater compared to the general population. However, it varies considerably with disease severity. It was only slightly increased in the majority of patients who did not receive treatment at the time of diagnosis, but was increased two-fold for individuals who received corticosteroids, methotrexate or azathioprine at the time of diagnosis, probably due to more severe disease. This subset of patients with a higher risk of poor outcomes should be prioritised for future interventions aiming to reduce the burden of sarcoidosis, and the effectiveness of treatments should be evaluated.10.1183/13993003.01815-2017.Supp1Please note: supplementary material is not edited by the Editorial Office, and is uploaded as it has been supplied by the author.ERJ-01815-2017_SupplementSupplementary tables 10.1183/13993003.01815-2017.Supp2ERJ-01815-2017_ArkemaE.V. Arkema


L1, with a 7,8-dihydro[5]helicene core structure- and L2, with a fully aromatic [5]helicene core structure) were synthesized. Despite their structural similarities, L1 and L2 exhibit particularly different characteristics in their use as chiral ligands. L1 was highly effective in the asymmetric allylation of indoles with 1,3-diphenylallyl acetate (up to 99% ee), and in the etherification of alcohols (up to 96% ee). In contrast, L2 was highly effective in the stereocontrol of helical chirality in Suzuki–Miyaura coupling (SMC) reaction (up to 99% ee). Density functional theory analysis was employed to propose a model that accounts for the origin of the enantioselectivity in these reactions.A series of novel optically active [5]helicene-derived phosphine ligands ( The rational design and development of new chiral ligands to enable stereocontrol in a wide variety of reactions is one of the most important topics in modern organic synthesis13457891011121314L1, with a 7,8-dihydro[5]helicene core structure; and L2, with a fully aromatic [5]helicene core structure) as rationally simplified molecules -1-bromo-7,8-dihydro[5]helicene rac-6 (54% yield over two steps). Lithiation of rac-6 and subsequent reaction with chlorodiphenylphosphine and treatment with hydrogen peroxide afforded the (±)-phosphine oxide (rac-7) in 63% yield. The optical resolution of rac-7 was successfully achieved by application of the Keglevich procedure using spiro-TADDOL (−)-8 as a resolving agentP)-7. Enantiomer (M)-7 could also be prepared using (+)-8 (see P)-7 was converted into the desired L1 using trichlorosilane and P(OEt)3 in 72% yield, while ligand L2 was prepared in 70% yield over two steps by the oxidative aromatization of (P)-7 with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), and subsequent reduction of (P)-9.Ligands +)-8 see . PhosphiL1 and L2, electronic circular dichroism (ECD) spectroscopy was carried out to give the spectra shown in L2 showed two bands with a first positive band at ~325 nm and a second negative band at ~280 nm, indicating P helicity (opposite signs were observed for M helicity). These results are in agreement with the absolute configurations previously reported for 1-funcionalized [5]helicenesP)-L1 and -L2 to compare them with the experimental ECD spectra (P)-L1 and -L2.To determine the absolute configuration of the resolved enantiomers of  spectra . The ligL1 and L2 were confirmed by X-ray crystallography  is longer than that of L2 (3.39–3.34 Å). To obtain further information regarding the phosphine-metal-arene interaction of the metal complex of L1, we prepared Pd(dba)[L1] complex 10 from Pd2(dba)3·CHCl3 and L1. As expected, results from X-ray crystallography showed that the double bond (C8a–C14b) of the helicene ligand was coordinated with the palladium center in a side-on (η2) fashion.The structures of lography . Based o11 with dimethyl malonate, using Cs2CO3 as the base and [PdCl(C3H5)]2 (0.5 mol%) as the palladium source, in the presence of a catalytic amount of L1 or L2 (1 mol%) in CH2Cl2 at room temperature (M)-L1 was highly effective in this reaction, affording (S)-12 in 99% yield with 94% ee, while (M)-L2 afforded (S)-12 in 99% yield with only 71% ee.With the phosphine ligands in hand, we investigated their effectiveness in Pd-catalyzed asymmetric allylic substitution reactions, as the mechanism of these reactions is fairly well understood4041perature . Ligand L1, the asymmetric allylation of indoles using 11 was subsequently investigated. In contrast to commonly studied nucleophiles, the Pd-catalyzed asymmetric allylation of indoles has been met with very limited success1013a-g) in excellent yields (95–99%) and enantioselectivities (96–99%) under optimized reaction conditions  in high yields (61–95%) and enantioselectivities palladium intermediates (IM-exo and IM-endo), DFT calculations using the B3PW91/6-31G* level of theory (LANL2DZ for the Pd atoms) were performed for geometry optimization. IM-endo being the most stable based on these calculations. Indeed, the IM-endo intermediate is favored because of the reduced steric repulsion between the helicene backbone and the allylic group. Assuming that nucleophilic attack on the allyl complex takes place at the allylic carbon atom (C3) due to the larger trans effect of phosphorusIM-endo affords products with S configuration. This is clearly reflected in the computed lower natural bond orbital (NBO)-charge of the C3 position.To elucidate the stereoselectivity of the L1 and L2 in the asymmetric SMC reaction45464748495015a and o-tolylboronic acid a16 under optimized reaction conditions (Pd(OAc)2/L*/toluene/50 °C, see P)-L1 , the use of (P)-L2 gave product (R)-a17a in excellent yield and enantioselectivity . We then moved on to further examine the substrate scope of this reaction. A coupling reaction between phosphonates 15a-d and o-tolylboronic acid a16 gave the corresponding axially chiral biaryls (a–17da17a) in excellent yields and moderate to high ee values . This confirmed that L2 afforded improved enantioselectivity over L1 -L1) and Pd((P)-L2) complexes providing a17b were performed using the B97-D/6-31G* level of theory (LANL2DZ for the Pd atom). As shown in IM1-A–D) could be formed after oxidative addition. As the arene substrate and phosphorus center bearing strong trans influences prefer the cis arrangement, we surveyed the transition states (TSs) for the reductive elimination steps of IM2-Aanti, IM2-Asyn, IM2-Bsyn, and IM2-Banti, derived from IM1-A and IM1-B.It was reported that the stereoselectivity of the SMC reaction is induced by reductive elimination from Pd(II)46L1 and L2, we optimized seven (for L1) and six (for L2) of the eight possible transition state structures and their energies were calculated as shown in TS6 and TS7 were the most stable for (S)-a17b and (R)-a17b, respectively. The energy gap between TS6 and TS7 could arise from differences in the hydrogen bonding partners of the phosphonate sp2-oxygen and the hydrogen atom on the tolyl group -H is a better hydrogen donor than C(sp3)-H48TS6 and TS7 . Since in the transition states, the average helical pitch of L2 (3.44 ± 0.06 Å) is smaller than that of L1 (3.52 ± 0.10 Å), additional CH/π interactions can form between the arene moiety and the helical aromatic framework -L1 and (P)-L2 as ligands predicted (R)-product formation with selectivities of 62% ee and 91% ee, respectively. These values correlated well with the experimental results.Using ligands yl group . TS7 wouramework , reflectL1, with a 7,8-dihydro[5]helicene core structure, and L2, with a fully aromatic [5]helicene core structure) were successfully synthesized, and the ligand structures were determined using X-ray crystallography. As expected, the X-ray crystallographic analysis of the Pd(dba)[L1] complex unambiguously showed that both the phosphorus atom and the double bond of the ligand are coordinated to the Pd center. These ligands were applied in Pd-catalyzed asymmetric allylic substitutions and Suzuki–Miyaura coupling (SMC) reactions. The newly developed ligands, in particular L1, were highly effective in asymmetric allylic substitutions. Moreover, we demonstrated that L2 serves as a highly enantioselective ligand in the asymmetric SMC reaction to yield axially chiral biaryl compounds. Our concept may therefore open a novel route toward the application of helicene-based phosphine ligands in Pd-catalyzed asymmetric reactions.In summary, two novel optically active [5]helicenylphosphine ligands (3H5)]2 (0.5 mol%), L1 (1.0 mol%), and CH2Cl2 (0.30 mL) under argon. The resulting