The aim of this manuscript is to describe radiological findings of extra-pulmonary sarcoidosis. Sarcoidosis is an immune-mediated systemic disease of unknown origin, characterized by non-caseating epitheliod granulomas. Ninety percent of patients show granulomas located in the lungs or in the related lymph nodes. However, lesions can affect any organ. Typical imaging features of liver and spleen sarcoidosis include visceromegaly, with multiple nodules hypodense on CT images and hypointense on T2-weighted MRI acquisitions. Main clinical and radiological manifestations of renal sarcoidosis are nephrolithiasis, nephrocalcinosis, and acute interstitial nephritis. Brain sarcoidosis shows multiple or solitary parenchymal nodules on MRI that enhance with a ring-like appearance after gadolinium. In spinal cord localization, MRI demonstrates enlargement and hyperintensity of spinal cord, with hypointense lesions on T2-weighted images. Skeletal involvement is mostly located in small bone, showing many lytic lesions; less frequently, bone lesions have a sclerotic appearance. Ocular involvement includes uveitis, conjunctivitis, optical nerve disease, chorioretinis. Erythema nodosum and lupus pernio represent the most common cutaneous manifestations encountered. Sarcoidosis in various organs can be very insidious for radiologists, showing different imaging features, often non-specific. Awareness of these imaging features helps radiologists to obtain the correct diagnosis.Systemic sarcoidosis can exhibit abdominal, neural, skeletal, ocular, and cutaneous manifestations.• T2 signal intensity of hepatosplenic nodules may reflect the disease activity.• Heerfordt’s syndrome includes facial nerve palsy, fever, parotid swelling, and uveitis.• • In the vertebrae, osteolytic and/or diffuse sclerotic lesions can be found.Erythema nodosum and lupus pernio represent the most common cutaneous manifestations.•  Sarcoidosis is a multi-systemic disorder of unknown cause, pathologically characterized by the accumulation of inflammatory cells forming non-caseating granulomas. Lesions can be located in any organ but in about 90 % of patients, granulomas affect the lungs or the related lymph nodes .According to Geize et al., sarcoidosis can be encountered in extra-pulmonary locations, in approximately 30 % of cases . MoreoveThe systemic location of the disease can be very insidious for radiologists: a wide variety of imaging features are found, often non-specific, which can mimic other pathological conditions , 7. TherThe disease affects young men and women aged between 25 and 40 years, without major differences in ethnicity. Some studies do show a greater incidence in females . In abouNormally, sarcoidosis shows a variable clinical course; it may also exhibit different extra-thoracic manifestations. These vary widely according to ethnicity: for example, involvement of the eye, liver, bone marrow, extra-thoracic lymph nodes, and skin has been more statistically associated with African Americans than with Caucasians , 5.Cardiac and ocular diseases are frequently encountered in Japanese patients, erythema nodosum in northern Europeans, and ocular and granulomatous skin involvement in black patients –13. In aExtra-thoracic sarcoidosis involves females more frequently than males .Extra-pulmonary sarcoidosis has been found in 30 % of patients with the disease . The mosThe cutaneous system and the eyes are involved with a frequency of 25 % , 15. FolThe following sections below describe more in detail clinical and radiological features of systemic sarcoidosis Table , focusinHepatic sarcoidosis occurs with a prevalence of 1-40/100,000; it generally involves young people . The disCommonly, imaging demonstrates hepatomegaly, with homogeneous appearance of parenchyma ; very ofGranulomatous lesions are located in the portal and periportal spaces of hepatic sinuses and generally exhibit “an identical stage of maturation” , 23. TheSplenic sarcoidosis is generally associated with lung disease; however, normal chest radiography is observed in one quarter to one third of patients with splenic sarcoidosis . SplenicPatients can be asymptomatic, or complain of fever, weight loss, and malaise , 32. In Splenic sarcoidosis can be present in a homogeneous fashion or in a nodular pattern , 27, 33.On MRI acquisitions, nodular lesions show low signal in all sequences ; the visParenchymal lesions show increased FDG uptake on PET-CT : low-denAmong abdominal sarcoidosis, autopsy revealed only a small percentage of cases (5 %) located in pancreatic tissue, intestinal tract, and testes . The gasThe biliary tree may be involved in extra-hepatic or intra-hepatic pattern. Granulomatous lesions develop in the portal triad, causing a cholestatic pattern.Extra-hepatic involvement of the biliary tree can be caused by multiple small granuloma of the wall; the radiological appearance in these cases may simulate a cholangiocarcinoma; in many cases, the biliary tree may be compressed by enlarged abdominal nodes.According to Koyama et al., in 6 % of cases parotid glands may have sarcoidosis involvement; generally, the disease is found bilaterally, with enlargement of glands . An inhoLymphatic abnormalities have been already described as abdominal manifestations of sarcoidosis, consisting of “lymphadenopathy or increased number of normal sized nodes” ; lymph nEnlarged nodes can be observed in different locations: in the periportal region, close to the liver hilus Fig. , or betwLymphatic involvement is frequently associated with pulmonary disease, and this can be very helpful for radiologists in order to achieve a correct diagnosis. When the disease is limited to the lymphatic system in mediastinum and in the abdomen, a differential diagnosis from other disorders, e.g. lymphoma, may be very difficult.Peritoneal sarcoidosis is extremely rare , 38–41. In sarcoidosis, the real incidence and prevalence of renal involvement is difficult to establish, because the disease can occur in a variable manner; in most cases, it has been found in 35 to 50 % of patients . Main clThe increased levels of 1,25-dihydroxyvitamin, produced by mononuclear cells trapped in pulmonary alveoli, determines a greater absorption of calcium: this leads to hypercalcemia and consequential nephrolithiasis and nephrocalcinosis .The prevalence of interstitial nephritis in sarcoidosis oscillates from 7 to 27 % , 45. TheEnhanced CT images may show signs of interstitial nephritis , 47, 48 Rarely, radiological features of renal sarcoidosis can be represented by granulomatous pseudotumour; these lesions are generally incidentally discovered during CT examination. They can appear as hypo-, iso-, or hypeattanuating areas on unenhanced CT scans; after contrast administration, they are hypodense to the surrounding renal parenchyma Fig.  48]. Di. Di48]. The urinary manifestations of sarcoid nephritis are nonspecific: the alterations of the urine analysis are similar to those of chronic tubular pathologies. Biopsy is the only way to make a diagnosis, showing normal glomeruli with interstitial infiltration of mononuclear cells and non-caseating granulomas; occasionally, it may be also depicted with glomerular disease , 50.Central nervous system (CNS) involvement is reported in 25 % of cases at autopsy; in a post-mortem series, neural sarcoidosis was detected with a frequency of 14–27 % –54. SympThe disease shows a special predilection for the basal cisterna of the brain; cranial nerve involvement is frequent, influencing the clinical manifestation of disease. Facial nerve palsy, for example, is a typical manifestation of brain sarcoidosis in young adults . It may Other symptoms associated with neural sarcoidosis are headache, seizure, and signs of meningeal irritation , 56. InvDiagnosis of sarcoidosis can be performed following the criteria reported in the study by Pawate et al., in which the presence of neurosarcoidosis is evaluated as definite, probable, and possible. According to their criteria , the disLesions appear hyperintense on T2-weighted MRI images, located in the superficial and deep white matter; sometimes, these imaging features may resemble lesions of multiple sclerosis . Also grDural thickening is generally isointense to the grey matter on T1-weighted acquisitions, hypointense on T2-weighted sequences, with contrast enhancement after gadolinium injection .Leptomeningeal localizations are more visible after contrast injection, showing increased enhancement Fig.  58]. As. As58]. Nodules can be solitary or multiple, showing enhancement after contrast injection with a ring-like appearance in the activity phase: this appearance can simulate glioblastoma or metastases. An intracranial mass presentation has been also reported in the literature, simulating a brain neoplasm .Spinal sarcoidosis is reported in about 4–28 % of cases; it occurs predominantly in old people and has a poor prognosis . The disMRI features of stage 1 and 2 disease include leptomeningeal thickening, with hyperintense signal on enhanced T1-weigheted images . In cervThe skeletal system represents a rare localization of sarcoidosis . RadioloThe disease generally involves hands and feet, which have been reported in several reviews as the most affected locations , 64. On Large bones and axial skeleton involvement is uncommon; however, the disease may be encountered in ribs, long bones, skull and vertebrae. Axial skeleton disease, without pulmonary lesions, is rarely reported .Lesions of large bone and axial skeleton can be detected as radiolucent Fig.  or sclerIn patients with sarcoidosis, skeletal FDG uptake can be observed Fig. ; these iOcular involvement occurs in approximately 25–60 % of patients with systematic sarcoidosis, more frequently in the third decade (first peak of incidence) and between the sixth and the seventh decade (second peak of incidence) . This maIn most cases (about 20 %), uveitis is the first common manifestation: symptoms that patients complain of include tearing, photophobia, pain, infection, lacrimation, redness. However, these symptoms can be absent: “silent uveitis” is very insidious because it may produce permanent ocular damage before treatment. The common type of uveitis is often anterior in black patients, posterior in white patients, specifically in elderly female patients –74. BlinIn the anterior segment, conjunctivitis occurs in 7–70 % of the patients with ocular sarcoidosis and, together with lacrimal gland involvement, is usually asymptomatic. The sarcoidosis granulomas in the eyes have the appearance of yellow “millet-seed” nodules. When the disease involves the posterior part of the eyes, it is frequently associated with neurological manifestations: optic nerve disease, cranial nerve palsies, encephalopathy, and disorders of the hypothalamus and pituitary gland , 78.Other manifestations of ocular sarcoidosis include granulomatous iridocyclitis, retinal periphlebitis and chorioretinis; foci of retinal involvement are generally revealed by fluorangiography as “punched-out” lesions Fig. . In 7 toIn sarcoidosis, cutaneous involvement occurs in 20–35 % of patients . The cutErythema nodosum is the most common nonspecific cutaneous lesion of sarcoidosis. In most cases it occurs with subcutaneous erythematous nodules Fig. , often oThe term “specific” is misleading, because clinically lesions are not specific to sarcoidosis, and the only way to make a diagnosis is biopsy. Papule Fig.  is the mA characteristic clinical manifestation of cutaneous sarcoidosis is lupus pernio Fig. : it appeThe diagnosis of cutaneous sarcoidosis is performed with histological examination of a cutaneous biopsy, which shows the presence of sarcoidal granuloma without any cornoid lamella.Cardiac involvement with clinical manifestations occurs in 2–7 % of patients, but the occult form is much more present –86.Sarcoidosis characteristic granulomatous lesion manifests in any part of the heart, but the myocardium is most frequently involved . In ordeGadolinium-enhanced cardiac MRI allows us to detect differences between normal tissue and altered tissue, the latter displaying areas of focal enhancement, particularly in the myocardial wall or subepicardial region Fig.  92, 93], 9392, 9Radionuclide scans with gallium, citrate, and thallium are used for diagnosis and follow-up in patients with cardiac sarcoidosis , 95. PETAbdominal, neural, musculo-skeletal, cutaneous, ocular and cardiac sarcoidosis has extremely variable clinical features; their imaging findings can often simulate other diseases, namely malignancies. Sarcoidosis should be considered in the differential diagnosis of multiple lesions of brain, abdomen and bones in patients with suspected or proved disease.In patients with pulmonary sarcoidosis, a careful evaluation of patients’ symptoms is mandatory in order to identify other locations of the disease. Radiologists, correlating imaging and clinical-pathologic findings, play an important role in diagnosis and follow-up of the disease, in order to reduce its morbidity and mortality.


