Spontaneous cancers are common diseases in dogs. Among these, some malignant cancers such as oral melanoma, osteosarcoma, hemangiosarcoma, and mast cell tumor are often recognized as clinical problems because, despite their high frequencies, current treatments for these cancers may not always achieve satisfying outcomes. The absence of effective systemic therapies against these cancers leads researchers to investigate novel therapeutic modalities, including immunotherapy. Programmed death 1 (PD-1) is a costimulatory receptor with immunosuppressive function. When it binds its ligands, PD-ligand 1 (PD-L1) or PD-L2, PD-1 on T cells negatively regulates activating signals from the T cell receptor, resulting in the inhibition of the effector function of cytotoxic T lymphocytes. Aberrant PD-L1 expression has been reported in many human cancers and is considered an immune escape mechanism for cancers. In clinical trials, anti-PD-1 or anti-PD-L1 antibodies induced tumor regression for several malignancies, including advanced melanoma, non-small cell lung carcinoma, and renal cell carcinoma. In this study, to assess the potential of the PD-1/PD-L1 axis as a novel therapeutic target for canine cancer immunotherapy, immunohistochemical analysis of PD-L1 expression in various malignant cancers of dogs was performed. Here, we show that dog oral melanoma, osteosarcoma, hemangiosarcoma, mast cell tumor, mammary adenocarcinoma, and prostate adenocarcinoma expressed PD-L1, whereas some other types of cancer did not. In addition, PD-1 was highly expressed on tumor-infiltrating lymphocytes obtained from oral melanoma, showing that lymphocytes in this cancer type might have been functionally exhausted. These results strongly encourage the clinical application of PD-1/PD-L1 inhibitors as novel therapeutic agents against these cancers in dogs. Dogs often develop spontaneous cancers, which may cause death  or reducImmunotherapies have potential as novel treatment options for cancer. Among those, immune checkpoint inhibitors such as anti-programmed death 1 (PD-1) and anti-PD-ligand 1 (PD-L1) antibodies showed promising effects on several malignancies in humans ,7. The iHowever, there are only few reports on the PD-1/PD-L1 axis in dogs, and its association with diseases remains to be elucidated. Previously, we have reported that PD-L1 is expressed in dog melanoma, MCT, and renal cell carcinoma and that PD-L1 blockade by an anti-PD-L1 antibody enhances IFN-γ production by tumor-infiltrating cells . These rAnimal use throughout this study was approved by the Institutional Animal Care and Use Committee , Hokkaido University. No animal was sacrificed for the purpose of this study. Clinical samples of dog tumor tissues were surgically excised at the Veterinary Teaching Hospital, Graduate School of Veterinary Medicine, Hokkaido University and at Veterinary Hospitals in Sapporo city in 2014–2015. Upon the use for this study, informed consent was obtained from all owners of the dogs. Peripheral blood samples of clinically healthy dogs were collected from beagles at the age of 12–15 years kept at the Experimental Animal Facility, Graduate School of Veterinary Medicine, Hokkaido University.To assess the binding specificity of a recently established anti-PD-L1 monoclonal antibody (mAb) , flow cy+ cells or CD8+ cells was evaluated.To assess whether PD-1 expression is upregulated in the context of canine cancers, flow cytometric analysis was performed with cross-reactive anti-human PD-1 antibody . Periphen = 40) used in this study originated from the oral cavity. The MCT samples (n = 5) were all classified into Patnaik grade III [n = 5) analysis, no inflammatory mammary carcinoma samples were included. In addition to the above-mentioned specimens, OS (n = 10), HSA (n = 10), prostate adenocarcinoma (n = 5), skin squamous cell carcinoma (n = 5), diffuse large B-cell lymphoma (n = 5), nasal adenocarcinoma (n = 5), soft tissue sarcoma (n = 5), histiocytic sarcoma (n = 5), transitional cell carcinoma (n = 5), and anal sac gland carcinoma with metastatic disease (n = 5) specimens were studied by immunohistochemistry.Specimens were randomly selected from formalin-fixed and paraffin-embedded tissues of dog malignant tumors , which had been submitted for histological diagnosis and kept in a commercial pathology laboratory . All the melanoma samples  procedure. Sections (4 μm) were dewaxed in xylene and hydrated through graded alcohols. To remove endogenous peroxidase, the sections were immersed in a 3% hydrogen peroxide solution at room temperature for 10 min. Following this, the sections were incubated with anti-PD-L1 antibody 6G7-E1 (10 μg/mL). After washing with phosphate-buffered saline, the sections were incubated in a secondary antibody solution (biotin-labeled goat anti-rat IgM)  at room temperature for 30 min. Following the incubation, the sections were reacted using the ABC in the VECTASTAIN Elite ABC Kit  at room temperature for 30 min. Visualization was accomplished using a 0.05% 3,3′-diaminobenzidine solution. Mayer’s hematoxylin stain was used as a counterstain. For the negative control, sections were subjected to the same procedures without the primary antibody or with the rat IgM isotype control antibody . The specimen was considered positive for PD-L1 if there was histologic evidence of cell staining. No obvious staining was observed in the negative control sections . To prepp < 0.05 was considered to be statistically significant.In the flow cytometric analysis of PD-1 expression, Mann–Whitney U test was conducted and The anti-PD-L1 antibodies that can bind to dog PD-L1 were established previously ,17. AmonImmunohistochemical analysis was performed to detect PD-L1 expression in various malignant cancers. In total, 110 specimens were tested, among which 36 oral melanoma, 7 OS, 6 HSA, 3 grade III MCT, 4 mammary adenocarcinoma, and 3 prostate adenocarcinoma specimens were PD-L1 positive Figs . Both cy+ and CD4+ TILs from oral melanoma (p < 0.05). The positive rate of PD-1 expression on CD8+ or CD4+ TILs was 70.9–96.6% or 80.2–96.8%, respectively Click here for additional data file.S2 FigTILs obtained from (A) hepatocellular adenoma and (B) hepatocellular adenocarcinoma were analyzed for PD-1 expression by flow cytometry (red line). Normal tissue-infiltrating lymphocytes (NILs) obtained from adjacent healthy liver tissue of the same individuals (blue line) were used as control. Black line, isotype control for TILs. Shaded area, isotype control for NILs.(TIF)Click here for additional data file.