mixture was stirred for 30 min at room temperature. Racemic 1,3-diphenyl-2-propenyl acetate 11 (0.15 mmol) in CH2Cl2 (0.45 mL), Cs2CO3 (0.3 mmol), and the corresponding indole (0.3 mmol) were added subsequently, and the reaction mixture was stirred at room temperature until all starting material had been consumed. The reaction mixture was then diluted with CH2Cl2, washed with water and brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel eluting with hexane–EtOAc (95:5) to afford 13. The absolute configuration was determined by comparing the specific optical rotations with literature data (see An oven-dried screw-capped vessel was charged with [PdCl(Cdata see .3H5)]2 (0.5 mol%), L1 (1.0 mol%), and CH2Cl2 (0.30 mL) under argon. The resulting mixture was stirred for 30 min at room temperature. Racemic 1,3-diphenyl-2-propenyl acetate 11 (0.15 mmol) in CH2Cl2 (0.45 mL), Cs2CO3 (0.45 mmol), and the corresponding alcohol (0.45 mmol) were added subsequently, and the reaction mixture was stirred at room temperature until all starting material had been consumed. The reaction mixture was then diluted with diethyl ether, washed with water and brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel eluting with hexane–EtOAc to afford 14. The absolute configuration was determined by comparing the specific optical rotations with literature data (see An oven-dried screw-capped vessel was charged with Helicene-Derived Phosphine Ligands and Their Application in Pd-Catalyzed Asymmetric Reactions. Sci. Rep.6, 36211; doi: 10.1038/srep36211 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


PD-L1 was expressed in TCs of 18.4% and in TIICs of 83.3% of these patients. PD-L1 expressions in TCs and TIICs were associated with significant infiltration of various TIIC types, especially CD8+ cells. PD-L1 expressions in both TCs and TIICs were significantly associated with favorable overall survival, and combining their levels enhanced prognostic accuracy. Prognostic impacts of PD-L1 expressions in TCs and TIICs, abundant PD-1+ cell infiltration, a high CD8+/FOXP3+ ratio, and the CD8+/CD204+ ratio remained significant after adjusting for clinicopathological factors. In conclusion, PD-L1 expression reflects anti-tumor immunity, and PD-1/PD-L1 expression and the ratio of infiltrating effector to immune suppressor cells have prognostic value. Therapeutic strategies inhibiting the PD-1/PD-L1 signal and immune suppressor cells are anticipated in ESCC patients.Immunotherapy with anti-PD-1 antibody preliminarily showed promising efficacy for treating esophageal squamous cell carcinoma (ESCC). Herein, we used tissue microarrays and immunohistochemically analyzed PD-L1 and various tumor infiltrating immune cells (TIICs) in specimens from 196 ESCC patients who had undergone curative resection without preoperative therapy. PD-L1 expressions in tumor cells (TCs) and TIICs, as well as infiltration of lymphocytes (CD4 Squamous cell carcinoma is the predominant histological type of esophageal cancer worldwide, though the incidence of esophageal adenocarcinoma exceeds that of squamous cell carcinoma in the UK, certain other western European countries, and the United States . Smoking+ T cell infiltration have been shown, in several clinical trials testing anti-PD-1 and anti-PD-L1 antibody therapy, to be potential predictive biomarkers of clinical efficacy ) on OS were first examined with univariate Cox regression models. Next, the impact of each immunological factor on OS was assessed by adding each factor to the multivariate model which contained the possibly confounding clinicopathological factors such as age, gender, smoking status, alcohol consumption, location, pT factor, lymph node metastasis, histological grade, lymphatic invasion, and venous invasion. All statistical analyses were performed using IBM SPSS statistics 20 . All P values were two-sided, with a significance level of 0.05.Univariate and multivariate Cox proportional hazards models were used to examine the associations of clinicopathological factors with OS. The impacts of immunological factors (PD-L1 expressions in TCs and TIICs, number of each type of TIIC, and TIIC ratios [CD8


Z = 0.31 T, ∇BZ = 90 T · m−1) has a capability of patterning graphene microstructures with a line width of 29 μm and lateral under-oxidation less than 4 μm. Our approach is applicable to patterning graphene field-effect transistor arrays, and it can be a promising solution toward resist-free, substrate non-damaging, and cost effective microscale patterning of graphene film.Developing an alternative method for fabricating microscale graphene patterns that overcomes the obstacles of organic contamination, linewidth resolution, and substrate damaging is paramount for applications in optoelectronics. Here we propose to pattern chemical vapor deposition grown graphene film through a stencil mask by magnetic-assisted ultraviolet (UV) ozonation under irradiation of a xenon excimer lamp. In this process, the paramagnetic oxygen molecules and photochemically generated oxygen radicals are magnetized and attracted in an inhomogenous external magnetic field. As a consequence, their random motions convert into directional, which can greatly modify or enhance the quality of graphene patterns. Using a ferromagnetic steel mask, an approximately vertical magnetic-field-assisted UV ozonation (B Patterning graphene film is a significant step in fabricating graphene-based elements for both fundamental studies and industrial applications123456789101112131415134567891011121314153456891011810112-based photocatalysis142-based photocatalysis and the subsequent complicating disposals prevent its development14In order to overcome these problems in graphene patterning, some alternative solutions have been proposed in the past few years, like TiO−1)20Z = 0.31 T, ∇BZ = 90 T · m−1) has a capability of patterning 29-μm-wide conformal graphene microstructures with the lateral under-oxidation less than four micrometers, and this approach is applicable to patterning graphene field-effect transistor (FET) arrays.In this study, we propose to pattern CVD grown graphene film through stencil masks by magnetic-assisted UV ozonation. An external inhomogenous magnetic field can magnetize the paramagnetic oxygen molecules and photochemically dissociated oxygen radicals, and induce strong attractive magnetic forces19202122232) atmosphere with the total pressure of 1 atm. A cube permanent magnet is placed underneath the SiO2/Si substrate in order to exert a vertical inhomogenous magnetic field . Nonmagnetic sapphire mask, and magnetic masks made of nickel grid  and molybdenum doped steel grid are used individually for patterning graphene film. The magnetization of nickel mask is along its surface when put in a magnetic field, and as a result it cannot be used to pattern graphene film in a vertical magnetic-field-assisted UV ozonation see . However3P) atoms, or called oxygen radicals under irradiation of the xenon excimer lamp centered at 172 nm. Then, these oxygen radicals oxidize graphene into carbon monoxide (CO) and carbon dioxide (CO2) molecules, or they combine with oxygen molecules to form instable ozone (O3). As confirmed by the X-ray photoelectron spectroscopy analyses  and the prefilled gaseous nitrogen behave as weak diamagnetic substances293031293031Supplementary Note 223In the UV ozonation process, oxygen molecule behaves as a strong paramagnetic substance, while the reactive products  is applied to UV ozonation, the paramagnetic oxygen molecule and radical, which have the magnetic moment μB equal to 2.0 and 1.67 Bohr magnetons, respectively, are magnetized as shown in Fig. 123Z exerted on one oxygen molecule and radical is deduced to be both in the order of 10−22 N toward grapheneZ = gJμB∇BZ, where gJ is the Landé g-factor taking values between 1 and 2. The paramagnetic-induced attractive magnetic force converts the random motions of oxygen molecules and radicals into directional ones as demonstrated by the longer orange arrows pointing to graphene film. On the contrary, all the diamagnetic molecules  experience weak repulsive magnetic forces away from graphene surface due to their two-order smaller negative magnetic susceptibilities21293031When an inhomogenous magnetic field , have much lower partial pressures. As a consequence, it may further enhance directionality due to decreasing probability of intermolecular collisions adjacent to graphene surface in UV ozonation.UV ozonation is used to pattern graphene film through a ten-micrometer-thick nickel mask without applying any magnetic field. The mask has a honeycomb structure of hexagonal holes with the lattice constant of 62 μm and rib width of 26 μm as shown in the SEM image of 14Y = 40 mT, ∇BOY = 2 T · m−1) can completely modify the graphene pattern even though the same nickel mask is used  can be intuitively demonstrated when patterning graphene by UV ozonation. Comparing the optical images in 19Using a non-magnetic 316-μm-thick sapphire mask shown in Z = 0.31 T, ∇BZ = 90 T · m−1). 