Pan troglodytes) use tools to probe for out-of-reach food, both in the wild and in captivity. Beyond gathering appropriately-sized materials to create tools, chimpanzees also perform secondary modifications in order to create an optimized tool. In this study, we recorded the behavior of a group of zoo-housed chimpanzees when presented with opportunities to use tools to probe for liquid foods in an artificial termite mound within their enclosure. Previous research with this group of chimpanzees has shown that they are proficient at gathering materials from within their environment in order to create tools to probe for the liquid food within the artificial mound. Extending beyond this basic question, we first asked whether they only made and modified probe tools when it was appropriate to do so (i.e. when the mound was baited with food). Second, by collecting continuous data on their behavior, we also asked whether the chimpanzees first  modified their tools prior to probing for food or whether such modifications occurred after tool use, possibly as a by-product of chewing and eating the food from the tools. Following our predictions, we found that tool modification predicted tool use; the chimpanzees began using their tools within a short delay of creating and modifying them, and the chimpanzees performed more tool modifying behaviors when food was available than when they could not gain food through the use of probe tools. We also discuss our results in terms of the chimpanzees’ acquisition of the skills, and their flexibility of tool use and learning. Am. J. Primatol. 77:162–170, 2015. © 2014 The Authors. American Journal of Primatology Published by Wiley Periodicals Inc.Chimpanzees ( Pan troglodytes) using tools to probe for out-of-reach food, both in the wild and in captivity [e.g., Boesch & Boesch, There are numerous accounts of chimpanzees . The group was comprised of three males and four females, with an average age of 10 years (range: 4–19 years). The chimpanzees inhabited an expansive indoor/outdoor exhibit that included climbing structures and deep-mulch bedding and an off-exhibit holding area. The indoor exhibit space also featured an artificial termite mound [Bonnie et al., This study was approved by the Lincoln Park Zoo Research Committee, which is the governing body for all animal research at the institution. No social group manipulations occurred as the result of this project. Food substances, amount, and frequency were reviewed and approved by veterinary and nutrition staff prior to the start of the project. No modifications were made to standard animal care routines. This research adhered to legal requirements in the United States of America and to the American Society of Primatologists’ Principles for the Ethical Treatment of Nonhuman Primates.For this study, liquid foods were presented to the chimpanzees via an artificial termite mound, mimicking the look of a natural termite mound found in wild ape habitats . The mouCrataegus spp.), serviceberry trees , red oak trees (Quercus rubra), and a bur oak tree (Q. macrocapra). Unfortunately, due to the degradation of the video footage collected in 2004 when these data were collected, for these current analyses, it was not possible to identify which species of plants were used by the chimpanzees to create their probe tools. However, contemporary observations of this group of chimpanzees revealed that they create tools from a number of species, including willow and poplar that are now provided to them by keeper staff, and they also use the young shoots of serviceberry trees that grow in their outdoor exhibit, which they modify by fraying the ends of the sticks , for this analysis we only include the chimpanzees’ responses in the first two months of the baited phase to enable a clearer comparison across the two periods. These data represent a subset of data previously collected at Lincoln Park Zoo as part of a wider investigation of ape social learning and tool use. The original findings from this study were published previously by Lonsdorf et al. , but theThe first two months (56 days) of the study period represented the “Baseline” phase. Behavioral data were collected on 35 of these 56 days (i.e. Monday–Friday every week) from June 7th 2004–August 2nd 2004. These Baseline sessions were spread evenly throughout the Baseline period and all Baseline sessions were filmed for later analysis. During this period, the termite mound was never baited with food. The chimpanzees had no prior experience with obtaining food from this artificial mound and so this period represented a true behavioral baseline . Spread evenly throughout the Experimental period were 19 “Bait” sessions, on which the tubes in the termite mound were baited with desirable viscous food (e.g. ketchup), at randomized times between 1100 and 1400 hr (Monday–Friday). No more than one Bait session was conducted on a given day. For Bait sessions, the chimpanzees did not know prior to testing what the termite mound was baited with, although, once baited, the chimpanzees could inspect the holes through smell and could also sample the food stuffs directly with probe tools [c.f. Bonnie et al., Video footage of the chimpanzees’ behavior, on, or within 1 m of, the artificial termite mound, was recorded for 35 Baseline sessions, 19 Bait sessions, and 19 Control sessions. Video footage was recorded using a stationary, ceiling-mounted security camera which captured the entire mound and a 1 m perimeter around the mound. This camera was connected to a time-lapse VCR (Panasonic AG-RT650) that was pre-set to record each session. Data were scored directly from the video footage in 2004–2006, wherein each group member’s behavior was scored continuously, as described by Lonsdorf et al.  (Table1)The recorded data captured each session entirely but the data presented here represent the first 15 min of each session. For these analyses, we focused on the ‘probe’ and ‘tool-modification’ behaviors , all behaviors were converted to average rates/session. Friedman’s tests and Wilcoxon’s signed-ranks tests were used to compare the chimpanzees’ responses across conditions. Firstly, we compared the rate of fishing and tool-modification behaviors in the Baseline phase to (a) the days on which the termite mound was baited (Bait sessions) and (b) days within the experimental phase when the termite mound was not baited (Control sessions). Secondly, once the chimpanzees had experienced food in the artificial mound (Bait sessions), to determine whether tool modifications were functional, we compared the chimpanzees’ rates of tool modification and tool use across the Control and Bait sessions. To further investigate the interplay between tool-modification and probing behaviors, we ran a Pearson’s correlation between rates of tool modification and fishing behaviors. All analyses were conducted in IBM SPSS, Version 20 .Z-test and likelihood ratio tests (LRTs) [Bates et al., We were also interested in determining if tool modification was more likely than other behaviors to be followed by tool usage (i.e. probing). For the purposes of this analysis, we simplified the continuous sampling dataset by noting whether or not, for each pair of behaviors observed, the first behavior was modification and whether or not the second behavior was tool use. All other behaviors described in TableThere were significant differences across all three phases  regarding both the rate of tool-modification behaviors as well as the rate of probing behaviors recorded  and we cX2(2) = 11.273, P = 0.04). The chimpanzees performed more tool modification when the termite mound was baited (Bait) than during the Baseline  or Control  sessions. While none of the chimpanzees ever modified tools in the Baseline sessions, six of the seven chimpanzees performed one or more tool-modification behaviors during the Bait phase  and all did so on the first Bait session . Emphasizing the low rate of tool-modification behaviors in Control sessions, there was no difference in the number of tool-modification behaviors seen during the Baseline and Control sessions, but they showed a trend towards performing more tool modification in the Control sessions  = 14.00, P = 0.001). Comparable with their tool-modification behaviors, the chimpanzees performed more probing behaviors when the termite mound was baited (Bait) than during the Baseline  or Control  sessions. The chimpanzees were observed to probe more during the Control sessions compared to during Baseline sessions , indicating comparably low levels of probing behaviors in both phases.During the Baseline sessions, five of the seven chimpanzees performed one or more probing behaviors and the first chimpanzee to do so was a five-year-old female during the 3rd Baseline session (June 10th 2004). In contrast, during the Bait sessions, all seven chimpanzees probed the artificial mound . There was a significant difference in the rate of probing behaviors observed across the three experimental phases , such that those chimpanzees that probed more, also performed more tool-modification behaviors. There was no significant correlation, however, between rates of probing behavior and tool modification in Control sessions .Chimpanzees were only recorded to modify tools in Bait and Control sessions. During the Bait sessions, there was a significant positive correlation between rates of probing and tool-modification behaviors (Pearson’s X2(1) = 862.32, P < 0.0001) and sessions  = 91.61, P < 0.0001), although among-individual variance was substantially higher than among-session variance . After accounting for this variation, the fitted values revealed that probing behaviors occurred more frequently after modification (47.6%) than after other behaviors (14.9%), and this difference was statistically significant . Indeed, in Bait sessions, the average delay between a chimpanzee modifying a tool and then performing a probing behavior was only 4.5 sec (range = 1–247 sec). The degree to which modification predicted probing did not significantly differ among chimpanzees or sessions .Overall, probing behaviors comprised 24.1% of the total behaviors observed. The rate of probing behaviors varied significantly among both chimpanzees  from a novel ‘artificial termite mound’ in their enclosure and, if so, to identify under what circumstances they modified tools for fluid dipping behaviors. Specifically, we wished to record whether the chimpanzees modified their tools prior to using them, and this is what we found. Expanding upon a previous study, which had investigated the learning of tool-use behavior by chimpanzees [Lonsdorf et al., Although captive chimpanzees have been documented to create and modify functionally-relevant tools, these studies also report individual differences across chimpanzees as a function of age or rearing history [Bania et al., Of the six chimpanzees that modified their tools, four did so in Control sessions when no food was available, perhaps due to “carry over” effects from Bait sessions . Indeed, Price et al.  noted thSix subjects began modifying tools as soon as there was available food (in the first Bait session), and because these chimpanzees made their first tool modification within 60 sec of each other, it is most likely that these behaviors were already known, but only elicited when it was appropriate for them to do so. However, we might consider too that the chimpanzees were able to  learn tool-modification skills rapidly. We evaluate both of these proposals in turn. First, although the task of probing an artificial termite mound was novel to this group of chimpanzees, it is likely that they had prior experience with probing given that this is a near-universal behavior among chimpanzees [Whiten et al., sensu Tennie et al., sensu Hrubesch et al., If probe tool manufacture and modification is within chimpanzees’ ‘Zone of Latent Solutions’ [Pongo pygmaeus abelii, Lehner et al., Corvus moneduloides, von Bayern et al., In our study, tool modification predicted tool use. Regardless of whether the subjects in our study already knew how to create, modify, and use probe tools, or if they had to acquire the skills at the start of the study, they all had to adapt and apply their skills to this novel task. This demonstrates their flexible foraging abilities. This is perhaps not surprising as, when presented with novel problem-solving tasks, chimpanzees [Hopper et al., We defined tool modification broadly, but a specific form of probe tool modification that is commonly reported is tool fraying. While even early studies of chimpanzee probing behavior reported chimpanzees’ proclivity to fray the end of probe tools [e.g., Teleki,