Sarcoidosis is an idiopathic multisystem disease characterized by the formation of noncaseating granulomas. It frequently presents with pulmonary infiltrates and bilateral hilar and mediastinal lymphadenopathy. Splenic involvement is common, but massive splenomegaly is a rare occurrence. Sarcoidosis is known as “the great mimicker” (or “the great imitator”) since it exhibits a myriad of symptoms, mimicking other inflammatory, infectious, and neoplastic conditions, including lymphoma. Herein, we report the case of a 44-year-old male patient who was found to have bicytopenia, hypercalcemia, diffuse lymphadenopathy, and massive splenomegaly, a constellation of findings suggestive of underlying lymphoma. Interestingly, lymph node biopsy showed noncaseating granulomas suggestive of sarcoidosis, without evidence of malignancy. Sarcoidosis is a chronic inflammatory disorder of unknown origin which occurs mainly in young people , 2. It iThe symptoms of sarcoidosis, if present, are nonspecific. The presence of noncaseating granulomas is also not pathognomonic of the disease, as it can be seen in malignancy . MoreoveHerein, we report the case of a 44-year-old male patient who was found to have bicytopenia, hypercalcemia, diffuse lymphadenopathy, and massive splenomegaly, a constellation of findings suggestive of underlying lymphoma. Surprisingly, lymph node biopsy showed noncaseating granulomas suggestive of sarcoidosis, without evidence of malignancy.We report the case of a 44-year-old Caucasian male who was referred to our hospital by his primary physician for abnormal outpatient laboratory test values. The patient had been healthy until 5 months prior to admission, when he started to have progressively worsening fatigue. Outpatient blood tests revealed kidney injury, hypercalcemia, and anemia, findings that required hospitalization.On the day of admission, the patient's only complaint was severe fatigue. Upon further questioning, he admitted having a 70-pound unintentional weight loss over the last 18 months. He denied any fever, chills, night sweats, cough, rash, or joint or abdominal pain. His prior medical history consisted of diabetes mellitus, gout, and hyperlipidemia. The patient was a nonsmoker and had no allergies. His family history was noncontributory.On physical exam, the patient's body temperature was 98.6°F, blood pressure was 159/92 mmHg, and heart rate was 100/min. Cardiovascular and pulmonary exams were unremarkable. Left upper quadrant tenderness was noted on the abdominal exam, as well as a firm and enlarged spleen, which was palpable below the umbilicus. No rash, cervical, or axillary lymphadenopathy was identified.μm3. The rest of the hematologic panel was as follows: white blood cell count of 3.93 × 109/L, platelet count of 254 × 109/L, and erythrocyte sedimentation rate (ESR) of 93 mm/h. A peripheral blood smear was within normal limits. In addition, hypercalcemia of 13.7 mg/dL was noted, along with a blood urea nitrogen (BUN) of 33 mg/dL and a serum creatinine of 2.39 mg/dL, findings consistent with kidney injury. The renal function was normal three years ago. Liver enzymes were normal. An abdominal sonogram showed a markedly enlarged and diffusely heterogeneous spleen measuring 30 cm in length  of 82.3 n length . Enlargen length , along wn length . A chestThe patient received packed red blood cells transfusions to maintain his hemoglobin level around 8 mg/dL. He was also given intravenous fluids to treat the hypercalcemia and received one dose of pamidronate, which helped to lower the calcium level to as low as 11 mg/dL over the next few days.2 levels (with low PTH levels). Anti-nuclear antibody, HIV test, monospot test, and purified protein derivative (PPD) were negative. Additional studies are listed in The presence of massive splenomegaly and diffuse lymph node enlargement was concerning lymphoma. Further workup showed anemia of chronic disease and elevated vitamin D 1,25(OH)Four weeks after the initiation of therapy, the patient's calcium level was 10.3 mg/dL, along with a hemoglobin of 9.4 g/dL and a creatinine of 1.38 mg/dL. 2 weeks later, ACE level was 31 mg/dL. 8 weeks after hospital discharge, a fluorine-18 fluorodeoxyglucose (FDG) PET imaging, done while the patient was still on treatment, showed no focal FDG avid lesions, along with stable non-FDG avid thoracic and abdominal adenopathy. The patient continued to be in good health 9 months after his diagnosis and had shown no progression of sarcoidosis.Sarcoidosis is a chronic idiopathic granulomatous disease which can affect all age groups . It has Sarcoidosis can be asymptomatic in some patients. If present, symptoms are both systemic  and/or organ-specific  . There iThe diagnosis of sarcoidosis is based on criteria from the American Thoracic Society (ATS), the European Respiratory Society (ERS), and the World Association of Sarcoidosis and Other Granulomatous Disorders (WASOG) . These cSome patients with sarcoidosis are not disabled by the illness and therefore do not require treatment . In gene Myelofibrosis (primary or secondary). Chronic myeloid leukemia. Lymphoma . Hairy cell leukemia. Gaucher disease. Amyloidosis. Beta thalassemia major. Schistosomiasis. Kala-azar . Sarcoidosis (rarely). Hyperreactive malarial splenomegaly syndrome . AIDS with mycobacterium avium complex. Splenic vein thrombosis.Splenic involvement in sarcoidosis is defined as the histologic presence of noncaseating granulomas in the spleen. Autopsy studies show that the spleen is the second most commonly affected organ in sarcoidosis, with the lung being first . ClinicaSplenic involvement in sarcoidosis is usually asymptomatic, although left upper quadrant pain is occasionally present. Patients with splenomegaly may have a higher incidence of constitutional symptoms and more disseminated disease . SplenicThe crux of sarcoidosis is its ability to masquerade as other diseases, most significantly lymphoma. As such, for clinicians, distinguishing these entities can make the difference between life and death for patients. In addition to the nonspecific clinical, radiological, and histological features in sarcoidosis, the lymphocyte activation and the reticuloendothelial system involvement  make the distinction of sarcoidosis from lymphoma extremely challenging. In fact, hypercalcemia and increased serum ACE levels have also been described in patients with lymphoma . MoreoveIn some cases, the coexistence of “true” sarcoidosis and lymphoproliferative disease has been reported in the literature. In most of these cases, sarcoidosis preceded the diagnosis of lymphoma, but in few other reports, lymphoproliferative disease occurred first . This poFDG PET imaging remains an essential modality in the management of lymphoma. One of many advantages it offers over conventional imaging is the ability to detect occult lesions. However, its specificity is limited by multiple false-positive conditions, including infections, inflammations, and sarcoidosis , 20. In This report illustrates an unusual case of sarcoidosis that presented as bicytopenia, hypercalcemia, diffuse lymphadenopathy, and massive splenomegaly, mimicking lymphoma. Physicians should be aware of this atypical presentation and accordingly should consider sarcoidosis in their differential diagnosis, after excluding other worrisome diagnoses, such as lymphoma.