34Using a magnetic steel mask, the quality of graphene patterning can be improved in the vertical magnetic-field-assisted UV ozonation  is applied, the quality of patterned graphene microstructure deteriorates rapidly after the same UV ozonation treatment  allows it to fabricate graphene electronic circuits , the conductivity curves varying with the gate bias for these three FETs are shown in 2 · V−1 · s−1 and ~1316 cm2 · V−1 · s−1, respectively3334353940At a fixed source-drain voltage VSD = 0. V, the cWhen lacking a vertical magnetic field, the above discussed 70-μm-wide graphene FET array cannot be successfully patterned by UV ozonation due to severe dissipation of oxidative reactants underneath the mask. Instead, using a 168-μm-wide steel mask, we can only obtain 129-μm-wide channels . MeanwhiCompared with the laser writing or reactive ion etching (RIE), the magnetic-assisted UV ozonation has the following characteristics10111214The magnetic-assisted UV ozonation approach manifests good sample-to-sample consistency and reproducibility for patterning graphene microstrustures. For seeking applications in the field of nanotechnology, it is important to explore the minimum line width that UV ozonation can realize to pattern graphene film. Put aside the quality of graphene film, a high-quality magnetic mask etched with micro/nano-structures may provide a solution to further improving graphene patterning. Besides, a precisely designed and controlled external magnetic field would facilitate improving  or intentionally modifying graphene patterns.In summary, we have proposed and demonstrated a new strategy to pattern CVD grown graphene film by magnetic-assisted UV ozonation. By virtue of the paramagnetic property of oxygen molecules/radicals, we can pattern 29-μm-wide graphene microstructure with the lateral under-oxidation less than four micrometers under irradiation of a xenon excimer lamp. The vertical magnetic-field-assisted UV ozonation approach is applicable to patterning graphene FET arrays, and it provides a resist-free, substrate non-damaging, and cost-effective solution to microscale graphene patterning.2 film (300 nm) on a highly doped p-type silicon substrate (0.001~0.004 ohm · cm) as follows33393 aqueous solution after 3 h immersion and then obtained the PMMA/graphene stack layer. Thirdly, the stack was etched by dipping in H2O/H2O2/HCl (20:1:1) and H2O/H2O2/NH4OH (20:1:1) solutions successively to remove possible metal residues. After each etching, it was rinsed sufficiently by deionized water and then scooped out onto a clean SiO2/Si substrate. Monolayer graphene, predominantly monolayer with randomly distributed multilayer flakes less than 5%, was finally obtained by solving the PMMA in acetone. In order to remove possible organic residues and enhance its contact with the SiO2/Si substrate, an extra disposal of annealing in a flow of gas mixture (Ar:H2 = 200 sccm:100 sccm) at 290 °C was carried out for three hours.A “PMMA-mediated” approach was used to transfer CVD grown monolayer graphene on a copper foil onto the thermally grown SiOex situ XPS spectra were collected using a Kratos Axis UltraDLD spectrometer (equipped with a monochromatic Al Kα X-ray source) with the anode power of 150 W. A gaussmeter was used to measure the strength and direction of a magnetic field adjacent to graphene surface. Its gradient was approximated by the ratio of the magnetic difference to a certain distance within one millimeter. All the electrical measurements were carried out in a high vacuum chamber (4.5 × 10−4 Pa) with a combination of Keithley 6430 and 2400 systems.An optical microscope (Leica DM 4000) was used for morphology imaging of the patterned graphene microstructures. A scanning electron microscope  under 5 kV and 3 kV biases was used to obtain highly resolved topographical images of masks and graphene patterns, respectively. A confocal micro-Raman spectroscopy (Senterra R200-L) was used to map graphene patterns under excitation of 532 nm laser  with the scanning step size of 1 μm. Relative to the sample positioning platform, there exists a shift of ~3-μm upward and ~0.5-μm rightward for the laser positioning system. The How to cite this article: Wu, Y. et al. Patterning Graphene Film by Magnetic-assisted UV Ozonation. Sci. Rep.7, 46583; doi: 10.1038/srep46583 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Kink turns (k-turns) are widespread structural elements that introduce an axial bend into duplex RNA with an included angle of 50°. These mediate key tertiary interactions and bind specific proteins including members of the L7Ae family. The standard k-turn comprises a three-nucleotide bulge followed by G·A and A·G pairs. The RNA kinks by an association of the two minor grooves, stabilized by the formation of a number of key cross-strand hydrogen bonds mostly involving the adenine bases of the G·A and A·G pairs. The k-turns may be divided into two conformational classes, depending on the receptor for one of these hydrogen bonds. k-turns become folded by one of three different processes. Some, but not all, k-turns become folded in the presence of metal ions. Whether or not a given k-turn is folded under these conditions is determined by its sequence. We present a set of rules for the prediction of folding properties and the structure adopted on local sequence.  •k-turns are widespread elements that kink the axis of duplex RNA.•k-turns mediate tertiary contacts and bind proteins.•Both the folding process and the structure adopted depend on local RNA sequence.•We have deduced a number of rules to associate sequence and folding.•The rules have strong predictive power. Secondary structure depictions of medium length to long RNA species are generally rather complex, with extensive basepairing and tertiary interactions. However, secondary structure can be reduced to a series of semi-rigid duplex segments connected by helical junctions of different kinds that determine the relative trajectories of the helices and that facilitate long-range tertiary contacts. Thus, it is really the junctions that generate the architecture of the molecules. If we understand the folding properties of the junctions, then we can go a long way to understand the overall fold of the complete RNA molecule. This applies to relatively small, autonomously folding molecules such as riboswitches up to long RNA species such as rRNA, and it is to be expected that long non-coding RNAs will be similar.et al. In the last decade, one helical junction has emerged as both important and widespread throughout functional RNAs; this is the kink turn, generally abbreviated as k-turn. k-turns are found in double-stranded RNA, most often comprising a three-nucleotide bulge followed on the 3′ side by successive G·A and A·G pairs . The dupThe structure and folding of k-turns has been extensively analyzed to the point that this is probably the best understood RNA structural motif. The goal in these studies is now to relate the properties of k-turns to their sequence and thus to generate a set of rules to predict the structure and folding from a knowledge of the sequence alone.Haloarcula marismortui has been especially well studied, and we shall subsequently refer to this as HmKt-7. k-turns play a key organizational role in the assembly of the box C/D and H/ACA snoRNP structures that carry out guided methylation and pseudouridylation of archaeal and eukaryotic RNAs While not quite universal, k-turns are extremely widespread in RNA structures, with examples in most classes of functional RNA. There are many different k-turns found throughout ribosomal RNA from bacteria Canonical) helix, while that containing the G·A pairs is called the NC  helix. We label the loop nucleotides L1, L2 and so on sequentially from the 5′ end and the remaining nucleotides