The aggregation of RNA-binding proteins is a pathological hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). RBM45 is an RNA-binding protein that forms cytoplasmic inclusions in neurons and glia in ALS and FTLD. To explore the role of RBM45 in ALS and FTLD, we examined the contribution of the protein’s domains to its function, subcellular localization, and interaction with itself and ALS-linked proteins. We find that RBM45 forms homo-oligomers and physically associates with the ALS-linked proteins TDP-43 and FUS in the nucleus. Nuclear localization of RBM45 is mediated by a bipartite nuclear-localization sequence (NLS) located at the C-terminus. RBM45 mutants that lack a functional NLS accumulate in the cytoplasm and form TDP-43 positive stress granules. Moreover, we identify a novel structural element, termed the homo-oligomer assembly (HOA) domain, that is highly conserved across species and promote homo-oligomerization of RBM45. RBM45 mutants that fail to form homo-oligomers exhibit significantly reduced association with ALS-linked proteins and inclusion into stress granules. These results show that RMB45 may function as a homo-oligomer and that its oligomerization contributes to ALS/FTLD RNA-binding protein aggregation. Amyotrophic lateral sclerosis (ALS) is a progressive, fatal neurodegenerative disorder resulting from the death of upper and lower motor neurons. Approximately 90% of all ALS cases are considered sporadic and of unknown etiology. Multiple cellular mechanisms have been proposed to contribute to motor neuron degeneration in sporadic ALS, including altered RNA processing, protein aggregation, mitochondrial defects and oxidative stress3456Studies of the normal and pathological functions of RNA-binding proteins such as TDP-43 and FUS have provided insight into the role of these proteins in ALS and FTLD. TDP-43 and FUS are multi-functional RNA-binding proteins that contribute extensively to the regulation of gene expression. Both proteins influence pre-mRNA splicing, participate in RNA transport, and modulate RNA stability910TDP-43 and FUS genes of patients with familial forms of ALS and FTLDStructure-function studies of TDP-43 and FUS have provided considerable insight into the mechanisms by which these and other RNA-binding proteins exert their normal functions, associate with stress granules, and aggregate into inclusion bodies in disorders such as FTLD and ALS. Both TDP-43 and FUS contain RNA-recognition motifs (RRMs) that are necessary for the nucleic acid binding functions of the protein1315One such protein is RBM45, an RNA-binding protein we found in inclusions in the cells of patients with ALS and FTLD. A proteomic analysis of cerebrospinal fluid (CSF) from sporadic ALS and healthy control subjects identified increased levels of RBM45 in the CSF of sporadic ALS patientsIn this study, we identify structural determinants of RBM45 for subcellular distribution and protein-protein interactions. Our results show that RBM45 localizes predominately to the nucleus via a bipartite nuclear-localization sequence located at the C-terminal region. In addition, we have identified a novel and evolutionary conserved structural element located within the linker region between RRM2 and RRM3 that is essential for self-association and oligomerization of RBM45. Importantly, the oligomerization of RBM45 mediates its association with TDP-43 as well as inclusion within stress granules.RBM45 was previously detected in cytoplasmic inclusions of neurons and glia in ALS and FTLD patient tissueTo identify the sequence(s) responsible for nuclear localization, we analyzed the amino acid sequence of RBM45 using the computational program cNLS MapperWithin the second NLS candidate (454–472) are additional basic residues  that may contribute to RBM45’s nuclear localization . To examin vivo2627ALS-linked proteins TDP-43 and FUS contain prion-like domains and are intrinsically prone to aggregationWe next sought to map the domain responsible for RBM45 self-association by screening a series of truncated HA-tagged RBM45 fragments for interaction with the full-length FLAG-RBM45 protein co-expressed. We initially focused on the uncharacterized linker region between RRM2 and RRM3 by expressing four HA-RBM45 truncation constructs, Linker-1, -2, -3 and -4 , in a HEin vivo cross-linking with disuccinimidyl suberate (DSS) to capture native RBM45 assemblies in live cells. DSS is an irreversible, cell membrane permeable cross-linking reagent containing bifunctional amine-reactive NHS-estersTo determine if RBM45 self-association leads to dimerization or oligomerization, we performed To determine the stoichiometry of RBM45 homo-oligomers, we analyzed the size of the cross-linked product of a truncated HA-RBM45 variant (107–474) that lacks RRM1. By SDS-PAGE, the monomer of the full-length and truncated HA-RBM45 migrated at 59 and 46 kD, similar to the calculated 56 and 45 kD, respectively  to visualize the co-localization of RBM45 and its binding proteins. PLA visualizes protein-protein interactions To further determine if RBM45 and TDP-43 associate in the cytoplasm, we co-expressed FLAG-TDP-43 together with the HA-RBM45-NLS mutant that predominately accumulates in the cytoplasm, followed by co-IP to detect physical interactions between RBM45 and TDP-43. We observed significant association between the RBM45-NLS mutant and TDP-43 . SimilarWe next asked whether the oligomerization of RBM45 is also necessary for its association with TDP-43 and FUS. Anti-FLAG co-IP analysis using cells co-expressing FLAG-TDP-43 and HA-RBM45 deletion mutants, D1 and D4, showed that wild-type and the D1 mutant HA-RBM45 co-purified with FLAG-TDP-43 . HoweverSince the RBM45-TDP-43 association is RNA-mediated, we examined the importance of the three RRM domains of RBM45 for TDP-43 association. We performed co-IP assays of FLAG-TDP-43 with different RBM45 constructs that lack individual RRM domains . Interesin vivo findings that RBM45 was found in the TDP-43-containing but not FUS-containing inclusions, in neurons of ALS and FTLD patientsOxidative stress has been implicated in the pathogenesis of ALS and FTLD233910In both ALS and FTLD, aggregation of RNA-binding proteins contributes to disease pathogenesis and generates neuropathologic inclusions within the brain and spinal cord. RBM45 was first described as a neuronal RNA-binding protein expressed under spatiotemporal control in the rat brainWe identified a novel HOA domain (residues 258–318) within RBM45 that is responsible for self-association and oligomerization . The HOAet al. reported that RBM45 binds to overexpressed C-terminal fragment (CTF) of TDP-43 in mammalian cellsMany known RNA chaperones contain RRM domains363739PARK2, SMYD3 and KCNIP4) are important for neuronal function and are down-regulated in sporadic ALS motor neurons harboring TDP-43 pathology, suggesting a shared mechanism of neurodegeneration caused by dysregulation of TDP-43 or FUS. As RBM45 and TDP-43 interactions are RNA-dependent, it suggests that RBM45 and TDP-43 share the regulation of specific RNA targets. Future studies are needed to identify the RNA targets bound by RBM45 to determine RNA targets co-regulated by RBM45 and TDP-43.Both TDP-43 and FUS bind pre-mRNA with long introns424344in vitro studies, future studies will determine the presence or absence of this alternative protein isoform in ALS and other neurologic diseases.Although RBM45 is a nuclear protein, overexpressed wild-type RBM45 can form cytoplasmic granules that resemble the inclusions observed in ALS/FTLD patient tissues. Disruption of the RBM45 NLS and cellular stress further exacerbate cytoplasmic accumulation and lead to increased granule formation that co-localize with TDP-43 and stress granule marker TIAR . In cell713We have identified structural domains in RBM45 that contribute to its function, subcellular localization, homo-oligomerization and its interactions with the ALS-associated proteins TDP-43 and FUS. Homo-oligomerization of RBM45 is mediated by a novel HOA domain that is conserved across species and important for its protein-protein interactions and association with cytoplasmic stress granules. These results provide new insights into the role that protein assemblies play in the normal and pathological functions of RNA-binding proteins and provide further evidence of the association of RBM45 and TDP-43 in both the nucleus and cytoplasm, a phenomenon first observed in cytoplasmic inclusions in ALS/FTLD patients.The plasmids of RBM45, TDP-43 and FUS cDNA clones, cGST-hRBM45 (HsCD00356971), pDONR221-TDP-43 (HsCD00079870), and pDNR-Dual-FUS (HsCD00002882), were obtained from the DNASU Plasmid Repository at Arizona State University, Tempe. The cDNA was amplified by PCR using Phusion High-Fidelity DNA Polymerase (NEB) and sub-cloned into the pcDNA3 vector (Invitrogen). The 3xFLAG tag (DYKDHDGDYKDHDIDYKDDDDK) or 2xHA tag (DYPYDVPDYAGGAAYPYDVPDYA) was appended to the N-terminus of specific proteins to generate the 3xFLAG- or 2xHA-tagged construct. The EGFP-RBM45 construct was generated by inserting the EGFP gene into the N-terminus of RBM45 gene in pcDNA3. The mutations were introduced by site-directed mutagenesis using overlap extension PCR2. SHSY5Y cells were cultured in DMEM/F12 medium with 15% FBS, 1% non-essential amino acids and 1% Pen-Strep at 37 °C with 5% CO2. Transfection was performed using the Lipofectamine 2000 (Life technologies) and transfected cells were harvested 48 or 72 hours post-transfection. Stable cell lines were selected in the presence of 500 μg/ml G418 (Life Technologies).HEK293 , Neuro2A (ATCC) and SK-N-SH (Sigma) cells were cultured in DMEM medium with 10% FBS and 1% Pen-Strep at 37 °C with 5% COCells were grown on 6-well plates till confluent and harvested. The cell pellet was gently resuspended in 150 μl cytoplasmic extraction buffer  and incubated on ice for 5 min. The cells were spun at 600 × g for 4 min at 4 °C and the supernatant was transferred to a new tube as crude cytoplasmic fraction (~150 μl). The pellet was gently washed with 100 μl cytoplasmic extraction buffer without NP40 and spun at 600 × g at 4 °C for 4 min. The supernatant was discarded and the pellet was resuspended with 150 μl nuclear extraction buffer  and incubated at 4 °C with rotation for 15 min. The nuclear extract and the crude cytoplasmic fraction were cleared by centrifugation at 16,000 × g at 4 °C for 10 min. Equal volume of the nuclear and cytoplasmic fractions were analyzed by SDS-PAGE gel and immunoblot. The band intensity was quantified with Image Studio software (LiCOR) and the relative abundance of the nuclear RBM45 in total RBM45 was calculated as [(nuclear fraction)/(nuclear fraction + cytoplasmic fraction)] × 100%. Statistical analysis was performed using two-tailed paired t-test.Cells were grown on Poly-D-Lysine coated coverslips to 60% confluent. Fixation was performed with 4% paraformaldehyde at room temperature for 15 min, followed by permeabilization with 0.2% Triton at room temperature for 10 min. Cells were blocked with Super Block (ScyTek) at room temperature for 1 hour. Primary antibodies see  were dilCells were fixed, permeabilized, and blocked as described above. A pair of primary antibodies see  producedProtein samples were mixed with 4× SDS loading buffer and heated at 90 °C for 7 min, resolved on the Bolt 4–12% Bis-Tris Plus Gel (Life Technologies) or 3–8% Tris-Acetate Gel (Life Technologies), and transferred to Immobilon-FL PVDF membrane (Millipore). The membranes were blocked with Odyssey Blocking Buffer (LiCOR) for 1 hr. The antibodies see  were dilDisuccinimidyl suberate (DSS) in-cell crosslinking was performed as previously described28Formaldehyde in-cell crosslinking was performed prior to immunoprecipitation. Formaldehyde is a mild and reversible crosslinker with very short spacer length (2.3–2.7 Å) and cross-links only closely associated proteinsCells were lysed with NP40 lysis buffer  at 4 °C for 15 min. The lysates were cleared by spinning at maximum speed at 4 °C for 10 min and subsequently used for IP. FLAG-IP was performed using anti-FLAG M2 Affinity Gel (Sigma A2220). IgG-IP control was performed using Mouse IgG-Agarose (Sigma A0919). IP was performed at 4 °C for 2 hr and the beads were washed in NP40 lysis buffer for six times. The proteins were eluted with SDS sample buffer and heated at 95 °C for 20 min to reverse formaldehyde crosslinking. For fractionation-IP, the KCl concentration in the cytoplasmic fraction was adjusted from 60 mM to 430 mM to approximate the salt concentration in the nuclear extract.Freshly harvested cells were permeabilized with 100 μl PBS containing 0.15% Triton, 3 μl RNase A (Sigma R4642) and proteinase inhibitor for 10 min at room temperature. 