It is well established that the vast majority of the population favors their right hand when performing complex manual tasks. However, the developmental and evolutionary underpinnings of human manual asymmetries remain contentious. One often overlooked suggestion is that right handedness may stem from an asymmetrical bias in attention, with the right hand being allocated more attentional resources during bimanual tasks than the left hand . This re Approximately 90% of humans consider themselves to be right handed . This unThe goal of this article is to provide an overview of the evidence for a rarely discussed hypothesis – that right handedness is a consequence of a rightward attentional bias. Proponents of this hypothesis claim that when attention must be divided between the hands during bimanual tasks, most individuals will allocate the majority of their attentional resources to the right hand or its task. First, we describe the evidence for an automatic link between the attentional system and manual actions. Next, we present the empirical studies which have found evidence for a rightward attentional bias in right-handed individuals’ during rhythmic and discrete bimanual tasks. Finally, we discuss the viability of the attentional bias explanation as a way to bridge the gap between performance and selection asymmetries.On the face of it, there would seem to be obvious advantages to having two equally skilled hands to complete twice as many tasks. Such a strategy, however, would not be easily compatible with humans’ attentional limitations: prior to commencing a typical reach toward a visual target, a saccade is used to aim the high-resolution foveal portion of the eye to the region of interest . AlthougThe link between attention and action in the context of physiology and neuropsychology is well-studied in the context of sequential unimanual movements  the right hand of right handers. However, these findings offer little insight into the causal relationship between attentional and manual asymmetries. Clearly, altering one’s hand preference is not simply a case overtly attending toward the non-dominant hand . It is, The attentional bias hypothesis posits that attention is biased toward the right hand in a substantial proportion of the population from birth , 1994. TWhile the link between consistent right hand selection and right hand performance advantages is easy to understand, it is worth considering why an attentional asymmetry is necessary in human motor coordination. Peters suggests that the key to the attentional bias hypothesis is bimanual coordination – the common factor linking the experiments described in this review. The ‘kinematic chain’ hypothesis, proposed by Another point which is worth consideration is how to reconcile the rightward attentional bias which right handers exhibit during bimanual tasks with the oft-reported left hand unimanual reaction time advantage during unimanual localization tasks , which mUp until this point, only evidence has been presented from right-handed individuals. The situation for the understudied left handers remains unclear, largely because studies examining adextrals in this context are rare. In fact, they are crucial, if establishing the relationship to cerebral asymmetries is desired see , 1996 noThe underlying cause of human handedness is the cause of much debate. Here, we have presented the evidence for a bias in attention which occurs during bimanual coordination which may drive hand preferences, and presented a plausible account of how this bias would lead to manual asymmetries across the population. Future work on relating these effects to cerebral asymmetries in right and left handers, and how attentional and manual asymmetries develop and interact over developmental trajectories, may help clarify these relationships.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Tremelimumab is a selective human IgG2 mAb inhibitor of cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) that has been combined with durvalumab in a Phase Ib study in patients with NSCLC (NCT02000947), with encouraging clinical activity and a manageable safety profile. While anti-PD-1/PD-L1 monotherapy may be associated with greater clinical benefit in patients with PD-L1+ tumors, combination therapy has clinical activity in both PD-L1+ and PD-L1– patients in several solid tumors, enhancing the antitumor activity of anti-PD-1/PD-L1 agents in patients with PD-L1– tumors. The PD-L1 and CTLA-4 pathways are non-redundant and preclinical data indicate targeting both may induce synergistic antitumor effects. Here we describe the CONDOR study (NCT02319044), a Phase II study to determine the efficacy and safety of durvalumab monotherapy, tremelimumab monotherapy, and their combination in PD-L1– R/M SCCHN patients who have failed prior platinumtherapy.First-line palliative treatment for patients with R/M SCCHN includes platinum-based chemotherapy. There are no standard second-line options upon relapse and median survival is limited. In SCCHN, tumors create a highly immunosuppressive environment and evade immune detection by exploiting inhibitory immune checkpoints such as the programmed cell death ligand-1 (PD-L1)/programmed cell death-1 (PD-1) axis, making immunotherapy an attractive option to study in this disease. Durvalumab (MEDI4736) is a selective, high affinity human IgG1 mAb that blocks PD-L1 binding to PD-1  will be randomized 1:1:2 to receive durvalumab (10 mg/kg IV) monotherapy; tremelimumab (10 mg/kg IV) monotherapy; or durvalumab (20 mg/kg IV) plus tremelimumab (1 mg/kg IV) combination therapy for up to 12 months (Figure This is a randomized, open-label, multicenter, global, Phase II study in immunotherapy-naïve patients with PD-L1ClinicalTrials.gov identifier NCT02319044.


Here we show that the growth of electrically insulating BNNTs on graphene can enable the use of graphene as effective digital switches. These graphene-BNNT heterojunctions were characterized at room temperature by four-probe scanning tunneling microscopy (4-probe STM) under real-time monitoring of scanning electron microscopy (SEM). A switching ratio as high as 105 at a turn-on voltage as low as 0.5 V were recorded. Simulation by density functional theory (DFT) suggests that mismatch of the density of states (DOS) is responsible for these novel switching behaviors.High electron mobility of graphene has enabled their application in high-frequency analogue devices but their gapless nature has hindered their use in digital switches. In contrast, the structural analogous,  Here we show that zero dimensional (0D) heterojunctions between BNNTs and graphene could switch current flows.Graphene is known for its high electron mobility and zero energy gap nature146The graphene-BNNT heterojunctions are prepared by chemical vapor deposition (CVD)17I-V) measurements with the in situ SEM. 1, while another STM probe 2 was in contact with the graphene surface. The distance, d, between probe 1 and the vertical graphene-BNNT heterojunction, was controlled by changing the contact point on the BNNT.The as-grown graphene-BNNT heterojunctions were characterized for their electronic properties at room temperature by using a four-probe scanning tunneling microscopy (4-probe STM) system (see Methods)Vb  with a threshold turn–on voltage, Von ~0.5 V (inset). The current level detected at d = 0.10 μm is even higher, up to μA level (as limited by the preset 1 μA current limit), with Von ~0.15 V (inset). These conducting behaviors with obvious “on” and “off” states are different from the reported insulating nature of BNNTs185.For the case of d = 1.23 μm, we hardly detected any current flow across the graphene-BNNT heterojunction for a range of bias voltages, Vb  between I-V curves were measured . As shown in −7A is detected. We believe that the absence of the gating effect is due to the shielding of the metallic graphene located between the gate oxide surface and the heterojunction. As the junction is small in diameter (~60 nm) and is potentially surrounded by additional graphene layers underneath, an electrostatic screening effect will be able to effectively shield the gating effect. Therefore, gating the junction should still be possible if a top gate is applied.A full series of measured . The detTo understand these experimental observations, calculations were performed by employing Density Functional Theory (DFT)21I-V characteristics of pristine graphene and graphene-BNNT heterojunction are shown in I-V behavior for the pristine graphene is predicted affirming its metallic nature. On the other hand, the calculations predict very little, if any, electron transport through the graphene-BNNT heterojunction within a range of low bias voltages (i.e. ±0.1 V). At higher bias voltage, however, the linear I-V behavior is recovered with slightly higher resistance. The calculated switching properties of the heterojunction are in qualitative agreement with our experimental results.Next we calculated the electron transport properties of the heterojunctions using density functional theory together with the non-equilibrium Greens Function method see . Fig. 3dd = 0.10 μm, the effective dimension of the junctions is approximately ~120 nm in diameter.We believe that the switching behaviors observed here is due to the DOS mismatch at the heterojunctiond = 0.64 μm away from the heterojunction. Another STM probe is in electrical contact with the graphene sheet (beyond the SEM imaging window). As shown in Vb (“off” state) for both cases. At higher Vb, the heterojunction turned “on” with thresholds Von ~7 V and Von ~4 V for d = 0.73 μm and 0.64 μm, respectively. Obviously, Von for this horizontal heterojunction is higher than those discussed for a vertical heterojunction shown in Von. Such a DOS mismatch can only be artificially formed if local compression or severe bending occurs to the BNNT. It is unlikely to find such a deformation in as-grown BNNTs.We further examined the switching behavior of a horizontal graphene-BNNT heterojunction. In conclusion, we have demonstrated the fabrication of the stable graphene-BNNT heterojunctions without the use of catalyst. A bias-dependent switching behavior has been observed at the heterojunctions. DOS mismatch between graphene and BNNT at the nanoscale heterojunction is suggested to prevent current flow at low bias voltages, and turn on the current flow at higher applied bias. The novel Graphene-BNNT heterojunctions would likely combine the advantage of the ballistic nature of electron mobility°C in Argon ambient. These graphene sheets are then dispersed in tetrahydrofuran or isopropyl alcohol by sonication (10 minutes in a sonication bath). The suspended graphene was then coated on cleaned oxidized Si substrates and dried in hydrogen flow at 800 °C for 30 min. These graphene coated substrates are then used for the subsequence growth of BNNTs to form the heterojunctions. The growth of BNNT on graphene is based on our reported growth vapor trapping (GVT) chemical vapor deposition (CVD) method without the use of catalyst. These substrates were placed on the top of an alumina combustion boat in which B, MgO, and FeO precursors are loaded . This setup was placed inside a closed-end quartz tube in the horizontal tube furnace with the graphene sheets facing upward. The precursors and substrates were then heated up to 1100–1200 °C with an ammonia flow of 200–350 sccm and kept for 30 min. Images of as-grown graphene-BNNTs heterojunctions are shown in Fig. S1 and Raman spectra of these heterojunctions are shown in Fig. S2. We believe that the growth of BNNTs is initiated by point defects, where dangling bonds serve as the nucleation sites. As some of the point defects are not round, the initially grown BNNTs became distorted  were heat shocked into multi-layered graphene at 1000 How to cite this article: Parashar, V. et al. Switching Behaviors of Graphene-Boron Nitride Nanotube Heterojunctions. Sci. Rep.5, 12238; doi: 10.1038/srep12238 (2015).