are given positive numbers in the 3′ direction through the NC helix and negative numbers in the 5′ direction through the C helix bulged strand have a suffix b and those on the non-bulged strand have a suffix n.At its simplest (but see the section on k-turn classification), the k-turn comprises a duplex RNA containing a bulge of most often three nucleotides, followed by G·A and A·G pairs immediately to its 3′ side  top. Thetrans G(sugar edge)·A(Hoogsteen edge) connected by hydrogen bonds from GN2 to AN7 and from AN6 to GN3  is well folded under the same conditions T. solenopsae is a rare example of a k-turn with an adenine at the 2n position, that is, where 2b,2n is an A·A pair, which is isosteric with the G·A pair The simple, standard k-turn is formed from two strands as a bulged duplex, with the standard G·A and A·G pairs at the 1b,1n and 2b,2n positions. Examples of such k-turns include HmKt-7 of 23S rRNA, the U4 snRNA, box C/D snoRNA and  classes . In non-T. thermophilus provides an example. In this k-turn, the non-bulged strand of the NC helix reverses direction to form an S-turn so that the 1n and 2n nucleotides are separated by two nucleotides in the primary sequence including a cytosine at the 3n position. Despite this, the A2b is located at its normal position within the structure, participating in the usual A-minor hydrogen bonding of the k-turn.In complex k-turns, the nucleotides of the G·A pairs do not map linearly onto the sequence of the RNA. Despite this, the RNA adopts a normal k-turn structure, with the standard hydrogen bonding features. For this reason, we apply our conventional nomenclature Escherichia coli and the plant Arabidopsis thaliana. The k-junction exhibits all the key features of a k-turn. The third helix of the junction is coaxially aligned with the C helix, while the k-turn loop forms the turn into the NC helix. Analysis of more than 10,000 TPP riboswitch sequences suggested that they all have this feature. Evidently, the k-turn is structurally well disposed to be incorporated into a three-way helical junction.The k-junction is another class, where the k-turn has been elaborated into a helical junction Bacillus subtilis riboswitch bound the L7Ae protein. However, when the structure of the lysine riboswitch of Thermotoga maritima was elucidated T. maritima pk-turn is globally very similar to a k-turn but lacks the standard features of a k-turn. These two elements achieve the same structural result, and they can functionally substitute for each other. Thus, a SAM-I riboswitch in which the k-turn has been replaced by the pk-turn retains the ability to bind SAM ligand, while T. maritima RNase P in which the pk-turn is replaced by a k-turn has ribozyme activity Although the major function of the k-turn is to kink the axis of RNA to enable tertiary interactions to form, it may not be a unique solution to the problem. A k-turn in a particular RNA within one organism may be functionally replaced by an alternative structure in another. Studying a series of lysine riboswitches, Blouin and Lafontaine identified a probable k-turn-forming sequence in the aptamer domain that should facilitate tertiary interaction Many k-turns, including most of the ribosomal k-turns, are bound by specific proteins in their cellular context. As a class, this includes proteins of very different sequence and structure that interact with different faces of the k-turn. However, the L7Ae family of proteins represents the archetypal k-turn binding proteins. These include the eukaryotic and archaeal proteins L7Ae, L30e and S12e H. marismortui large ribosomal subunit Archaeoglobus fulgidus L7Ae bound to HmKt-7 The structure of L7Ae bound to Kt-15 was determined in the context of the The α-helix of the first element is located in the major groove of the NC helix  in a man2  plus G·U all fold weakly or not at all, with G·C and C·G being especially bad. The U4 k-turn follows this rule.2 + ions. Natural HmKt-7 follows this rule.Rule 2. The presence of 3n = G (third column) confers an ability to fold in Mg2 + ions.Rule 3. The presence of 3b = C (second row) confers an ability to fold in MgRule 4. Rule 1 takes precedent over Rules 2 and 3 because 3b,3n = C·G is unfolded in metal ions.2 +) had clear octahedral symmetry, with inner-sphere ligands that included both water molecules and guanine O6 atoms . This offers a partial explanation of Rule 1.We can understand some of these rules at a molecular level at present. A high-resolution crystal structure of HmKt-7 offers an explanation of Rule 2. A structure of HmKt-7 as a simple duplex was solved at 2.0 Å resolution O6 atoms b. One io2 + ions. Mutation of the U4 k-turn sequence to convert 3b,3n from G·C to A·G conferred an ability to fold in response to addition of Mg2 + ions, that is, changing the 3b,3n sequence is sufficient to switch the folding behavior completely. It appears that the SAM-I riboswitch k-turns have been selected for predisposition to fold in metal ions alone. Given that the riboswitch is not known to bind a protein, it must be able to fold unaided to generate a functional structure. The U4 k-turn binds the 15.5-kDa protein in vivo; thus, this will remain unfolded until the protein binds. Until this occurs, the k-turn is likely to be more flexible; this may allow the formation of other interactions during the biogenesis of this complex and dynamic assembly that only become fixed on binding the 15.5-kDa protein. Thus, we see that there is a correlation between the folding properties conferred by the 3b,3n sequence and the biological function of specific k-turns.Our deduced folding rules correlate with biological function. We analyzed the distribution of 3b,3n sequences in several thousand examples of two functional RNA species, the k-turns of the SAM-I riboswitch and U4 snRNA H. marismortui 50S ribosomal subunit Application of the folding rules to the k-turns of the Is this the whole story? Preliminary evidence indicates that other parts of the k-turn, including the − 1b,− 1n sequence and the loop, also have a role to play in determining the folding characteristics in response to metal ions. These studies are ongoing and will hopefully lead to a refinement of the folding rules in due course.T. thermophilusSaccharomyces cerevisiae L30e protein H. marismortui ribosome, which adopts the N1 structure. However, this has 3b,3n = A·G, which leads to an N3 conformation in every other case. Indeed, in all other circumstances we have examined, Kt-7 adopts the N3 structure, including when it was inserted into the SAM-I riboswitch H. marismortui ribosome is the very rare exception to this. Nevertheless, apart from this single exception, the association between the 3b,3n sequence and the conformation adopted by the k-turn remains good.In addition to the folding properties, it is also clear that the equilibrium structure of the k-turn must also be sequence dependent. As noted above, some k-turns adopt the N3 structure while others form the N1 structure. Examination of the sequences of 18 natural k-turns of known structure in a variety of environments reveals that, like folding, the conformation also correlates with the 3b,3n sequence. This is depicted in array form in We see that k-turns play a key conformational role in the formation of medium- and large-scale RNA structures and thus are important in many of the functions of cellular RNA. Probably, the single-most important role of k-turns is to mediate tertiary interactions; very often one or other of the helical arms is involved in a loop-receptor interaction of some kind. Many k-turns are also the targets of specific protein binding. During the biogenesis of functional assemblies such as the ribosome, it is expected that k-turns will initially be relatively flexible, allowing the exploration of conformational space. However, the formation of successful tertiary contacts will begin to reduce this flexibility, and the structure could then be fixed by the binding of protein to the k-turn. Both the folding process and the eventual structure adopted depend on the local nucleotide sequence, especially at the 3b,3n sequence, and these will clearly affect the function. It is therefore extremely important to understand the rules that associate the sequence with the folding and structural properties. A set of rules that have strongly predictive power are beginning to emerge, and it can be applied to new RNA sequences as they emerge.