400 μl 0.25% formaldehyde/PBS was added to the 100 μl permeabilized mixture so that the final formaldehyde concentration is 0.1%. The subsequent crosslinking and immunoprecipitation were performed as described above.The RBM45 sequences from human (NP_694453.2), chimpanzee (XP_515938.2), bovine (AAI05263.2), mouse (AAH57890.1), rat (NP_695218.1), chicken (NP_001026423.1), lizard (XP_008116425.1), African clawed frog (NP_001080090.1), rainbow trout (CDQ81595.1), zebrafish (NP_001120874.1), sea urchin (XP_785181.2), honey bee (XP_006566988.1), fruit fly (AAF47879.2), and mosquito (XP_315504.4) were aligned with ClustalW algorithm using the BioEdit software.How to cite this article: Li, Y. et al. RBM45 homo-oligomerization mediates association with ALS-linked proteins and stress granules. Sci. Rep.5, 14262; doi: 10.1038/srep14262 (2015).


Here, NiFe2O4 nanoparticles and nanohybrids using large-diameter graphene oxide (GO) sheets are prepared via solvothermal synthesis for low-frequency wide bandwidth shielding (L and S radar bands). The synthesized materials were characterized using XRD, SEM, FTIR and microwave magneto dielectric spectroscopy. The dimension of these solvothermally synthesized pristine particles and hybrids lies within 30–58 nm. Microwave magneto-dielectric spectroscopy was performed in the low-frequency region in the 1 MHz-3 GHz spectrum. The as-synthesized pristine nanoparticles and hybrids were found to be highly absorbing for microwaves throughout the L and S radar bands (< −10 dB from 1 MHz to 3 GHz). This excellent microwave absorbing property induced by graphene sheet coupling shows application of these materials with absorption bandwidth which is tailored such that these could be used for low frequency. Previously, these were used for high frequency absorptions  with limited selective bandwidth.The magnetic nanoparticle composite NiFe The role of multifunctional materials as absorbers is significant in both antenna engineering and electromagnetic interference shielding/radar absorption. Several strategies have been used to minimize the effective radar cross section and decrease the reflection and emissions as well ,2. SurfaMicrowave absorber materials are characterized normally based on the absorption mechanism, i.e., dielectric absorbers, magnetic absorber and hybrid materials . Dielect2O4,have been widely studied for its high microwave absorption [2O4or its composite with rare earth elements; polymers; carbon nanostructures, especially CNTs; and reduced graphene oxide (rGO) for high microwave frequencies (>4 GHz) [2O4is its limited absorption bandwidth and selective absorption frequencies [2O4nanoparticles were prepared via novel modified solvothermal route and the influence of large-diameter rGO addition is analyzed by using in situ reduction and hybrid formulation utilizing the benefits of solvothermal treatment. Their magneto-dielectric properties were tailored with an aim to get high microwave absorption in the low-frequency region (mixed L and S bands) that can cover their entire bandwidth. Here, in this research work, NiFe2O4-rGO hybrid absorbers were prepared that showed excellent microwave absorption in the low-frequency region covering the entire UHF/L/S bandwidth instead of selective frequency resonances.Soft ferrites, especially NiFesorption . Most of(>4 GHz) ,12. Addiquencies –15. Cons4 in 400ml acidic mixture (320ml:80ml H2SO4:H3PO4). The reaction mixture was stirred rapidly and continuously until the color of solution turned brown. After ceasing the oxidation, the suspension containing highly oxidized flake graphite was washed multiple times with 5M HCl and double-distilled deionized water. Nanocrystals of NiFe2O4 were synthesized via modified solvothermal approach using ethanol and DI in a definite proportion[2O4. Thesame method was carried out for hybrid preparation using a different weight percentage of GO powder [Graphene oxide was used as a source of graphene sheets through modified solvothermal reduction process. All the chemicals used as precursors at any stage were of AR grade. Graphene oxide was synthesized using a simplified Hummer method;typicallroportion. In a tyO powder .2O4 nanoparticles and the NiFe2O4-rGO hybrid to analyze the structure of the synthesized nanopowder. 2O4 were fccstructurednanoferrites. Their structure fully matches with powder diffraction file JCPDS 10–0325. Similarly, the XRD pattern of hybrid samples NFG5, NFG7 and NFG10also agreed well with the structure of NiFe2O4. Moreover, there was no peak of graphene oxide in all the three synthesized composite samples. The absence of the characteristic peak (001) associated with graphene oxide in samples shows the complete reduction under solvothermal condition mentioned in experimental synthesis.X-ray diffraction (XRD) was carried out for both the NiFe2O4and NiFe2O4-graphene composite samples NG5, NG7 and NG10, along with their chemical compositions. 2O4to graft on. The composition of graphene oxide analyzed through EDX shows that the synthesized graphene sheets have high oxygen content (around 42%) attached with individual carbon layers. 2O4and NFG5, NFG7 and NFG10. The SEM micrographs of NiFe2O4show that the synthesized nanoparticles via solvothermal medium are spherical and have grown in a leaflike pattern with a uniform size distribution, as shown in 2O4hybrid samples i.e. Scanning electron microscopy (SEM) coupled with energy dispersive x-ray spectroscopy (EDX) was used to analyze the microstructures, mainly the sizes, shapes, and distributions, and the oxidation contents of as-synthesized graphene oxide sheets, NiFe2O4 nanoparticles, graphene oxide powder and the hybrid NFG10. It is clear from the transmission spectra of NiFe2O4 nanoparticles that two majors peaks around the wave numbers 384 and 587 cm−1 correspond to the ν1 and ν2 bands, respectively, which are typical of ferrites[−1 corresponds to octahedral Ni-O stretching vibrations, while the bond lying around 587cm−1 shows the stretching vibrations due to tetrahedral Fe-O. The peak at 1110and 1383cm−1 are the result of asymmetric stretching vibrations of CO32−/NO3− adsorbed on the ferrite matrix or of the impurities from reaction precursors[−1 and at 1609cm−1 arethe result of O-H stretching and bending vibrations attached with iron ions and of moisture adsorbed on ferrite nanoparticles[−1. The peak at 1107cm−1 shows the in-plane bending vibrations of C-H bonds. The band around 1636 cm−1 shows the C = O stretching vibrations of the oxidized carbon backbones. The peak at 1386 cm−1 shows the C-OH stretching vibrations. The broad peak-like feature shown at 3446 cm−1 shows the presence of moisture in graphene oxide powder[−1. The major peak at 1571cm−1 shows the vibrations from oxygen-depleted carbon lattice showing the fair reduction process during solvothermal conditions. The peaks at 1224and 1382cm−1 appear because of CO32− in the composite sample[Fourier transform infrared spectroscopy (FTIR) was performed to analyze and confirm the bonding behavior, functionalization of graphene lattice with nanoferrite crystals and reduction of graphene oxide in composite.  ferrites. The banrecursors. The banparticles,23,24. Ide powder. In the te sample,24. Abser), respectively; μ′ and μ″ are the real and imaginary parts of complex permeability μr, respectively. 2O4 and hybrid samples NFG5, NFG7 and NFG10. 2O4, the real parts of permittivity are 4.47, 4.82, 5.57 and 8.12 at 10MHz and these decreases to 1.61, 2.19, 1.65 and 2.24 at 1GHz for NiFe2O4, NFG5, NFG7 and NFG10, respectively. Magnetic losses are smaller than the dielectric losses in all the samples because of the dielectric polarization, which shows that the higher contribution is from dielectric loss along with the magnetic loss.It is clear from the pattern of the Microwave magneto-dielectric properties usually depend on a large number of factors such asthemethod of synthesizing graphene and pristine nanoferrite particles, their attachment to carbon lattice, defects in nature and distribution, sizes and shapes of graphene sheets and nanoparticles, edge structure or chirality, the nature of dielectric relaxation and the motion of electrons,27,29–312O4, NFG5, NFG7 and NFG10 are below −10dB for the entire range of frequency from 10MHz to 3GHz. There are two absorption vortexesthat appear around 200 MHz and 900 MHz for pristine NiFe2O4 nanoparticles. With the increase in graphene concentration, the vortexes for all of samples radiate away from the center absorption frequencies of pure Ni-ferrites synthesized through modified solvothermal route, with lower vortex shifting to 100, 72 and 50 MHZ and higher vortex to 1.11, 1.36 and 1.46 GHz for NFG5, NFG7 and NFG10, respectively. The results of microwave absorption shows the nanoparticles of NiFe2O4and their composites show ultrahigh performance with absorber thickness around 2 mm in the vast frequency range of 10 MHz-3 GHz as compared to the already reported absorption with high absorber thicknesses of typically 10 mm and at high frequency (>4 GHz) [It is clear from (>4 GHz) –15,27.The dielectric permittivity shows the dipolar and electric polarization at the microwave spectrum and with the increases in reduced graphene oxide percentage in hybrid sample and the strained lattice causes change in electric and dipolar polarization. The change in dielectric properties can also be explained by hopping between Fe and Fe & spin/charge polarization . In case2O4anchored with graphene sheets. This type of hybrid multilayer stacking thereby presents a continuous path for the incident energy of microwave to be absorbed via repeated magneto-dielectric losses coupled with the nanohybrid system. The reason of absorption peaks in reflection losses are mainly due to resonance phenomenon and charge hopping in ferrites based samples i.e. domain wall resonance and defects polarization centers and its coupling. [This unusually high absorption and large absorption bandwidth can be attributed to the low dimension and uniform dispersion of nanoparticles of NiFeoupling. ,35. Thes2O4and its composites with large-diameter rGO sheets were prepared via solvothermal synthesis in mixed ethanol/DI media with an average particle size in the range of 30–58 nm. Microwave materials analysis shows that the both the NiFe2O4and the composite with rGO sheets show high absorbance (−10 dB) of microwave from 1 MHz to 3 GHz with absorption bandwidth comprising whole L and S bands instead of usual selective absorptions of microwaves. The maximum reflection loss was −68 dB at 1.11 GHz.NiFeS1 Fig2O4, NFG5, NFG7 and NFG10.Microwave magneto-dielectric properties (a) real part (b) imaginary part of complex permittivity (c) real (d) imaginary parts of complex permeability of NiFe(TIF)Click here for additional data file.