In spoken interactions, interlocutors carefully plan, and time their utterances, minimizing gaps and overlaps between consecutive turns. Cross-linguistic comparison has indicated that spoken languages vary only minimally in terms of turn-timing, and language acquisition research has shown pre-linguistic vocal turn-taking in the first half year of life. These observations suggest that the turn-taking system may provide a fundamental basis for our linguistic capacities. The question remains, however, to what extent our capacity for rapid turn-taking is determined by modality constraints. The avoidance of overlapping turns could be motivated by the difficulty of hearing and speaking at the same time. If so, turn-taking in sign might show greater toleration for overlap. Alternatively, signed conversations may show a similar distribution of turn-timing as spoken languages, thus avoiding both gaps and overlaps. To address this question we look at turn-timing in question–answer sequences in spontaneous conversations of Sign Language of the Netherlands. The findings indicate that although there is considerable overlap in two or more signers’ articulators in conversation, when proper allowance is made for onset preparation, post-utterance retraction and the intentional holding of signs for response, turn-taking latencies in sign look remarkably like those reported for spoken language. This is consistent with the possibility that, at least with regard to responses to questions, speakers and signers follow similar time courses in planning and producing their utterances in on-going conversation. This suggests that turn-taking systems may well be a shared cognitive infrastructure underlying all modern human languages, both spoken and signed. Spontaneous conversations among speakers often run smoothly with slight overlaps and gaps between consecutive turns . ComparaOne of the substantial discoveries of the last 50 years is that sign languages show all the properties of full natural languages on all relevant levels of linguistic structure, including, for instance, sublexical structure at the phonological level . HoweverConversation analytic work on sign languages has been sparse, yet early studies of signed conversations have observed some remarkable features of signed interactions. terminal overlap  moving toward the position where a sign will be made as the other interactant is making a sign.” However, in considering the comparison between spoken and signed languages, we must bear in mind that the preparation for vocalization in spoken languages is mostly inaccessible to other participants, consisting apart from inbreaths in early motor preparation of the vocal organs  as the turn beginning as it most directly reflects the phonological content of a sign.In spoken conversation, addressees sometimes initiate a response at a possible point of completion while the initial speaker continues his turn, thus resulting in  overlap . Similarl organs . Therefol organs . In our Overlap at signed turn transitions may also result from turn-final holds, which are typically released as soon as the relevant response has been recognized . CrucialIf turn-taking lies deep in our communicational instincts as has been suggested , then itTo address this question we analyze turn-timing in 190 question–answer sequences captured from spontaneous conversations of Sign Language of the Netherlands Nederlandse Gebarentaal (NGT). Questions–answer sequences provide a particularly well-suited conversational context in which to investigate turn-timing, as questions make due a conditionally relevant and timely response cf. . The sigThe paper is structured as follows. Section “Materials and Methods” provides details on our data collection as well as the annotation scheme. Section “Results” presents a statistical comparison of turn-timing in the NGT sample to the spoken languages as reported by De structuur en ontwikkeling van conversaties in gebarentaal .This study exploits the NGT Interactive corpus, which consists of spontaneous conversations of native NGT signers in informal settings, which have been collected, and analyzed by Merel van Zuilen, Stephen C. Levinson and Connie de Vos (Max Planck Institute for Psycholinguistics), and Onno Crasborn (Radboud University) from early 2011 onward. All data and analyses have been ethically approved by the Radboud University Ethical committee under the research program The recording sessions of the NGT Interactive corpus took place in participants’ homes, at various deaf clubs, as well as a small restaurant, between participants who were long-term acquaintances and friends. These signers were also very familiar with the research assistant who recorded them, and who is a deaf native signer of NGT herself. The data therefore has the character of natural conversation. All conversations were recorded using two HD cameras from different camera angles. The data on which the present study is based features 16 signers  in one triadic and six dyadic interactions totaling 11 h and 2 min of raw video data. In one of the dyadic interactions, a third person occasionally joined the conversation, but did not participate in any of the question–answer sequences in our analysis. For this reason, this recording is treated as a dyadic interaction. All but one of the signers included in this study had acquired NGT early in life, before the age of ten and all three variants of NGT – Northern, Western, and Southern – are represented in the sample. Conversation topics ranged, unprompted by the investigators, from a work meeting regarding the write-up of a professional paper, home improvement activities, the history of the deaf club, and interpersonal relations.The video recordings were compressed into MPEG2 format at 1920 × 1080 resolution and 25 fps. The relevant sections were then translated into written Dutch and annotated further using ELAN video annotation software . As is cIn order to ensure a diverse sample of question–answer sequences, we selected 30 min segments from each video file that were dense in turn transitions. In identifying these sequences, we adopted the selection criteria which were originally developed as part of the MPI Coding Scheme for Question–Response Sequences in Conversation at the MPI for Psycholinguistics  and formhuh?). Similarly, in some contexts, polar questions evoked a minimal response such as a head nod (yes) or a side-to-side headshake (no). Importantly, sign languages are essentially multi-modal in nature in the sense that signers do not only use their hands but also their facial expressions and body postures to express meaning at the linguistic and paralinguistic level. The non-manual components are sometimes considered as the equivalent of intonation or prosody in sign . The stroke most directly represents the phonological form of a sign and includes the internal movement of a sign, in this case touching the contra-lateral upper arm . Subsequently to the stroke, a signer might hold a sign during interaction, for example to mobilize a response in their interlocutor . Finally, the sign may be retracted and the hands move into resting position .Our analysis of turn-timing is based on the coding of the various movement phases that make up a sign. Specifically, we adopted the movement phase coding developed for Sign Language of the Netherlands and co-speech gesture . This coImportantly, each sign is minimally associated with a single stroke, but the other three movement phases do not always occur . This anFor a subset of items (59 questions) a second coder, who is also a native signer of Sign Language of the Netherlands, applied the same Gesture Phase coding system. Subsequently, any items that showed discrepancies of more than two video frames were discussed, and adjusted when necessary. In a few cases these differences were based in a distinct phonological analysis of the signs that were being used and these two interpretations could not be reconciled. After these discussion sessions the overall correlations between these two coders was 0.9 for the sign-naive boundary measures and 0.98 for the stroke-to-stroke boundary measures.Figure 2 illustrates each of the reported phonetic measures schematically. In section “Results,” each of these phonetic measures of signed turn-timing are compared to findings from the spoken turn-timing in cross-linguistic study presented by In our study we report on three phonetic measures of turn transition times based on the coding of gesture phases. The first measure looks at sign-naive turn-boundaries and includes all manual actions, that is, all movement phases that make up a signer’s utterance. The second measure looks at stroke-to-stroke turn boundaries, which run from the start of the initial stroke of a turn till the end of the turn-final stroke. For each signer, gestural movement phases from both hands were taken into account. The third and final measure calculates the offset of the addressee’s preparation phase with respect to the end of the question’s final stroke, and is called signed utterance launch. Figure 3 displays an excerpt from the corpus to exemplify the three phonetic measures. This question–answer sequence stems from a conversation between two males who are close friends living in the North of the Netherlands. They are discussing remodeling activities while enjoying lunch on a roof top terrace. Directly preceding the excerpt, the signer to the viewer’s right (R) indicates his misunderstanding using a minimal repair initiator by furrowing his brows and leaning forward similar to a spoken ‘huh?’ (cf. hier), which is formed by a downward index finger point, and co-produced with the Dutch mouth movement nu ‘now.’ At the end of his question, the sign HERE is immediately retracted over 430 ms without a sentence-final hold, presumably because the addressee (signer R) has already raised his hand to produce an appropriate response. Signer R’s answer starts off with the sign NOW, and he initiates the preparation of this initial sign before signer L has initiated the start of the preparation of his final sign. As a result, both signers overlap by 690 ms according to the sign-naive turn boundary measure and the launch of R’s sentence articulation has a negative value of -260 ms. According to the stroke-to-stroke measurement, however, there is a slight gap of 30 ms between both turns. This turn transition is detailed in  which displays snapshots from both camera angels of the final frame of Signer L’s turn-final stroke. On the left-hand side there is a clear view of signer L and the end state of the sign HERE. The camera view on the right-hand side clearly shows signer R who is in the middle of the preparation phase of his turn-initial NOW; the white arrow indicates the trajectory of its stroke. The exact timing of this turn transition is illustrated by a scaled representation of the gestural movements of this turn transition in Figure 3B.uh?’ cf. . As explFigure 4A shows a density plot of turn transition offset for the overall data set according to the sign-naive turn boundary measure. The sign-naive boundary measure calculates turn-timing by including all gestural movement phases of the hands. According to this phonetic measure, the average offset of answers to questions was -812 ms, the median was -607 ms, and the mode  was -361 ms. These negative values of central tendency indicate that addressees generally start signing well before the question has fully ended. The average value of -687 ms was 6.18 SDs below the cross-linguistic average turn transition time as estimated from m = 229 ms, SD = 168 ms). Assuming that average turn transition times across spoken languages are normally distributed, and using the data reported in p < 0.0001). The sign-naive turn boundary measure thus suggests that the timing of responses to questions in the visual modality deviates substantially from oral-auditory turn-taking in that it exhibits both more and more extended overlap.To test whether signers optimize turn-taking on the basis of stroke-to-stroke turn boundaries, we compared turn transition times in NGT with turn transition times in spoken question–answer sequences in ten languages as reported in Figure 4B shows a density plot of the stroke-to-stroke turn boundary measure. According to this measure, turn transition times in NGT now exhibit a positive gap, with an average of 307 ms, a median of 269 ms, and an estimated mode of 227 ms. The average value of 307 ms was only 0.46 SD above the cross-linguistic average turn transition time as estimated from p = 0.32). When we exclude utterance-initial preparatory movements, and utterance-final holds and decays, turn-timing in signed interaction thus falls within range of oral-auditory turn-taking as reported by Figure 5, which shows the average turn transition times in Secondly, we calculated turn-timing following the stroke-to-stroke boundary measure, which is based on the hypothesis that signers observe stroke-to-stroke turn boundaries. This phonetic measure calculates the offset of the answerer’s initial stroke with respect to the end of the final stroke of the questioner. Another way of looking at signed versus spoken turn-timing is by comparing the distributions of overlapping and delayed turn transitions. Figure 6 presents an overview of the percentages of overlapping answers to questions, including the data reported in this study. As estimated by p = 0.28). According to the sign-naive measure, however, 82.2% of answers overlap with the respective question. This percentage clearly falls outside the cross-linguistic range for spoken languages. The probability of observing this value or a higher value in the cross-linguistic distribution is extremely low (p < 0.00001).Figure 7 presents an overview of the percentages of transitions between answers and questions involving a gap of more than 120 ms, including the data reported in this study. As estimated by p = 0.54). According to the sign-naive measure, however, only 17.8% of answers overlap with the respective question, which would be three times less than the spoken language which allows for the smallest number of delays in question–answer sequences. The probability of observing this value or a lower value in the cross-linguistic distribution is much lower (p < 0.00001).Table 1 summarizes all main results from the present corpus analysis.Unlike spoken languages, signed languages offer us the unique opportunity to examine turn preparation using non-invasive methods. In our data, the preparatory phase of utterances had an average duration of 474 ms, a median of 280 ms, and an estimated mode of 255 ms. We computed a third turn transition timing measure corresponding to the latency between the end of the final stroke of the question and the onset of the initial preparatory movements of answer articulation. Relative to the end of the final stroke of the question, response articulation in our data starts with an average latency of -86 ms, a median latency of -78 ms and a modal latency of -53 ms. The timing of preparatory movements in sign is thus slightly earlier than the initiation of pre-utterance inbreaths in answers to questions in spoken Dutch 15 ms; . We retup > 0.1 in all three cases), indicating that the triadic conversation was not significantly different from the dyadic conversations in terms of turn-timing.As explained in Section “The NGT Interactive Corpus,” one of the recordings in our corpus consisted of a triadic interaction, the other six involving dyadic conversations. Because turn-timing might differ between triadic and dyadic interactions , we examined each of the timing measures presented in the previous subsections  as a function of the number of participants in the interaction. We fitted a series of mixed-effects regression models with each of the timing measures as the response, number of participants (dyadic vs. triadic) as a fixed factor, and conversation as a random factor. None of the three models yielded a statistical effect for the fixed factor number of participants  in order to address the question as to what degree turn-timing in signed conversation differs from turn-timing in spoken conversation. The present study has focused on responses to questions, as questions require a timely and contingently relevant response, and could therefore serve as a baseline measure as to how much overlap or gap might be allowed in a given language. Moreover, the timing of responses to questions has been documented in a wide range of spoken languages , thus alAs noted in the introduction, it has previously been claimed that signed conversation exhibits more overlap than spoken conversation. We have also seen that, using the stroke-to-stroke measures, the proportions of turn transitions with noticeable gaps and overlaps in NGT is within the same range as spoken languages according to the stroke-to-stroke boundary measure. These comparisons were carried out on the assumption that the threshold for noticeable gaps and overlaps lies at FTOs of 120 ms and -120 ms, respectively cf. . At presFigure 8 shows a schematic representation of typical time courses in a signed question–answer sequence based on modal values in our data , and, for the sake of comparison, and as reported by Figure 8B), and in a sequence in which a preparatory inbreath was produced . Notice that, in spoken answers too, in line with our findings for sign language, the typical onset of the physical response, in the form of a preparatory inbreath, or of speech proper in answers not preceded by an inbreath, typically starts briefly after the end of the question.In sign, the articulators are large and heavy, and reaching the articulatory targets of hand strokes from an inactive state will require more time than a vocal articulatory gesture. For this reason, the timing of preparatory hand movements preceding signed turns-at-talk may provide a crucial insight into the time course of signed utterance planning. In our question–answer sequences, we have observed that initial preparatory hand movements of responses are typically launched during the second half of the last stroke of the question, and that the preparatory phase typically ends a couple of 100 ms after the last stroke of the question. On a par with the findings of The time course of turn production critically depends on the perception and comprehension of the preceding turn . Recent A related issue concerns the types of non-manual signals that might enable the accurate projection of TRPs by sign-perceivers. Reaction time experiments are currently being run to establish sign-perceivers’ sensitivity to TRPs as defined by stroke-to-stroke turn boundaries . That study also aims to identify the visual information signers rely on to determine utterance boundaries online, on the basis of linguistic annotations of the visible cues in this additional data set. On a par with previous work on spoken languages , we hypoThis paper has centered on question–answer sequences within a relatively limited data set. Our findings are in accordance with the hypothesis that there may be a single turn-taking system underlying both signed and spoken interactions . If thisIn sum, the observed patterns in signed turn-timing in NGT are within the range observed for spoken languages in terms of response latency to questions once we exclude preparatory movements from turn beginnings