Designing a versatile and rational method for the tactical tuning of the surface and interfacial properties of graphene is an essential yet challenging task of many scientific areas including health care, sensors, energy, and the environment. A method was designed herein to tackle the challenge and tune the surface and interfacial properties of graphene using a simple electrochemical tethering of arylamines that provides diverse reactive end groups to graphene. This method resulted in the preparation of graphenes with thiol, hydroxy, amine, carboxyl, and sulfonate surface functionalities respectively. X-ray photoelectron spectroscopy, scanning electron microscopy, and cyclic voltammetry were used to study the chemical, morphological, and electrochemical properties of the modified graphenes. The results show the promising scope of the reported method towards the tactical tuning of the surface and interfacial properties of graphene. Also, this method can give fundamental insights of the surface tuning of graphene and its structurally similar materials. Hence, this approach can be used to advantageously tune the surface properties of the other structurally similar nanocarbons and their hybrid materials to make them potential candidates for many applications. Very often tuning of the surface and interfacial properties of graphene become a prerequisite to making the material an effective candidate for the given application6. In general, researchers have been adopting protocols similar to that employed for the covalent functionalization of its allotropes graphite, fullerene and CNTs8. Most of these methodologies which employ aryl diazonium species are spontaneous on metals and semiconductors than on the graphene11. Hence, the procedures may not be suitable for the applications that require direct tuning of graphene in the case of lab on chip devices, chemical and biosensor arrays. The issue of controllable and versatile tuning of the surface properties of graphene is still a great challenge to the research community. The tuning of carbon surfaces by electrochemical methods is more efficient than other procedures as these routes are simple to adopt, require lesser time and can be performed under eco-friendly conditions14. The electrochemical tethering of amines to carbon surface reported by Blandine Barbier has been utilized herein12.In recent times, the material graphene is being considered as an obvious alternative of carbon allotropes2, -COOH and -SO3H). The five arylamine derivatives utilized herein are aminothiophenol (ATP), aminophenol (AP), para-phenylenediamine (PPDA), aminobenzoic acid (ABA) and sulfanilic acid (SA) which possess the reactive end groups -SH, -OH, -NH2, -COOH and -SO3H respectively. The amine functional group present in the corresponding derivative is electro-oxidized to form a covalent bond between amine and graphene yielding graphenes with thiol, hydroxy, amine, carboxyl, and sulfonate surface functionalities. The anodic oxidation of primary amine forms an amine radical cation that loses a proton and forms an amine radical. Then, the formed amine radical attacks the sp2 carbon of the graphene and forms a C-N covalent bond between the amine and graphene. This mechanism has been well established with the other allotropes of carbon including fullerenes, carbon nanotubes, carbon fibers, graphite, etc15. This versatile protocol leads to the formation of a stable and compact monolayer of the modifier molecule on the graphene surface. The surface decorated graphene thus obtained would be of great interest to researchers of chemical sensors, biosensors, immunosensors, and DNA sensors for tethering enzymes, redox mediators, DNA, proteins, antigens, and antibodies16. The surface tuned graphenes thus prepared are characterized by x-ray photoelectron spectroscopy (XPS), field emission scanning electron microscopy (FE-SEM) and cyclic voltammetry (CV). The interfacial electron transfer properties of the graphenes are investigated by cyclic voltammetry using a positively charged, [Ru(NH3)6]Cl3, and a negatively charged, K4[Fe(CN)6], redox system. Besides, the electrochemical behavior of two essential bioanalytes dopamine (DA) and ascorbic acid (AA) is also investigated. The above studies provide the fundamental insights about the influence of the terminal functional groups in tuning the surface and interfacial properties of graphene. Figure Herein, a versatile and rational electrochemical method has been designed for the tactical tuning of the surface and interfacial properties of graphene by covalently linking arylamine derivatives containing different reactive terminal groups , LiClO4, potassium chloride, sodium hydroxide, ruthenium hexamine chloride, dopamine, p-aminothiophenol, p-aminophenol, p-phenylenediamine, p-aminobenzoic acid and sulfanilic acid were purchased from Sigma-Aldrich. Sulfuric acid was purchased from Fisher-Scientific. Potassium ferrocyanide and potassium hydrogen phosphate were purchased from Hi-Media. Acetonitrile was purchased from SRL. All the chemicals were used as received without any purification. Ultrapure water from Siemens LaboStar (resistivity ~18.2 MΩ.cm) system was used to prepare phosphate buffer and other aqueous solutions.Graphite powder (≤20 µM), α-Al4[Fe(CN)6] in 1 M KCl and (2) positively charged redox probe 1 mM [Ru(NH3)6]Cl3 in 1 M KCl to get the fundamental insights and electrochemical properties of the surface tuned graphenes. Also, the voltammetric response of two bioanalytes dopamine  and ascorbic acid  were studied to check the feasibility of the surface tuned graphenes towards bioanalytical applications. The changes in peak current and peak-to-peak separations were tracked to evaluate the electrochemical properties of the surface tuned graphenes in the above cases. All the electrochemical experiments were carried out at room temperature and in dissolved O2 condition with a stationary working electrode.All the electrochemical experiments were carried out using Autolab PGSTAT-302N from Metrohm. A three electrode system consisting of glassy carbon electrode  or surface tuned graphene modified glassy carbon electrode as a working electrode, spiral platinum wire as a counter electrode and normal calomel electrode (NCE) as a reference electrode was used. A silver wire was used as a pseudo reference electrode in non-aqueous solution based electrochemical experiments. Cyclic voltammograms were recorded using (1) negatively charged redox probe 1 mM Kα X-Ray source operated at 150 W and the spectra were deconvoluted by CASA XPS using Shirley baseline with a combination of 20% Gaussian and 80% Lorentzian function. In all the cases, the secondary electron background was subtracted using the Shirley function. Also, the XPS spectra were referenced to the C1s peak from the adventitious carbon at 284.8 eV to eliminate any positive charge-induced binding energy shift. All the topographical studies were done using FE-SEM Zeiss Supra 55VP from CARL ZEISS equipped with a high brightness conical FE gun.All the XPS studies were done with MULTILAB 2000 based X-Ray photoelectron spectrometer using monochromatic Al K17. Graphene (Gr) was synthesized using the previously reported method by our team18. In brief, carrot extract was obtained by boiling 20 g of carrot in 100 mL deionized water at 100 °C for 50 min, then filtered using whatman-40 filter paper. Then the 100 mL of the carrot extract was added to the flask containing 20 mL of GO (1 mg/mL) and 200 mL of 1 M NaOH. The resulting solution was stirred for 2 h at 90 °C. The black suspension obtained was filtered, centrifuged and washed with water for 5 times. Finally, the obtained black precipitate was lyophilized to get the graphene powder.Hummer’s method was adopted to synthesize graphene oxide (GO)2O3 and abrasive papers and then sonicated in alcohol and double distilled water respectively. Then, 10 µl of the graphene dispersion (1 mg/mL) was drop casted on the cleaned GCE and allowed to dry at room temperature for 3 h.Graphene suspension was prepared by dispersing 5 mg in 5 mL DMF for two hours by ultrasonication. Before the covalent functionalization, GCE