The PNA molecule, if used parallel with hydrolysis probe in QPCR analysis, greatly facilitates the detection efficiency of MSRV even if ERVWE1 is present abundantly in respect to MSRV in the analysed sample. We achieved a wide and measurable range from 1 × 10 e2 to 1 × 10 e8 copies/reaction; the linearity of the technique was maintained even at the low MSRV level of 1 % in respect to ERVWE1. Using our newly developed method we confirmed that the expression of MSRV takes place in normal human astrocytes and in human umbilical vein endothelial cells in vitro. We also found that the stimulation of human monocytes did not influence the specific expression of MSRV but it caused changes in mRNA level of distinct HERV-W templates.HERV-W is a multi-locus family of human endogenous retroviruses (HERVs) that has been found to play an important role in human physiology and pathology. Two particular members of HERV-W family are of special interests: ERVWE1  and MSRV (multiple sclerosis-associated retrovirus that is thought to play a significant role in human pathology as a result of its increased expression in the brain tissue and blood cells derived from patients with multiple sclerosis (MS)). Both ERVWE1 and MSRV mRNA share high level of similarity and hence a method that allows to exclusively quantify the MSRV expression in clinical samples would be desirable. We developed a quantitative polymerase chain reaction (QPCR) technique for the detection and quantification of the multiple sclerosis-associated retrovirus. The assay utilises fluorescently labelled oligonucleotide probe, which is complementary to the conservative fragment of MSRV  Accordigression . Brudek ividuals . Anotherividuals . Both muilarity) . On the  in 81 % , 11.Because an increased level of HERV-W RNA has been reported in brain tissue from MS patients , a preciPeptide nucleic acid (PNA) is a DNA mimicking synthetic oligonucleotide with peptide back-bones instead of sugar groups and was first reported in 1991 by Nielsen et al. from the University of Copenhagen . By compAs mentioned above, PNA-mediated PCR clamping method is especially desired if DNA of interest is present in a very low amount relative to the wild type one . This sienv mRNA copy number differed in respect of the presence or absence of the ERVWE1-specific PNA, therefore we concluded that MSRV is present predominantly over syncytin-1 in human astrocytic U-87 MG cell line. Additionally, compared to the intercalating dyes (e.g. SYBR Green I), our novel method incorporated the specific fluorescent probe that is much more suitable for virology/microbiology experiments, due to higher pathogen-specificity of the procedure primers, the utilisation of a third sequence-specific element, such as oligonucleotide probe is preferred over an intercalating dye in the virology/microbiology fields due to higher pathogen-specificity of the method. In the probe-based chemistry, both primers and probe hybridise to the common target, thus as much as three events must occur independently to give a specific fluorescent signal [In our previous report, we described a real-Time polymerase chain reaction/PNA-mediated methodology that allows the specific quantitative amplification of multiple sclerosis- associated retrovirus (MSRV). The assay is based on two sequence-specific oligonucleotides (PCR primers) that are common for both MSRV and ERVWE1 gene fragment, an ERVWE1- specific PNA probe and SYBR Green 1 intercalating dye that was essential in the quantitative assessment. Those results showed that it is possible to estimate the MSRV level in a biological sample if the ERVWE1 template is excluded from the reaction by means of PNA-mediated PCR clamping. According to our results with the PNA inhibition of ERVWE1, we showed that the overall expression of HERV-W was similar in samples treated with PNA or without PNA clamping, therefore we concluded that the expression of MSRV is predominant over syncytin-1 in human astrocytic U-87 MG cell line. Because the calculated HERV-W t signal .env sequences were derived from the GenBank database according to previously published data [env sequences denoted as MSRV with four env sequences denoted as coding for syncytin-1. Accession numbers of the investigated entries were as follows: AF331500, AF127229, AF072498 for MSRV and AY101582, AF513360, AF208161 and NM_014590 for syncytin-1. In both sequence types, a consensus sequence was built using CONS software from the EMBOSS package [HERV-W hed data , 11. We Peptide nucleic acids, 15-nucleotides in length, were synthesised and purified at the Department of Chemistry, University of Gdańsk, Poland (please see “Synthesis of peptide nucleic acids complementary to ERVWE1” caption for details). Lyophilised PNA was resolved in 5 % DMSO and left for 20 min at room temperature (RT) to obtain a stock concentration of 10 μM. PNA was then aliquoted and stored at −20 °C.env gene sequence are shown in Fig. Oligonucleotide primers for polymerase chain reaction were designed according to previously published guidelines . Three rTotal RNA was extracted from normal human astrocytes (NHA) cell line , human umbilical vein endothelial cells (HUVECs) . Peripheral blood monocytic cells (PBMCs) were obtained after centrifugation on blood in the Histopaque . Afterwards monocytes were isolated from the PBMCs by negative immunomagnetic separation using Pan-T and Pan-B Dynabeads  . Monocyt2, 2.0 μl of 10 mM dNTP mixture, 0.5 μl of RNasin Ribonuclease Inhibitor, 15 units of AMV Reverse Transcriptase and 0.5 μg of Random hexamers. Reaction mixture was incubated for 10 min at room temperature and then for 30 min at 42 °C followed by thermal inactivation of reverse transcriptase at 95 °C for 5 min. According to the manufacturer’s recommendations, reverse transcription reaction mixture was further diluted 1:4 with nuclease-free water before the use in QPCR. Five μl of diluted RT reaction mixture  was then used as a template for subsequent QPCR analysis.For NHA and HUVECs, 3 ml of TRI reagent  was used to lyse cells from one 10 mm in diameter tissue culture plate after the >80 %-confluence was achieved. Before TRI reagent was added, culture medium was removed but without washing the cells with PBS. Monocyte cultures were lysed using one ml of TRI reagent, which was added directly into wells of a 24-well culture plate after removing the culture medium. Three wells were then pooled together. After cell lysis and complete disruption of nucleoprotein complexes, total RNA was precipitated and further purified according to the one-step extraction method described by Chomczynski et al. . FinallyIn our assay, we used random hexamers as primers for the reverse transcriptase enzyme. As it is stated in Promega’s instruction for use of their product # A3500 “Choose Oligo(dT)15 when priming at the 3′poly(A) region is desired. Choose Random Primers when priming throughout the length of the RNA is desired”.15 and random primers may, in theory, improve the quality of cDNA pool. However, the use of oligo(dT)15 concomitantly with random hexamers/nonamers is problematic because both solutions require slightly different thermal conditions and finally the resulting cDNA library may not be more representative than when using random primers alone.It should be emphasised that the choice of optimal RT priming is crucial to get the most representative cDNA pool. Human endogenous retroviral-derived particles make even more complex issue because they are broadly dispersed thorough the genome and are mostly truncated; e.g. a significant part of HERV-W possesses a poly-A tail (such as syncytin-1) while other retroviral members does not. If so, a mixture of oligo(dT)For cloning purposes, DNA extract obtained from human placenta was used .Polymerase chain reaction was performed using Qiagen Taq PCR Master Mix kit . The reaction mixture in a total volume of 25 μl consisted of 12.5 μl of 2× Qiagen PCR Master Mix, 200 nM of each Syncytin_out_F and Syncytin_out_R primer and 0.5 μl of placental DNA extract as a template. After reaction, amplicons were resolved by means of agarose gel electrophoresis, excised from the gel and extracted/purified using silica-based spin columns . Four separately purified amplimers were then sequenced . BLAST analysis of obtained sequences confirmed that the specific amplification of ERVWE1 locus took place, giving a 100 % similarity to the reference sequence (GenBank: NM_014590). For cloning purposes, PCR reaction was repeated at identical conditions as described above, but less error-prone Pfu polymerase was used instead of less accurate Taq polymerase . Furthermore, Pfu polymerase generated blunt-ended amplicons that were essential for the subsequent blunt-end cloning procedure.4, 200 nM of each deoxyribonucleotide, 1 U of Pfu polymerase and of 25 pmol of each syncytin_mut_F and a mutagenic_MRSV_R primer .Both ERVWE1–derived and mutated MSRV-like PCR products were cloned into pSC-B-amp/kan plasmid using StrataClone Ultra Blunt PCR Cloning Kit according to the manufacturer’s guide . Mini preps were prepared in 3.5 ml of Luria Broth medium and were purified by NucleoSpin Plasmid kit . Obtained plasmids were given an initial examination to confirm the presence of the insert (regardless of their orientation) by PCR with a plasmid-specific universal T3/T7 primer pair. Finally, pSC-B-MSRV and pSC-B-ERVWE1 positive clones were sequenced in both directions by means of dideoxy-sequencing with the use of insert-specific primers, i.e. Syncytin_out_F and Syncytin_out_R . The mini-prep extracts were quantitatively measured using SYBR Green 1 fluorescent dye  against the known amounts of lambda DNA as a reference  in a fluorometer . The excitation wavelength of the dye was 495 nm, and emission was filtered using a 520-nm barrier filter. Plasmid DNA copy number was then calculated using dsDNA copy number calculator  and filtered with a 0.22-μm syringe filter before use. Fmoc-XAL-PEG-PS resin for the PNA synthesis was obtained from Merck KGaA . Fmoc/Bhoc-protected PNA monomers were purchased from Panagene . DNA oligonucleotides were obtained from Future Synthesis . The other reagents and solvents were obtained from Sigma-Aldrich Co. .PNA sequence: TACCAGTTTGGGTGA-amide complementary to ERVWE1 was synthesised using a Labortec AG SP-650 Peptide Synthesizer on a 3.8 µmol scale using a 2.5-fold molar excess of the Fmoc/Bhoc-protected monomers and Fmoc-XAL-PEG-PS resin (amine groups loading of 0.19 mmol/g) . MonomerThe crude oligomer obtained was lyophilised and purified by semi-preparative reverse phase, high performance liquid chromatography RP-HPLC using a Knauer system with a Kromasil C8 column  . The mobile phase consisted of 0.08 % TFA in acetonitrile (solvent A) and 0.1 % TFA in water (solvent B). The gradient profile for the mobile phase was as 0–30 % of solvent A held for 90 min. The column was maintained at ambient temperature. The flow rate was 4.5 ml/min, and the eluate was monitored with a UV detector