and retraction phases from turn ends. Moreover, unlike previously argued by The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Gypsies, Travellers and Roma (referred to here as Travellers) experience significantly poorer health and have shorter life expectancy than the general population. They are also less likely to access health services including immunisation. To improve immunisation rates, we need to understand what helps and hinders individuals in these communities in taking up immunisations. This study has two aims: (1) Investigate the barriers and facilitators to acceptability and uptake of immunisations among six Traveller communities in the UK; (2) Identify potential interventions to increase uptake in these Traveller communities.A three-phase qualitative study with six Traveller communities. PHASE 1: In each community, we will explore up to 45 Travellers’ views about the influences on their immunisation behaviours and ideas for improving uptake in their community. PHASE 2: In each community, we will investigate 6–8 service providers’ perspectives on barriers and facilitators to childhood and adult immunisations for Traveller communities with whom they work, and ideas to improve uptake. Interview data will be analysed using the Framework approach. PHASE 3: The findings will be discussed and interventions prioritised in six workshops, each with 10–12 phase 1 and 3–4 phase 2 participants.This research received approval from NRES Committee Yorkshire and The Humber-Leeds East (Ref. 13/YH/02). It will produce (1) findings on the barriers and facilitators to uptake of immunisations in six Traveller communities; (2) a prioritised list of potentially feasible and acceptable interventions for increasing uptake in these communities; and (3) methodological development in undertaking research with diverse Traveller communities. The study has the potential to inform new ways of delivering services to ensure high immunisation uptake. Findings will be disseminated to participants, relevant UK organisations with responsibility for the implementation of immunisation policy and Traveller health/welfare; and submitted for publication in academic journals.ISRCTN20019630. This is the first large-scale multi-community qualitative study exploring barriers and facilitators to childhood and adult immunisation for Travellers in the UK.The findings will have the potential to inform new ways of delivering accessible immunisation services to socially excluded, marginalised communities.Our research team may be perceived as ‘outsiders’, resulting in difficulties in engaging with Travellers to participate.Note. Throughout this paper, we use the term Traveller in its broadest sense to include distinct and diverse Gypsy, Traveller and Roma communities, who may be settled or nomadic, and may live on authorised or unauthorised sites or in houses). Despite a greater health need, there is low uptake of health services by Travellers, including preventive healthcare.7Travellers typically experience significantly poorer health and shorter life expectancy compared to the general population.The public health benefits and cost-effectiveness of immunisation are well established.19A large body of literature27Many of the issues identified in this literature are likely to be similar for Travellers. However to develop interventions that are tailored to the needs of diverse Traveller communities, further research with these communities is required. To date, a small number of studiesThe effectiveness of interventions to increase immunisation uptake among childrenInvestigate the barriers and facilitators to acceptability and uptake of immunisations among six Traveller communities  across four UK cities;Identify possible interventions to increase uptake of immunisations in these Traveller communities, which could be tested in a subsequent feasibility study.Investigate the views of Travellers on the barriers and facilitators to acceptability and uptake of immunisations and explore their ideas for improving immunisation uptake;Examine whether and how these responses vary across and within communities, and for different vaccines (childhood and adult);Investigate the views of service providers on the barriers and facilitators to uptake of immunisations within the Traveller communities with whom they work, and explore their ideas for improving immunisation uptake;Examine whether and how these responses vary within and across communities, for different vaccines (childhood and adult) and for different service provider roles;Use the data collected from (1 to 4) to identify possible interventions to increase uptake of immunisations in the six Traveller communities;Conduct Feedback Workshops in each community to discuss findings and to produce a prioritised list of potentially feasible and acceptable interventions to be considered for testing in a subsequent feasibility study.This three-phase qualitative study is the first ‘intervention development’ stage of the Medical Research Council Framework for developing and evaluating complex interventions.43The study will focus on six Traveller communities and be undertaken in four UK cities  and human papilloma virus (HPV) vaccine (given at 12–13 years); and adults who are pregnant, over 65 years or have long-term conditions for views on flu and pertussis vaccines). Typically, decisions on childhood immunisation are made by mothers;within a community across gender and age as well as draw out meaningful comparisons across Traveller communities, to allow robust conclusions to be made.Phase 2: Service providers in the four cities will be recruited to the study. We will purposively sample to ensure that we interview a mix of ‘frontline workers’  and those working in more strategic/commissioning roles . We will aim to interview six to eight service providers in each city . Examples of organisations and providers that we intend to approach are presented in Phase 3: A subsample of participants from phases 1 and 2 will be invited to take part in the ‘Feedback Workshops’, specifically between 10 and 12 Traveller participants per community and three to four service providers . Ideally, we will attract a mix of Traveller men and women, across ages (including teenage girls) with different experiences of immunisations; and a mix of frontline service providers and those with a more strategic role.This is a complex, multisite project working with socially excluded, marginalised communities who are traditionally hard to engage in research.47Phase 1: In each community, we will use a multipronged approach to access and recruitment. We are aware that our position of researchers as ‘outsiders’ with few similarities of experience and no ‘network connections’ with participant Traveller communities could result in difficulties in gaining access.49The study will be discussed with members of the community and written information provided for people to read or have read to them by others to aid discussions with family members and peers. For the two European Roma communities, information will be in English and Romanian/Slovakian. We will return a week later to establish who might wish to take part, and to arrange a time and place for the interview. We will regularly meet with the community during the recruitment phase.Phase 2: In each city, we will establish a list of contacts of the relevant service providers. We will approach these people by email, in the first instance, with a participant information sheet (PIS) and then follow this up with a telephone call a week later. Our existing links to the multiple agencies working in each city will also be used to facilitate introductions.Phase 3: Participants in phases 1 and 2 who agree to be contacted about phase 3 will be reapproached using their preferred method of contact and provided with a PIS about the Feedback Workshop. Where insufficient phase 1 participants are able to attend, we will seek to recruit new members of the community to attend the workshop.Phase 1: We will conduct small group interviews with members of the same family to elicit a cross-generational/lifespan perspective. We also plan to run group interviews with teenage girls, and with young women planning families, pregnant women and those with preschool children, to capture peer influence on immunisation decisions. While immunisation may not seem a particularly sensitive issue (with the exception of HPV), group interviews may not be appropriate or favoured. We will, therefore be flexible about whom and how many participants take part in an interview. Individual and group interviews will be conducted face to face in participants’ choice of setting. With the consent of participants, interviews will be recorded digitally and transcribed verbatim. Interpreters will be employed for Roma interviews.Phase 2: We will conduct individual interviews with the service providers face to face in participants’ choice of setting. With their consent, interviews will be recorded digitally and transcribed verbatim.In phases 1 and 2, we will use topic guides for the interviews to ensure consistency; however, the format will be flexible to allow participants to generate naturalistic data on topics they view as important. We will explicitly pursue negative cases (‘elements in the data that appear to contradict the emerging view’) ref. , p. 51 tAt the end of phases 1 and 2, for each of the six Traveller communities, we will (1) understand the potential barriers and facilitators for take-up of immunisations  and (2) have ideas for the content and delivery of potentially feasible and acceptable interventions to increase immunisation uptake for all five levels of the SEM. We will have insight