was polished using 0.05 µm α-Al4 containing 5 mM of AP (for Gr-OH), PPDA (for Gr-NH2), ABA (for Gr-COOH), and SA (for Gr-SO3H) separately for five cycles at a scan rate of 20 mVs−1 by cyclic voltammetry to obtain the surface tuned graphenes. As ATP is water insoluble, thiolation of graphene (Gr-SH) was done in an acetonitrile solution of 0.1 M LiClO4 containing 5 mM ATP by the procedure mentioned above.Four graphene modified GCEs were scanned from 0.0 V to 1.0 V in aqueous solutions of 0.1 M LiClO2, -COOH and -SO3H to the graphene surface using cyclic voltammetry. Figures 3H) and electron donating  reactive end groups respectively. The irreversible peak A1 appearing in Figs 2 carbon of graphene forming a covalent bond between amine and graphene15. It is worth noting that the potential required to generate the corresponding amine cation radical of the arylamine derivative depends on the electron withdrawing tendency of the reactive end group present. The Epa (of A1) of the arylamine derivatives is as follows 125 mV (for -OH substituted) < 145 mV (for -NH2 substituted) < 815 mV (for –SH substituted) < 830 mV (for -COOH substituted) < 905 mV (for -SO3H substituted). This trend might be explained by the availability of electrons at amine group. i.e. the more the electron donating tendency of the reactive end group, the more will be the availability of the electrons at amine group of the molecule, which leads to the easy oxidation of the amine functional group of the molecule at lower overpotential.Electrochemical oxidation of arylamines at graphene surface tuned the surface and interfacial properties of graphene by tethering different reactive end groups -SH, -OH, -NH2 and C1) might be attributed to the formation of the head to tail type aminodiphenylamine dimer, which is known for its redox activity during this kind of electrochemical cycling process19. The second oxidation peak A2 appearing in Fig. 20. From Figs 1 in successive cycles represents the formation of an irreversible layer of the corresponding arylamine derivative on graphene. Hence, the layer formed either stop the oxidation process or kinetically decelerates the electron transfer as a function of time21. The density of molecules bonded to graphene in each case was calculated from the charge consumed in the first anodic sweep of the corresponding molecule using the equation (221 (background subtracted), n is the number of electrons involved (n = 1), F is the Faraday constant, A is the effective surface area of the graphene modified GCE , NA is the Avogadro number and Γ is surface density of the molecules attached on graphene assuming that all the radicals generated in the first anodic scan were attached to graphene. The density of the aryl amines substituted with the terminal functionalities -SH, -OH, -NH2, -COOH and -SO3H respectively on graphenes are 45.23 × 1015 molecules cm−2, 38.43 × 1015 molecules cm−2, 40.99 × 1015 molecules cm−2, 44.1 × 1015 molecules cm−2 and 39.68 × 1015 molecules cm−2, which is higher than the reported values with aryl diazonium salts22. The standard deviation of the surface density of molecules bonded to graphene is found to be ±3 for different modifications. The slight differences in their molecular densities might be due to the different chemistry  of the reactive end group of the arylamines and their interactions with graphene during the surface tuning.As shown in Fig. 2) C-N and (sp3) C-N covalent bonds23. The (sp2) C-N and (sp3) C-N bonds could be between the aryl carbon and nitrogen from the same arylamine derivative and sp3 carbon of the graphene and the nitrogen of the arylamine respectively. The same (sp3) C-N covalent bond has been reflected again by the peak centered at 287.0 ± 1 eV in the deconvolution of spectra of C1s of the surface tuned graphenes25 shown in the supporting information Figs XPS characterization is one of the most reliable techniques to analyze aryl layers bonded to graphene. As shown in the survey spectra of graphene and surface tuned graphenes in Fig. 2 demonstrates the presence of -C-N and protonated amine on the surfaces, as illustrated in Fig. 27 as shown in the Fig. 30. The surface elemental composition of all the surface tuned graphenes and the relative distribution of sp3 C-N, sp2 C-N and protonated amines present in the same are tabulated in Tables The deconvoluted high-resolution N1s spectrum of Gr-NH31.The topography of the graphene before and after the surface tuning with the arylamine derivatives is scrutinized by FE-SEM as shown in Fig. 3)6]Cl3 and K4[Fe(CN)6] respectively to get the fundamental insights of the interfacial properties of the surface tuned graphenes. Figure 3)6]Cl3 on Gr-OH, Gr-COOH, and Gr-SO3H. The peak potential difference (ΔEp) values of the positively charged redox probe, [Ru(NH3)6]Cl3 on Gr-OH (74 mV), Gr-COOH (85 mV) and Gr-SO3H (83 mV) do not significantly deviate from that of untuned graphene (78 mV). The behavior is a common observation in the case of outer sphere redox probes like [Ru(NH3)6]Cl3. The cathodic or anodic peak currents observed for the [Ru(NH3)6]Cl3 are in the order GCE < Gr-SO3H < Gr-COOH < Gr < Gr-OH. The higher currents realized at Gr and Gr-OH might be attributed to the high surface area, higher conductivity of Gr and some charge based interactions between the redox probe and the Gr-OH. The lower currents observed at Gr-SO3H and Gr-COOH might be due to the high molecular density and steric hindrance exhibited by the large reactive end groups of the aryl layer present on the Gr-SO3H and Gr-COOH.Cyclic voltammetry studies were carried out with a positively and negatively charged redox probes, [Ru(NH3)6]Cl3 is completely blocked on Gr-SH and Gr-NH2 6], i.e. the redox response of the probe is completely blocked at Gr-SH and Gr-NH2 , Gr-COOH (138 mV) and Gr-SO3H (123 mV) significantly increased when compared to that of untuned graphene (94 mV). This behavior can be due to the charge based repulsions between the probe and the negatively charged reactive end groups present on the graphene. The peak current and peak potential difference values are tabulated in Table As shown in Fig.  Gr-COOH 38 mV and34. The same has been realized in the case of Gr-COOH and Gr-SO3H in the presence of DA and AA as shown in the Fig. −) and sulfonate (-SO3−) functionalities suppressed the response of AA while increasing response of DA by charge-based interactions. Figures 2, Gr-COOH, and Gr-SO3H. The peak potentials and redox currents obtained are presented in Table In addition to the redox probes, two essential bioanalytes dopamine and ascorbic acid are also examined to demonstrate the suitability of the surface tuned graphenes towards the selective detection of dopamine. DA and AA oxidize at similar potentials at bare GCE and Gr as shown in Fig. 3H when compared to the rest. In the case of Gr-COOH and Gr-SO3H, the oxidation of DA is kinetically favorable as reflected by their peak to peak separation values, as shown in Fig. 3H (63 mV) < Gr-COOH (90 mV) < Gr-OH (138 mV) < Gr-NH2 (147 mV) < Gr-SH (163 mV). Hence, this substantiates that the Gr-COOH and Gr-SO3H have better electrocatalytic activity towards DA. The redox response of AA is obtained only on Gr-OH, while the others show no or negligible response, which manifests that the reactive end groups except -OH have enough ability to block the AA from reaching the electrode by electrostatic repulsion.From Table 2, -COOH and -SO3H by the versatile and rational electrochemical approach. The surface tuned graphenes exhibit terminal group dependent interfacial properties which can be useful for numerous applications in the fields of chemical sensors, biosensors, immunosensors, and DNA sensors. Also, the feasibility of post-functionalization of the reactive end groups of the surface tuned graphenes further springs the applications of graphene in different fields.These results together with XPS and FE-SEM analysis confirm the tactical surface tuning of graphene with desired reactive end groups such as -SH, -OH, -NH2, -COOH and -SO3H) on graphene. As one can anticipate, the surface tuned graphenes exhibited reactive end group dependent interfacial properties as realized by CV studies with the redox probes as well as bioanalytes. The reported protocol offers a way to understand the fundamental properties and useful applications of the surface tuned graphenes with desired reactive end groups. Besides, the post-functionalization feasibility of reactive end groups of the surface tuned graphenes further springs the scope of usefulness of the thus prepared material. Further research should be directed towards the control of the coverage, the density of the reactive end groups, and the effect of other reactive end groups on the graphene and their impact on the physicochemical and electrochemical properties of the surface tuned graphenes towards the desired chemical and electrochemical applications.The tactical tuning of the surface and interfacial properties of graphene using different arylamines has been realized by the versatile and rational electrochemical method. The protocol and the functional groups of the surface tuned graphenes are well examined by CV, XPS, and FE-SEM. The interfacial properties of graphene are tactically tuned by tethering the desired reactive end groups (-SH, -OH, -NHElectronic supplementary information