at 254 nm. Fractions, which had purity greater than 98 % were collected and lyophilised.All analytical RP-HPLC analyses were performed on a Phenomenex Kinetex XB-C18 column   using a Beckman Gold System. The mobile phase consisted of 0.08 % TFA in acetonitrile (solvent A) and 0.1 % TFA in water (solvent B). The gradient profile for the mobile phase was as 0–30 % of solvent A held for 30 min. The column was maintained at ambient temperature. The flow rate was 1 ml/min, and the eluate was monitored with a UV detector at 254 nm.Finally, PNA was characterised by MALDI-TOF mass spectrometry , which confirmed the identity of the product synthesised.The specificity of PNA hybridisation to the complementary ERVWE1 ssDNA fragment and not complementary MRSV ssDNA fragment was analyzed and confirmed by capillary electrophoresis . Separat5 copies of pSC-B-ERVWE1 and of pSC-B-MRSV plasmids were subjected, in parallel, as the template into polymerase chain reactions. Reaction mixtures contained 1× Qiagen PCR Master Mix, 200 nM of Syncytin_out_F and 200nM of an appropriate reverse primer . A hydrolysis probe, complementary to MSRV but not to ERVWE-1 gene fragment was designed using EPRIMER3 software available from the EMBOSS package . The tar®-1, BHQ-1) at the 5′ and 3′ ends, respectively. The reporter dye’s emission is quenched when the probe remains intact. Only if the probe hybridised with its complementary target (i.e. MSRV env gene), the fluorescence of a reporter dye becomes detectable due to 5′ → 3′ exonuclease activity of Taq polymerase. In the case of MSRV, the hybridisation process of the probe is not impeded by PNA because of its non-complementarity to MSRV sequence , 2.5 mM MgCl2, 200 nM of each Syncytin_out_F forward- and sync1_REV/-1 reverse primer, 400nM of MSRV-complementary hydrolysis probe, 300nM of PNA (or an appropriate volume of 5 % DMSO).The hydrolysis probe used in the assay possessed a reporter dye  and a non-fluorescent quencher  beforehand were used for tenfold serial dilutions from 1 × 10 eIn our previous experiments, we have documented that the amplification of ERVWE1 gene fragment by means of polymerase chain reaction (PCR) can be specifically inhibited by complementary peptide nucleic acids (PNA) while no inhibition occurred when MSRV clones were used as the template. Furthermore, we also found that slight differences in PNA design or in primers localization altered drastically the efficiency of PCR blocking. A PNA probe, 14-monomers in length that hybridise at the position 1370–1383 in respect to the reference gene (GenBank: NM_014590) efficiently clamped the amplification of ERVWE1 at the concentration of at least 4 μM while we noted that its longer analogue  worked effectively even at tenfold lower concentration of 400nM or less  by the competition for a common target site between a PNA and one of the PCR primers; (2) by the interference of primer elongation and/or 3) by the product elongation arrest. According to our observations, primer elongation stoppage was the only efficient way to inhibit the reaction. Neither the competition between PNA and one of the PCR primer for a common target nor the product elongation arrest was efficient to inhibit the reaction even at the final PNA concentration of as much as 10 μM. Even in the case of an inhibition caused by primer elongation stoppage, the efficiency of the clamping was strongly influenced by the exact reciprocal primer-to-PNA localization . If PNA was not present in the reaction, the target MSRV sequence was correctly measured only at the highest concentration of the MSRV compared to ERVWE1 and an estimated molecule number was similar to that theoretically calculated only in these experimental conditions. Measurements were underestimated when the concentration of MSRV template was lower  and in human umbilical vein endothelial cells (HUVECs) cultured in vitro. We also examined whether the MSRV expression level changed after the lipopolysaccharide (LPS) - induced activation of human monocytes in vitro.3 ± 2.88 × 10 e2 and 7.47 × 10 e2 ± 3.4 × 10 e1 mRNA copies number/μg RNA, respectively. For beta actin gene expression, the results were as follow: 1.29 × 10 e5 ± 1.54 × 10 e4 and 2.52 × 10 e4 ± 1.4 × 10 e3 mRNA copies per one microgram of total RNA, respectively. As we expected, the measurement values of MSRV mRNA copy number change if ERVWE1- specific PNA was added into the QPCR reaction mixture simultaneously with MSRV-specific fluorescent probe. In the control reactions where no PNA was added but only probe was present, the obtained results were lower in comparison with that after the addition of PNA  . Interestingly, an inhibitory effect of LPS on the expression of HERV-W was observed in samples without PNA against ERVWE1 , indicating that LPS may influence the ratio between MSRV and HERV-W env expression in human monocytes, especially by the diminishing of non-MSRV-derived HERV-W copy number  did not alter the expression of MSRV because no differences in HERV-W expression were observed after addition of PNA into the reaction  patients provided by Mameli et al. showed that envelope gene intracellular domain of MSRV and ERVWE1 (syncytin-1) RNA share >89 % similarity while whole envelope gene showed >93 % similarity in both MSRV and ERVWE1.env and syncytin-1 gave rise to speculations regarding their possible physiologic and/or pathogenic effects. A major difference is that only MSRV has been detected as an extracellular virus  RNA containing terminal repeats, gag, pol and env sequences) while the syncytin-1 protein has been found only intracellularly or on the plasma membrane [The homology between MSRV membrane .env and syncytin-1 on the protein level. Thus, any results based on immunostaining or Western blot could reflect either MSRV env or syncytin-1 expression, or both.As a result of high similarity at the protein level, no specific antibody for unique HERV-W family members has been identified so far and it is not possible to discriminate between MSRV env and syncytin-1 in research assays is to utilise PCR, reverse transcriptase (RT) PCR and/or real-time PCR assays that could selectively amplify either MSRV or syncytin-1 mRNA sequences. Reliability of the assays with respect to specificity, sensitivity and inter- and intra-assay variations has been published previously [env sequences harbour a 12-nucleotide insertion in the transmembrane moiety. Based on this insertion, discriminatory real-time PCR assay was developed that can selectively amplify either MSRV env or ERVWE1. The selectivity of the method was achieved by the use of allele-specific primer pairs and appropriate probes, TaqMan-type for MSRV and ERVWE1 and locked nucleic acid (LNA) probe for the generic HERV-W env [Another approach to distinguish between the expression of MSRV eviously . With reRV-W env .env-encoding DNA and RNA exhibited statistically significant increased detection and expression in the brains of MS patients in comparison with control. Moreover, ERVWE1 env transcripts were inducible in glial cells as well. Conversely, such changes in MSRV expression were not reported in that work [A similar approach was demonstrated by Antony et al., where authors employed a real-time PCR approach using MRSV or syncytin-1-selective oligonucleotide primers and fluorescent, intercalating SYBR Green I dye which allowed the quantitative analysis of gene expression in brain tissue samples, peripheral blood leukocytes (PBLs) and monocyte-derived macrophages (MDM) from multiple sclerosis (MS) and non-MS patients as well as in an astrocytic U373 cell line. Analyses provided by Antony et al. revealed that ERVWE1 hat work .2 etc.) may result in possible primers-to-template mishybridisation and, subsequently, to the synthesis of unspecific amplimers [Due to an error-prone nature of Taq polymerase, a reverse transcriptase-quantitative polymerase chain reaction based on intercalating dyes is not recommended for the detection and quantification of mutant nucleic acids, especially when slight differences among investigated sequences are expected . Any suboptimal reaction conditions  and generates fluorescent signal, which theoretically is proportional to the number of double-stranded DNA molecules that are produced during PCR. This dye is suitable in most typical, quantitative, nucleic acid- based assays. On the contrary, if the reaction conditions are not optimal and/or if any double-stranded DNA molecules other than that from desired template arise in the reaction mixture, SYBR Green I gives inappropriate, false-positive results. Indeed, in respect to HERV-W research, Garson et al. in his Letter to the Editor of “AIDS Research and Human Retroviruses” journal ascertain some technical flaws in the real-time PCRs previously employed in the studies performed by Antony et al. , 39. ThuPeptide nucleic acid (PNA) is an artificially synthesised polymer that has properties of both nucleic acids and proteins. Importantly, PNA binds to the complementary sequence of nucleic acid with much stronger binding capacity than DNA does, because of the lack of electrostatic repulsion.Thus, in our assay, PNA oligomer is intended to bind specifically to the cDNA of the ERVWE1 locus and prevent them from being amplified by polymerase action. Our methodology allows the detection and measurement of discrete MSRV expression level due to MSRV-specific fluorescent oligonucleotide. What is more, any ERVWE1- derived templates are excluded because of their specific clamping by PNA. These factors improve greatly the analysis of MSRV quantity even if it is present in minority in respect to other similar molecules.In our previously published data, we found that SYBR Green I-based Real-Time PCR and its specific clamping by ERVWE-1-specific PNA favour the phenomenon in which virtually all “non-ERVWE1-derived” amplimers could be synthesised. Because only two HERV-W members, namely MSRV and ERVWE1 comprise the majority of HERV-W expression products at all, the rest of amplimers that arose and that are quantified in course of the Real-Time PCR are, in theory, considered as being of MSRV origin. However, any possible expression from distinct HERV-W loci cannot be excluded. The principle of an assay changes if non-specific SYBR Green-I dye is replaced by a MSRV-specific oligonucleotide probe. Thus, the fluorescent signal source for the quantification of the results is derived exclusively from the amplimers synthesised on MSRV template due to the reciprocal, independent hybridisation of both primers and the probe. To sum up, PNA molecules play a role as “scavenger inhibitors” that alleviate the impact of substantial ERVWE-1 overload that could interfere with the reaction efficiency -specific PNA oligomer and MSRV-specific fluorescent probe. This technique is intended for the discrete detection and measurement of MSRV (multiple sclerosis-associated retrovirus) expression level in biological samples regardless of syncytin-1 expression from ERVWE-1 locus.