into whether the barriers and facilitators and interventions are similar or different dependent on the gender, age and self-reported immunisation history of Traveller participants, the professional role of service providers and across different vaccines (childhood/adult). These outputs will be presented at the Feedback Workshops in Phase 3.Phase 3: A Feedback Workshop will be held locally for each Traveller community. The aim of the workshops is to disseminate the findings of phases 1 and 2 and to discuss and ‘co-produce’Phases 1 and 2: Interviews will be fully transcribed and data subjected to thematic analysis using the Framework approach,53Familiarisation—The interview transcripts will be read and emerging ideas and recurrent themes recorded.Identifying a thematic framework—A thematic framework will be set up in which the interview data will be organised. This will be informed by the SEM. Additional themes representing emerging issues from the data will be added. The framework will be applied to three to four transcripts to refine. The same thematic framework will be used to analyse the data for all the six Traveller communities and associated service providers.Indexing—The thematic framework will be systematically applied to the interview data.Charting—Charts will be drawn up for each theme and summaries of responses from participants (and verbatim quotes) entered. This will enable us to consider the range of views within each theme, that is, on (1) barriers, (2) facilitators to immunisation, (3) ideas for intervention across the five levels of the SEM; and (4) other emerging issues.Mapping and interpretation—The charts will be reviewed and interrogated to compare and contrast views, as well as to seek patterns, connections and explanations within the data.The five stages of analysis, specified below, will first be undertaken independently for each of the six Traveller communities and for both phase 1 and phase 2 data. This ‘within-community’ analysis will be led by CJ, LD and CE. Members of the research team based in each city will contribute at stages 2 and 5. This will enhance rigour and ensure that the local context in which the data are collected and where participants live is appropriately considered. NVivo software will facilitate data management.The final outputs of the analysis of phase 1 and 2 data will be a thematic synthesis that takes account of the inferences derived from all the interview data for the sample as a whole.This research has received approval from NRES Committee Yorkshire and The Humber—Leeds East Ref. 13/YH/02). Drawing on the British Psychological Society's Code of Human Research Ethics,3/YH/02. Comprehensive, in-depth findings on the barriers and facilitators to uptake and acceptability of childhood/adult immunisation both within and across six Traveller communities living in four cities across England and Scotland.A prioritised list of potentially feasible and acceptable interventions for increasing immunisation uptake across the five levels of the SEM for these Traveller communities.Methodological development in undertaking research with diverse Traveller communities living in different localities. Historically, research has been small scale, localised and with one community. Lessons learnt from this large-scale multi-city qualitative study can be used to improve the quality of future research in Traveller communities of a similar scale.There will be three key outputs:The findings will be published within the final report to the National Institute for Health Research Health Technology Assessment (NIHR HTA) programme. A local summary will be disseminated to the community and local organisations with whom we have worked to access/recruit Traveller participants to the study and to service providers in that locality who took part. A summary of the findings on the barriers and facilitators to uptake and acceptability of immunisation for all six Traveller communities will be sent to relevant national UK agencies and organisations responsible for the implementation of immunisation policy and Traveller health and welfare including professional bodies. We will also submit research papers to relevant professional journals and conferences. Academic papers will be submitted for publication in high-impact peer-reviewed journals.


Freezing, melting, evaporation and condensation of water are essential ingredients for climate and eventually life on Earth. In the present work, we show how surface freezing of supercooled water in an open container is conditioned and triggered—exclusively—by humidity in air. Additionally, a change of phase is demonstrated to be triggered on the water surface forming surface ice crystals prior to freezing of bulk. The symmetry of the surface crystal, as well as the freezing point, depend on humidity, presenting at least three different types of surface crystals. Humidity triggers surface freezing as soon as it overpasses a defined value for a given temperature, generating a plurality of nucleation nodes. An evidence of simultaneous nucleation of surface ice crystals is also provided. Nakaya demonstrated that snow crystals grow in cold humid air as a large variety of mainly plane crystals depending on both temperature and humidity of air 80 years ago  specifically design to control both temperature from −13.0 ± 0.1 up to 30.0 ± 0.1 °C and humidity from 5 ± 1 to 99 ± 1 %. A schematic of the experimental layout is shown in Fig. In all cases freezing is triggered at the certain point as shown in Fig. In all cases freezing is always nucleated at the surface. Plane single crystals are nucleated and grow on the very surface of the liquid supercooled water. Figures Figure As it can be seen in the figures and videos above, surface crystals frontline growth velocity strongly depends on temperature (and corresponding humidity as shown in Fig. The experiments described in the present paper demonstrate that the dependence of the freezing point of deionized water on humidity in air is intrinsic to the existence of the liquid water–air interface. The freezing temperature-humidity curves for open containers and for droplets are the same. For a given temperature, surface freezing is nucleated at the liquid water–air interface by a high enough humidity, forming a surface ice crystal prior to freezing of bulk. The symmetry of the surface crystal, as well as the freezing point, depend on humidity, presenting at least three different types of surface crystals. An evidence of simultaneous nucleation of Surface ice single crystals is also provided.


Control over the film-substrate interaction is key to the exploitation of graphene’s unique electronic properties. Typically, a buffer layer is irreversibly intercalated “from above” to ensure decoupling. For graphene/Ni(111) we instead tune the film interaction “from below”. By temperature controlling the formation/dissolution of a carbide layer under rotated graphene domains, we reversibly switch graphene’s electronic structure from semi-metallic to metallic. Our results are relevant for the design of controllable graphene/metal interfaces in functional devices. Interfacial interactions play a key role in tailoring the properties of graphene and other surface-supported two-dimensional 2D) materials. Remarkably, the effect of such interactions is not limited to the film, but extends to the structures in contact with it, e.g. adsorbed atoms1D materia3678Nickel is an appealing choice in the attempt to engineer such a model system, as it is one of the most technologically relevant substrates for graphene production. Among transition metals, it has a low-cost and is widely available; further, it can readily sustain graphene growth by chemical vapour deposition (CVD), with the additional advantage of requiring lower temperatures than other catalysts10111317182021620A striking feature of the nickel-graphene system is the formation of interfacial nickel carbides, which segregate from bulk Ni upon cooling from growth to room temperature (RT) and form exclusively below rotated graphene domainsTo optimize the relative coverage and the size of the rotational domains, we grew graphene on clean Ni(111) by exposing the sample to ethylene at 540–610  °C. Subsequently, the sample was cooled to RT with a cooling rate of about 1 K/s. Following this procedure, graphene displays coexisting EG, RG and RGC domains. Our microprobe low energy electron diffraction (μ-LEED) data show that in micron-sized RG domains, the lattice is preferentially oriented ± 17° from one of the Ni main crystallographic directions, even though domains with different rotation angles are also observed. A statistical analysis of the distribution of rotational orientation of RG regions can be found in the The brightfield (BF) LEEM image in To resolve each phase in real space, we imaged the film using darkfield (DF) LEEM, a method that uses secondary diffracted beams to map the lateral extent of a given surface phase. As shown in A  - surface nickel carbide; Cdis  - interstitial carbon dissolved into the near surface Ni layers; Cgr  - weakly-interacting graphene, identified with the RG domains; and CB  - epitaxial graphene, used as binding energy scale reference. We note that both components originating from below graphene (Cdis and CA) have very low intensity, limited by the low effective photoelectron attenuation length (~4.4 Å) at the kinetic energy usedB, thus indicating that carbon has a similar interaction with the substrate in the two cases. The apparent larger width of the CB peak (~28%) in RG regions is to be expected, considering that, while only two distinct sites contribute to the EG phaseB peak, originating from the adjacent