Few studies have reported the diagnostic variability in patients with a possible usual interstitial pneumonia (UIP) pattern on high-resolution CT (HRCT) who underwent surgical lung biopsy (SLB), and the prognostic factors for these patients have not been fully evaluated. We retrospectively investigated the frequency of idiopathic pulmonary fibrosis (IPF) and prognostic factors in patients with possible UIP pattern on HRCT.Consecutive patients who had a possible UIP pattern on HRCT, underwent SLB, and had a diagnosis of IIPs before SLB were retrospectively recruited from 10 hospitals. Diagnoses were made based on multidisciplinary discussion using the criteria for current IPF guidelines and multidisciplinary classification for IIPs in each hospital.179 patients who underwent SLB were enrolled. The diagnoses were IPF in 91 patients (51%), unclassifiable IIPs in 47 (26%), idiopathic NSIP in 18 (10%), and chronic hypersensitivity pneumonia in 17 (9%). One-year FVC changes showed significant differences between IPF and non-IPF . Patients with IPF had a worse mortality than those with non-IPF . Multivariable Cox regression analysis demonstrated that diagnoses of IPF , high modified MRC score , and low %FVC .About a half of patients with a possible UIP pattern on HRCT had diagnoses other than IPF, and patients with IPF had a worse mortality than those with an alternative diagnosis. We reaffirmed that multidisciplinary discussion is crucial in patients with possible UIP pattern on HRCT. Idiopathic pulmonary fibrosis (IPF) is an intractable disease with a poor prognosis. Therefore, the accuracy of a diagnosis of IPF is crucial for making therapeutic decisions and predicting patient’s survival ,2. In reAntifibrotic drugs, pirfenidone and nintedanib, are now regarded as “conditional” recommendation drugs for IPF –9, so an, Sildenafil Trial of Exercise Performance in Idiopathic Pulmonary Fibrosis (STEP-IPF) and Anticoagulant Effectiveness in Idiopathic Pulmonary Fibrosis (ACE-IPF)), and reported that among 64 patients with possible UIP pattern on HRCT, 60 patients had a pathological UIP pattern and 4 patients had a pathological probable UIP pattern . The Kaplan-Meire survival curves showed patients with IPF had a significantly worse prognosis than those with non-IPF  . SimilarUnivariate Cox regression analysis showed that cough, modified MRC score, percent predicted FVC, percent predicted diffusing capacity of lung for carbon monoxide (DLco), and diagnosis of IPF were significant prognostic factors . MultivaIn this retrospective study, we studied a total of 179 ILD patients with a surgical lung biopsy and a possible UIP pattern on HRCT. Non-IPF was identified in about a half of the patients with possible UIP pattern on HRCT. Patients with IPF diagnosed by MDD have more disease progression and worse prognosis than those with non-IPF. Multivariate analysis demonstrated that the diagnosis of IPF, high modified MRC score, and low %FVC were independent significant prognostic factors in our cohort. These results indicate that a possible UIP pattern on HRCT did not necessarily indicate a diagnosis of IPF, and patients with IPF diagnosed through MDD with SLB have worse prognosis than those with an alternative diagnosis. We reaffirmed that multidisciplinary discussion is crucial in patients with possible UIP pattern on HRCT.Our study cohort consisted of 179 SLB patients with possible UIP pattern on HRCT from 10 Japanese hospitals that have experts in the field of ILD. This is the largest cohort to date of consecutive patients with possible UIP pattern on HRCT who underwent SLB. This cohort also had a similar gender and age distribution, and despite some mild pulmonary function impairment compared with other cohorts ,13. BecaA variety of diseases other than IPF, such as unclassifiable IIP, idiopathic NSIP, CHP, and CTD-ILD were observed in our cohort. In addition, significant differences were observed in the disease progression and the prognosis between IPF and non-IPF. These results support the importance of a diagnosis of IPF in this population. Because SLB is occasionally difficult to carry out for a variety of reasons, such as poor pulmonary function testing, severe fibrotic changes, and patient unwillingness because of the invasive nature and associated risks, consideration should be given to the differential diagnosis in patients with a possible UIP pattern on HRCT. Because patients with non-IPF showed significantly higher % lymphocytes in BALF than those with IPF, lymphocytosis in BALF may suggest a diagnosis of non-IPF.A quarter of patients were diagnosed with unclassifiable IIPs in this study, which is compatible with previous studies that reported unclassifiable IIPs in approximately 10 to 25% of all patients with ILD . The reaOur study did not demonstrate a strong association between a possible UIP pattern on HRCT and a diagnosis of IPF, which contradicts a few recent reports ,12,13. WAlthough age and sex were predictive factors for IPF in another cohort , they arThere are several limitations in this study. First, the diagnoses examined in this study were made in individual hospitals; therefore, there may be some biases in diagnosis between hospitals. However, we suppose this is less likely because all participating hospitals had experts in the field of ILD, which helps to ensure the accuracy of diagnostic evaluations. In addition, the finding in a recent study that agreement in MDD diagnosis of expert groups is acceptable in IPF  may lendIn conclusion, we studied the frequency of IPF and prognostic factors in a biopsy-proven ILD cohort of patients with a possible UIP pattern on HRCT. Although previous studies have reported high positive predictive values of possible UIP patterns on HRCT for predicting IPF, this study demonstrated that patients with possible UIP patterns on HRCT did not necessarily have IPF; the PPV for IPF in patients with possible UIP pattern on HRCT over 65 y.o. was only 51%. Because patients with IPF had a worse prognosis than those with an alternative diagnosis, identification of IPF in patients with a possible UIP pattern on HRCT is crucial. Further studies are needed to define clinically appropriate predictors for IPF.S1 FileTable A in S1 File. Positive predictive value, negative predictive value, sensitivity, and specificity when classifying patients with IPF based on being at least as old as the age indicated.Table B in S1 File. Positive predictive value, negative predictive value, sensitivity, and specificity when classifying patients with broader definition of histological UIP pattern based on being at least as old as the age indicate.Table C in S1 File. The reasons for, broader definition of pathologic UIP pattern in, and most suspected diagnosis of unclassifiable IIP.Fig A in S1 File. Relationships between age and diagnosis.(DOCX)Click here for additional data file.S1 Datasheet(XLSX)Click here for additional data file.