This enables a voltage responsivity as high as 23,000 mV mW−1 with a low-frequency input signal. Taking advantage of the output channels being orthogonal to the input terminals, the noise is found to be not strongly influenced by the input. Hence, the corresponding noise-equivalent power is as low as 0.64 pW Hz−1/2. Such performance is even comparable to superconducting bolometers, which however need to operate at cryogenic temperatures. Furthermore, output oscillations are observed at low temperatures, the period of which agrees with the lateral size quantization.Although graphene has the longest mean free path of carriers of any known electronic material, very few novel devices have been reported to harness this extraordinary property. Here we demonstrate a ballistic nano-rectifier fabricated by creating an asymmetric cross-junction in single-layer graphene sandwiched between boron nitride flakes. A mobility ∼200,000 cm The high mobility of graphene has the potential to enable design of devices working in a collisionless regime of carrier transport. Here the authors fabricate a ballistic nano-rectifier based on encapsulated graphene, showing intrinsic performances comparable to those of superconducting bolometers. Despite this, even the most promising applications to commercialize graphene in the electronics industry do not take advantage of this property. Instead, they often attempt to tackle the problem that graphene has no bandgap by using, for example, vertical tunnelling transistorsλMFP. This means that the carriers would be more likely to scatter off the edges in the device than off randomly distributed phonons or defects. Hence, a simplistic billiard ball model could be used for the transport of carriers through the device. One such planar, ballistic device that can take advantage of graphene is the ballistic rectifier (BR), which is an asymmetric four-terminal cross-junction that relies on the ‘billiard ball' motion of carriers to rectify a.c. signals into d.c. outputs582, which leaves charge puddles in the graphene that scatter the carriers and lower the mobility to about 1,810 cm2 V−1 s−1, making the device not truly in the ballistic regime. This resulted in a responsivity of 110 mV mW−1 and a noise-equivalent power (NEP) of 10−9 W Hz−1/2  the neutrality point. It is useful to first check if the voltage output from these devices could be a result of the Seebeck effect. The Seebeck effect describes the process of hot carriers, travelling towards cool regions and it is one of the origins of thermo-electric effect. When the current is passed from S to D, the active region of the device will heat up due to carrier interactions with the lattice. In turn, the asymmetric nature of the device may create a temperature gradient in the vertical direction of the cross-junction and therefore a net voltage difference. This effect has been studied in graphene and other materials, and does produce a quadratic response13VLU, due to the asymmetric nature of the device. We have observed that the output is in fact negative (or positive) VLU. Therefore, any thermal rectification effect that may be present in our devices is at least less dominant than the ballistic rectifying effect at room temperature.VG=+5 V for the electrons and around VG=−8 V for the holes. The output further away from the neutrality point becomes lower, which is a direct result of increased carrier density and Fermi energy12VLU. Furthermore, the output is generally stronger in the electron transport region than for holes, which reflects the different carrier mobilities. PSD, which are in good agreement. The dependence is quite linear at low input power, as expected from the extended Büttiker–Landauer formalismJ∝Eα in the non-linear regime in graphene, where 1>α>1.5 and E is the electric field strength15VLU dependence on VG at a low-a.c. input of 1 μA at different temperatures. It can be seen that the device output in general increases as the temperature decreases, since the value of λMFP is larger at lower temperatures. When electrons are the majority carriers, the output shows more dependence on temperature than in the hole transport region, which may be a result of the different dependences in carrier mobility and mean free path with temperature as shown in VG is higher than +13 V or lower than −12 V. Both the Seebeck effect and ballistic rectifying effect are expected to present in the device, and the latter is known to weaken at high-gate voltages because of high carrier concentrations. So, the output reversal is likely due to the Seebeck effect that becomes more dominant at large gate biases14VLU/dVG against Fermi energy (EF) in E) fluctuates around a mean value of 15.1 meV. The lateral quantization fulfils k⊥WSD=πm, where k⊥ is the wave vector perpendicular to the direction of transport, WSD is the narrowest part of the injection leads and m is the number of conducting modesE=ħvFπ/WSD, where vF is the Fermi velocity (106 ms−1 in graphene). The average ΔE for this device predicts a WSD of 71 nm. The width is in the same order of magnitude as, but less than those measured with atomic-force micrograph, 122 nm for the narrowest region of the source lead and 137 nm for the drain. It is likely that the ballistically conducting region of the graphene does not extend all the way to the edge due to edge states and traps (Coulomb blockade and single-electron effect of graphene constrictions)n and hence One prominent feature that the ballistic model does not explain is the oscillations at 50 K. They may look like mesoscopic conductance fluctuations at first glance, but they are observed also at 150 K. Conductance fluctuations are typically washed out at 5 K ref. . Shubnik1920VG at room temperature by applying an a.c. to the device. The responsivity is determined by the output d.c. voltage divided by the input a.c. power (the a.c. voltage drop across S and D is measured simultaneously). The optimum intrinsic responsivity was measured to be 23,000 mV mW−1, which is to the best of our knowledge, one of the highest in diode detectors to date9252627282 had a responsivity of just 67 mV mW−1, >300 times lower2 V−1 s−1 and a responsivity ∼10,000 mV mW−1.One of the advantages of the BR is its high speed, which was measured up to 50 GHz and simulated at terahertz (THz) frequencies even when using a conventional semiconductor InGaAsf noise) and, in the case of a tunnelling diode, shot noise are both proportional to the applied current. Furthermore, the BR has an intrinsic zero threshold, and it hence does not need any biasing circuit that are not only troublesome at very high frequencies, but also may introduce significant parasitic elements. Operating at zero bias also greatly reduces the flicker noise, which often practically limits microwave detection capability of diode detectorsSV, as a function of frequency at different input currents. At zero input, the BR generated a white noise that is measured at 13.3 nV Hz−1/2. As RLU is around 15 kΩ when no gate bias was applied  can be as low as 2 kHz, which is significantly lower compared with normal detector diodesIt is useful to note that the corner frequency −1/2, which is more three orders magnitude better than the previous device made of low-mobility graphene on SiO2 without BN encapsulationThe minimum power that a microwave sensor can detect is determined by the NEP, which is given by the measured noise divided by the responsivity. In RSD≈22 kΩ, as shown in Currently, the BR still has a relatively large input resistance, To fabricate a graphene BR on an insulating substrate with a desirable carrier concentration for high-frequency applications, it is possible to use an insulating layer on top with a local gate to shift the Fermi energy. However, this will introduce some parasitic capacitance, and may hamper the high-frequency performance. Alternatively, it is possible to carry out chemical doping in various ways, such as using nitric acidet al.For certain applications, such as radio-astronomy or THz detectors, the cost of exfoliated devices might be acceptable. Devices based on chemical vapor deposition (CVD) graphene would significantly reduce the cost and therefore have a much wider range of applications. Currently, the carrier mobility in CVD graphene is generally much lower than exfoliated graphene, but it is improving constantly. The most recent paper by Banszerus −1/2 at room temperature, a value that is even comparable to superconducting bolometers, which however need to be cooled to cryogenic temperatures. The encapsulation of the device in BN layers also enables operation in atmospheric conditions and shows promise for a range of applications in high-frequency electronics.In summary, a very high responsivity has been achieved at low frequency in an asymmetric cross-junction by using graphene that has a carrier mobility close to the theoretical limit. Having an intrinsic zero threshold and being nearly immune to the noise of input current due to the orthogonal arrangement of the output and input channels, the BR exhibits very low flicker noise and the low-frequency noise is mainly limited by thermal noise. The thus obtained intrinsic noise-equivalent power is found to be as low as 0.64 pW Hz2 (ref. Graphene flakes for the device are fabricated using the exfoliation technique on 290 nm SiO2 ref.  and then2 ref.  ref. 1)2 .Supplementary Figures 1-4, Supplementary Note 1 and Supplementary References


The interaction between two different materials can present novel phenomena that are quite different from the physical properties observed when each material stands alone. Strong electronic correlations, such as magnetism and superconductivity, can be produced as the result of enhanced Coulomb interactions between electrons. Two-dimensional materials are powerful candidates to search for the novel phenomena because of the easiness of arranging them and modifying their properties accordingly. In this work, we report magnetic effects in graphene, a prototypical non-magnetic two-dimensional semi-metal, in the proximity with sulfur, a diamagnetic insulator. In contrast to the well-defined metallic behaviour of clean graphene, an energy gap develops at the Fermi energy for the graphene/sulfur compound with decreasing temperature. This is accompanied by a steep increase of the resistance, a sign change of the slope in the magneto-resistance between high and low fields, and magnetic hysteresis. A possible origin of the observed electronic and magnetic responses is discussed in terms of the onset of low-temperature magnetic ordering. These results provide intriguing insights on the search for novel quantum phases in graphene-based compounds. Due to the two-dimensional nature and Dirac fermionic behaviour of charge carriers, the electronic and magnetic properties of graphene can be easily modified, making it one of the most appealing materials for a variety of disparate applications1236789101113141516171819n-doped 6 H-SiC(0001) and undoped 4 H-SiC−6 Torr. The ampule is annealed at 230 °C in a furnace for 60 hours, while the pressure inside the ampule increased by vapourized sulfur was ~360 Torr. The presence of sulfur in the samples is confirmed by the observation of: a) sulfur 2 p core electrons in the photoemission spectra and b) Auger electrons corresponding to the sulfur LMM transition in the Auger electron spectroscopy (AES) spectraIn this work, we have combined two different but complementary probes such as angle-resolved photoemission spectroscopy (ARPES) to study the evolution of the graphene band structure upon sulfur introduction and magneto-transport to explore the electro-magnetic properties of the graphene/sulfur (G/S) system. Changes in the electronic structure are monitored by ARPES experiments. 2829ntensity , a quantntensity  that shiT is lowered, a clear depletion of states at T-dependence of G/S from G excludes the possibility of thermal smearing as the origin of the depletion of states at −1, which is far away from 0.05 Å−1 where the energy gap is observed  versus T-dependence observed at low temperatures is typical of disordered systems in the variable range hopping regime (VRH)4041T range.To gain better insight on the origin of the gap opening, we have performed magneto-transport measurements  and 4. Iwering T . Figure R versus external magnetic field H) for both samples. The most striking difference is the behaviour of the magneto-resistance at low fields, where its complete reversal is observed upon sulfur introduction. This reversal can be explained with a transition from a weak antilocalization regime to a weak localization regime as previously reported for pure graphene43R near zero field. While the magneto-resistance of the G sample (R upon increasing H) over the whole field range, the magneto-resistance of the G/S sample (R upon increasing H) at low fields, similar to the case of weak localizations. We note, however, that the crossover field is three orders of magnitude higher than the previous results for weak localizations, where the strength of localization is suppressed very quickly in the presence of magnetic fieldIn G sample  is positS sample  shows a H = 0 T reveals another surprising phenomena of G/S, magnetic hysteresis with coercive fields of 0.12 T and 0.02 T  exhibits spin-polarized states via the interaction with the buffer layer . The structural optimisation and spin-polarized calculation were performed.Calculations. How to cite this article: Hwang, C. et al. Magnetic effects in sulfur-decorated graphene. Sci. Rep.6, 21460; doi: 10.1038/srep21460 (2016).