In a magnetic field, combination of quantum Hall physics and the characteristic transport across p–n junctions leads to a fractionally quantized conductance associated with the mixing of electron-like and hole-like modes and their subsequent partitioning. The mixing and partitioning suggest that a p–n junction could be used as an electronic beam splitter. Here we report the shot noise study of the mode-mixing process and demonstrate the crucial role of the p–n junction length. For short p–n junctions, the amplitude of the noise is consistent with an electronic beam-splitter behaviour, whereas, for longer p–n junctions, it is reduced by the energy relaxation. Remarkably, the relaxation length is much larger than typical size of mesoscopic devices, encouraging using graphene for electron quantum optics and quantum information processing.Graphene offers a unique system to investigate transport of Dirac Fermions at  Dirac fermions at a p–n junction can exhibit a wide variety of unusual properties. Here, the authors investigate the dynamics of such fermions in a graphene junction using shot noise measurements and demonstrate the crucial role of junction length. Investigations of charge carrier transport across such a p–n junction (PNJ) have revealed unique phenomena reflecting the Dirac Fermion character in graphene, such as Klein tunneling125B, the conductance across a PNJ shows plateaus at GPNJ=G0|ν1||ν2|/(|ν1|+|ν2|), where G0=e2/h is the conductance quantum (h is Planck's constant), ν1=2, 6, 10, … and ν2=−2, −6, −10, … are the Landau level filling factor in the n and p regions, respectively7810ν1 and ν2, and then partitioned at the exit of the PNJ  becomes out-of-equilibrium and the subsequent partitioning of the modes gives rise to the shot noise. If the mode mixing is quasielastic, fPNJ(E) is a double-step function. At zero temperature, the shot noise generated by the partitioning of the modes with double-step fPNJ(E) is expected to be =. Energy losses towards external degrees of freedom can drive fPNJ(E) towards a Fermi distribution with a chemical potential eVsd/2 (1516fPNJ(E) to relax towards a Fermi distribution with a finite temperature Teff(Vsd) given by the balance between the Joule power dissipated in the PNJ and the heat flowing along the outgoing electronic channels10ν1, ν2)=]. Note that standard transport measurements yield the same value of GPNJ for all cases, and thus cannot distinguish them.characterized by the Fano factor l eVsd/2 151617, csd/2 1516. InelastfPNJ(E) with carrier dwell time in the PNJ, which can be tuned by changing the length of the PNJ. We demonstrate that the amplitude of the noise is consistent with an electronic beam-splitter behaviour when PNJ is short and the energy losses are negligible.In the following, we investigate the evolution of VG, while that in the ungated region is fixed to electron by the doping (Methods). Therefore, the PNJ is formed at the interface between the gated and ungated regions when the carrier type in the gated region is hole for ΔVG≡VG−VCNP<0 . We prepared five samples with different interface lengths L=5, 10, 20, 50, and 100 μm. The direction of B is chosen so that electron and hole modes from the ohmic contacts Vsd is applied to either Cdet = is formed for ΔVG<−10 V, the current injected from SI appears in this regime. As Vsd applied to SI≡SI−SI(Vsd=0) increases = for 20<ΔVG<50 V, on the other hand, the shot noise is zero (solid black circles), proving that the shot noise is indeed generated at the PNJ.The inset of F by fitting ΔSI as a function of Vsd using the relation including temperature broadeningQuantitatively, we extracted GPNJ is obtained by average current measurements. The fit yields F=0.015, which is one order of magnitude smaller than F=0.25 expected for the noise from the double-step energy distribution. This indicates that fPNJ(E) evolves during the charge propagation for L=50 μm, reducing the shot noise.where fPNJ(E) can be investigated using samples with different L. νug, νg)= for the five samples with L between 5 and 100 μm. The data show that the shot noise decreases with increasing L and almost disappears at L=100 μm (fPNJ(E) relaxes to the thermal equilibrium through interactions with external degrees of freedom. An exponential fit of the data yields a relaxation length L0=15 μm. The extrapolation to L=0 gives F∼0.27, consistent with the limit of quasielastic scattering F=0.25. Furthermore, the decrease is well reproduced by a model gradually coupling the modes propagating in the PNJ to cold external states = is formed for B>4 T and between νg=0 and −4 (the region indicated by dashed lines), in which the current is almost constant, consistent with the quantized conductance78L=10 μm becomes small , generating shot noise. For a short PNJ (L<<15 μm), the energy loss towards external states is negligible and the noise is consistent with a quasielastic mode mixing. This suggests that a graphene PNJ can act as a beam splitter. Since 15 μm is much larger than typical length scale of mesoscopic devices, our results encourage using graphene for electron quantum optics experiments and quantum information.In conclusion, we showed that the mode mixing at PNJ in graphene bipolar QH states leads to non-equilibrium 2 atmosphere. After the etching, the surface was covered with 100-nm-thick hydrogen silsesquioxane (HSQ) and 60-nm-thick SiO2 insulating layers. As a result of doping from the SiC substrate and the HSQ layer, graphene has n-type carriers with the density of about 5 × 1011 cm−2. The width of the PNJ roughly corresponds to the thickness of the insulating layers and estimated to be 200 nm at most. In the QH effect regime, because of the Landau level quantization, the width becomes smaller with B. An important advantage of the SiC graphene is its size: it is single domain for 1 cm2, allowing us to investigate the effect of PNJ length.We prepared a graphene wafer by thermal decomposition of a 6H-SiC(0001) substrate. SiC substrates were annealed at around 1,800 °C in Ar at a pressure of <100 torr. For the fabrication of devices, graphene was etched in an Oν=2 and the temperature of the system.For the noise measurement, the current noise is converted into voltage fluctuations across one 2.5 kΩ resistor in series with the sample. A 500-kHz bandwidth 3-MHz tank circuit combined with a homemade cryogenic amplifier is used. After further amplification and digitization, the autocorrelation voltage noise spectra is calculated in real-time by a computer. Accurate calibration of the noise is done using Johnson–Nyquist noise that relies on the quantification of the resistance at How to cite this article: Kumada, N. et al. Shot noise generated by graphene p–n junctions in the quantum Hall effect regime. Nat. Commun. 6:8068 doi: 10.1038/ncomms9068 (2015).Supplementary Figures 1-5, Supplementary Note 1, Supplementary Methods and Supplementary References