In addition, the frequency of PD-L1 immunoreactivity was significantly higher in IL-6-positive esophageal cancer specimens. When IL-6 signaling was inhibited in vitro, the level of PD-L1 is significantly down-regulated. PD-L1 is a significant predictor for poor treatment response and shorter survival. As demonstrated through in vitro experiments, Irradiation increased PD-L1 expression in human esophageal cancer cells. The inhibition of T cell functions including proliferation and cytotoxicity against tumor cells might be the mechanisms responsible to the role of PD-L1 in radiation response. In conclusion, PD-L1 is important in determining the radiation response and could predict the prognosis of patients with esophageal SCC. Therefore, we suggest inhibition of PD-L1 as a potential strategy for the treatment of esophageal SCC.The aim of this study was to assess the significance of programmed cell death 1 ligand 1 (PD-L1) in esophageal squamous cell carcinoma (ESCC) and its association with IL-6 and radiation response. Weretrospectively enrolled 162 patients with ESCC, and examined the correlation between PD-L1 levels and clinical outcomes in esophageal cancer patients. Furthermore, the human esophageal SCC cell line CE81T and TE2 were selected for cellular experiments to investigate the role of PD-L1 in T cell functions and radiation response. Here we demonstrated that PD-L1 expression was significantly higher in esophageal cancer specimens than in non-malignant epithelium. In clinical outcome analysis, this staining of PD-L1 was positively linked to the clinical T4 stage ( Esophageal cancer is one of the most common types of human cancer and a difficult gastrointestinal tumor to treat and cure . Most paTumor-induced immune suppression in cancer patients is a major issue that not only promotes tumor progression but also inhibits the efficiency of anti-cancer treatment , 5. One Increasing evidence suggests that the inflammatory response can be rerouted into a tumor-promoting direction association with poor prognosis in various type of cancer, and proinflammatory cytokines could promote PD-L1 expression in tumor microenvironment –12. We pversus 50% (37/74) in T4, P=0.004). Furthermore, the positivity of PD-L1 was significantly associated with the risk of LN metastasis and developing loco-regional failure (Table The IHC data for TMA slides demonstrated that PD-L1 was over-expressed at the membrane or in the cytoplasm (or both) of tumor cells in the surgically resected tumor specimens compared to adjacent non-malignant epithelial tissues Figure . Figure p < 0.001). Given the positive association between IL-6 and PD-L1 expression in ESCC tumors, we examined the expression of PD-L1 in esophageal cancer cell lines whose IL-6 was regulated. Flow cytometric analysis and IF data revealed that IL-6 neutralizing antibody significantly decreased the level of PD-L1 expression at the cell surface and the cytoplasm  in non-responders, P<0.001). Furthermore, 47 among these patients received esophagectomy after neoadjuvant CCRT, PD-L1 staininig linked with lower complete pathologic response rate (pCR) (16% (3/18) in PD-L1(+) patients versus 31% (9/29) in PD-L1 (−) patients)). The role of PD-L1 in radioresistance and its underlying mechanisms were further examined in vitro. As shown in Figure For esophageal SCC, radiotherapy is a well-established therapeutic modality and provides survival benefits for responders. As shown in Table Table Malignant tumors possess mechanisms for evading host immune responses. A novel mechanism that tumor may evade host immune response through the expression of PD-L1. PD-L1 expression has been found in various human cancers, and was associated with patients' prognoses –19. In tPD-L1/PD-1 has been shown to be an important mechanism underlying T-cell dysfunction during chronic inflammation and chronic infection –22. SeveFor ESCC, treatment response is an independent prognostic factor. The identification and inhibition of key drivers of immunosuppression have the potential to improve patient outcome combined with radiotherapy. PD-L1 expression has been reported to link with the resistance to anticancer therapies , 9. Our It is known that infiltration of T cells into tumors correlates with improved prognosis in several types of GI cancers –36. FurtOur study has some limitations. First, we examined PD-L1 expression mainly in biopsy tissue specimen to evaluate its prognostic value. Biopsies only sampled a small esophageal volume, which might result in undersampling of esophageal cancer tissue. Also, to examine the prognostic value of PD-L1 in esophageal cancer patients, it is a retrospective analysis of a population with different stages from a single institution. Therefore, the issue should be best answered in context of a prospective study in a more patient population.These findings indicate that PD-L1-positive esophageal cancer provides a suitable microenvironment for the development of tumor growth and treatment resistance mediated by induction of activated IL-6 signaling. Novel immune-based therapies for the treatment of cancer are currently under development. Therefore, targeting PD-L1 could be a promising strategy for the treatment of esophageal cancer.This study was approved by the institutional review board of our hospital. Patients who did not comply with the treatment regimen and those who received surgery alone for early-stage esophageal cancer were excluded from the study. A total of 162 patients with ESCC were enrolled in the study. The curative treatment for esophageal cancer included neoadjuvant CCRT combined with surgery or definitive CCRT according to the guidelines proposed by oncology team at our hospital. On completion of neoadjuvant CCRT, patients underwent a repeat CT scan and endoscopic examination to determine the response to treatment. Specimens collected from the 162 patients at diagnosis were subjected to immunochemical analysis. The main end points were overall survival (OS), disease-free survival (DFS) and treatment response. Survival probability was analyzed statistically using the Kaplan—Meier method. Significant differences between groups were assessed using the Spearman-rank test. Multivariate analyses were performed using a Cox regression model for survival.Formalin-fixed, paraffin-embedded tissues from 162 patients with esophageal cancer were subjected to IHC staining. Dissected esophageal cancer specimens from 21 of these patients were converted into tissue microarray (TMA) blocks using an AutoTiss 1000 arrayer . The TMA block contained esophageal SCCs and the adjacent non-malignant epithelium. The quality of the TMA slides was confirmed by the pathologist using hematoxylin- and eosin-stained slides. The IHC data for the specimens were assessed using the semi-quantitative immunoreactive score (IRS), as described previously . For thePeripheral blood (PB) samples were obtained from 56 of these patients before treatment. IL-6 levels in human serum samples were analyzed using an IL-6 human Quantikine ELISA kit (R&D system). The samples were frozen and stored before assaying.The human cancer cell lines CE81T, which is derived from a well-differentiated esophageal SCC and obtained from Bioresource Collection and Research Center , and TE2+CD8+ was sorted from freshly obtained PB.To obtain single-cell suspensions, tumor tissues were digested by 1 mg/ml collagenase IV (Sigma-Aldrich) and 0.2 mg/ml DNase I (Sigma-Aldrich) for 45 minutes at 37°C. Cells were blocked and then stained with antibodies against PD-L1. For apoptosis assays, tumor cells were stained with antibodies against 7-AAD and annexin V after the removal of CD8+ cells. To isolate cytotoxic T cells from peripheral blood mononuclear cells (PBMC) in the patients, multicolor fluorescence-activated cell sorting (FACS) was performed using a FACS caliber flow cytometer (BD Biosciences). The human cytotoxic T cells subset characterized as CD3The suppressive function of tumor cells was measured by their ability to inhibit the proliferation of autologous T cells in the following Suppression Assay, as described previously . The isoThe significance of differences between samples was determined using Student's t-tests. Data are presented as the means ± standard error of the mean (SEM). All experiments, comprising three replicates, were performed at least twice independently. A probability level of p < 0.05 was adopted throughout to determine statistical significance, unless otherwise stated.


Human cancer cells may suppress the adaptive immune response by expressing PD-L1 and down-regulating T cell activity through PD-L1/PD-1 and PD-L1/B7.1 interactions. Disruption of PD-L1 signaling restores anti-tumor immunity, resulting in durable responses across multiple human tumor types. Here we describe the clinical activity and development of predictive biomarkers for MPDL3280A, a human monoclonal antibody with an engineered Fc-domain designed to optimize safety and efficacy, that targets PD-L1 and prevents binding to receptors PD-1 and B7.1.We evaluated a multitude of biomarkers from pretreatment tumor specimens collected during clinical study of MPDL3280A. MPDL3280A has been administered as a monotherapy in over 300 pts with locally advanced or metastatic solid tumors in a phase 1 study.To date, MPDL3280A has been well tolerated across multiple dose levels and no G3-5 pneumonitis has been reported. Responses were observed in multiple tumor types, including NSCLC, RCC, and melanoma, with ongoing responses seen in the majority of responders. We report the association between pretreatment immune-related markers and response to MPDL3280A.Overall, our assessment of tumor specimens not only has resulted in markers that may potentially identify response to MPDL3280A but also has furthered our understanding of the biologic activity of PD-L1 inhibition on treatment.