RG regions, which contribute to the spectrum due to the limited lateral resolution of the microscope. However, the main peak is of Cgr type, with a small but evident (see inset) carbide component (CA) on the low-binding energy side. Such a component, absent in the spectrum from region II (see inset), confirms that region III corresponds to the RGC phase. Notably, the lower value of the CGr binding energy with respect to CB is typical of systems characterized by a weak interaction between graphene and the metal supportThe identification of the small patches labelled III is less straightforward. Their localization within the RG phase, small lateral size  ≤ 250 nm) and irregular shape suggest that they correspond to RGC areas, as previously imaged by STM150 nm and 2) comprising domains of both RG and RGC phases.The present results correct our previous indication of a different binding energy for C atoms in the EG and RG phasesF, plus a number of minor features at lower binding energies, already attributed to nickel and hybrid graphene-nickel states7F, as suggested by experiments performed at 40 KThe π band dispersion of the three different graphene phases was investigated by microprobe angle-resolved photoelectron spectroscopy (μ-ARPES). Since our data are collected from a region measuring about 2 microns in diameter, the contributions of the RG or RGC phases in the rotated regions cannot be separated. In 1927F, to image the surface. In the micrographs on the right hand side of In the MDC from mixed RG and RGC regions (D = EF – Ekin = 0.01 ± 0.06 eV). The Dirac energy ED was determined after calculating the intersection point of lines fitting the two branches of the π band, calculated in an energy interval extending 2 eV below EF. These lines were obtained after determining the exact position of each point of the π band branches in the  space. The error bar on ED (95% confidence level) is a consequence of the statistical errors on the intercept and slope of the lines, not of the limited energy resolution of the SPELEEM.The question now arises whether it is possible to control the population of the RGC phase in order to obtain a uniform carbidic buffer and whether this process is reversible. Clearly, such a capability would allow a drastic change in the electronic properties of graphene to be obtained and finely controlled. As shown in our previous work, the RGC phase is not present during growth, but forms only upon cooling towards RTUpon annealing , dissolves once more when it is raised (panels i-l), and so on. It is therefore possible to reversibly switch between the decoupled/coupled states by simply changing the temperature. We note that the switch between RGC and RG takes place only in the (220 °C–370 °C) temperature range. The carbide coverage remains constant upon further cooling below this range (not shown), while annealing in excess may lead to nucleation of bilayer graphene, as occurs for the thin bright stripe in panel (l). The fact that the conversion is active only in a narrow temperature range implies that rapid cooling of the sample from growth to room temperature yields an almost completely carbide free graphene layer. The temperature range where the switch is active is in agreement with previous investigations on the kinetics of C segregation on Ni samples. Indeed, in pioneering experiments, Blakely same rotated regions exist in two states: either decoupled or coupled to the substrate.The microprobe ARPES MDCs for the homogeneous RGC and RG phases (panels n and p) provide unequivocal evidence of the effect of the presence of the surface carbide: while direct strong interaction of graphene with Ni induces the formation of hybrid states and a pronounced modification of its electronic structure, carbide formation underneath the graphene sheet decouples microscopic regions of the layer from the substrateFinally, we have measured at high temperature the MDCs of the EG and RG phases see , observiIn summary, a thorough investigation of the electronic structure of graphene grown on Ni(111) and of its temperature dependence allowed us to devise a strategy to control the graphene-metal coupling. If a surface carbide buffer forms above the topmost Ni layer underneath rotated graphene, the 2D carbon network decouples from the substrate and its semi-metal nature is almost completely restored. Clear evidence is provided that micro-scale carbide domains can be reversibly formed and dissolved by just changing the temperature and deciding the time spent by the sample in the narrow temperature range where the conversion is active. A simple way to vary the graphene/substrate coupling is therefore provided, with significant implications for functional device fabrication and spintronics in particular. The lateral dimension of the switchable regions is only limited by the size of the rotated domains. Systematic studies are ongoing to explore the growth parameter space with the aim of obtaining rotated domains on the millimetre scale. + sputtering at 2 keV and annealing at 600 °C. The cleaning cycles were stopped when the sample did not show any C structure at RT in LEEM and μ-LEED measurements. Growth studies were performed in-situ by maintaining the sample at 540-610 °C while back-filling the chamber with C2H4 (p = 3∙10−6 mbar). The surface evolution during growth was monitored with LEEM.The Ni(111) surface was cleaned with Arstart. The kinetic energy of the electrons scattered (or emitted) by the sample is equal to Ekin = eVstart – δWi-s, the latter being the work function difference between the instrument and the specimen. In our measurements, LEEM was used in both bright and dark-field modes, utilizing, respectively, the zero or first order diffraction beam for imaging. The microscope lateral resolution approaches a few tens of nanometres in XPEEM mode, 9 nm in LEEM; the energy resolution is better than 0.3 eV. Besides imaging, the SPELEEM allows diffraction operation mode. Depending whether the beamline photons or low energy electrons are used as probe, μ-ARPES or LEED measurements can also be carried out. The probed area is defined by inserting a field limiting or illumination aperture in the first image plane along the optical path of the instrument. A 2 μm diameter field limiting aperture was used for μ-ARPES, while for μ-LEED three different illumination apertures were employed . Part of the ARPES measurements was performed using the darkfield PEEM methodThe photoemission microscopy measurements were carried out using the spectroscopic photoemission and low energy electron microscope (SPELEEM) at the Nanospectroscopy beamline of the Elettra synchrotron.How to cite this article: Africh, C. et al. Switchable graphene-substrate coupling through formation/dissolution of an intercalated Ni- carbide layer. Sci. Rep.6, 19734; doi: 10.1038/srep19734 (2016).


PD-L1 is expressed in many cancers, including non-small cell lung cancer (NSCLC). Immune cells play an important part in blocking the “cancer immunity cycle” by binding PD-1Cancer evades host immune surveillance by using immune checkpoints, which are inhibitory pathways crucial for maintaining self-toleranceEGFR) and translocated anaplastic lymphoma kinase (ALK). Majority of patients do not attain prolonged disease control, and 5-year survival rates remain lowPTEN leads to PD-L1 up-regulation in lung squamous cell carcinomaEGFR-mutant NSCLC may be susceptible to PD-1 blockade immunotherapyEGFR-mutant lung tumors inhibit antitumor immunity by activating the PD-1/PD-L1 pathway to suppress T-cell function and increase levels of proinflammatory cytokines; exposure to EGFR-TKIs leads to PD-L1 downregulationTreatment strategies for NSCLC include chemotherapy regimens based on histology and targeted agents for patients who carry somatic activated oncogenes, such as epidermal growth factor receptor  and the other 95 were EGFR-mutant, KRAS-mutant, or ALK translocated. All cases with moderate (+2) or strong staining (+3) in more than 5% of tumor cellsKRAS-mutant adenocarcinoma. By contrast, patients with PD-L1 positive tumors were more likely to be female and/or never or former smokers with EGFR-mutant or ALK-translocated adenocarcinoma histologyA recent study in the EGFR mutations remain controversial. Gettinger et al.EGFR or KRAS mutations did not correlate with response rate to nivolumab for NSCLC patients. Some researchers found that activation of the EGFR pathway induced PD-L1 expression to help NSCLC tumors evade the antitumor immune response,et al.EGFR, KRAS, BRAF, or ALK status in stage I NSCLC patients. Similarly, Zhang et al.EGFR or KRAS in lung adenocarcinoma. Most recently, Ansen et al.EGFR, KRAS, and STK11 in the two most common histological NSCLC subtypes  in the 2014 ASCO Annual Meeting.Until now, the results of the correlation between PD-1/PD-L1 expression and et al.KRAS mutations, whereas PD-L1 positive status was significantly associated with presence of EGFR mutations. Recent data presented by Rizvi et al.D’Incecco POLD1, POLE, and MSH2. Some of these mutations occur in never-smokers with high mutational burden; this finding may explain why some never-smokers may also respond to therapy with PD-1 inhibitorsBesides smoking-related changes, the researchers were also able to identify other mutations present in lung cancer that may contribute to a high mutation burden and response to PD-1 inhibition. Specifically, they noted deleterious mutations in DNA repair and replication genes that have high mutation burden response and high response to pembrolizumab, such as et al.EGFR-mutant patients, TTP to EGFR TKI was significantly longer in PD-L1 positive than negative tumorset al.et al.D’Incecco et al.+ TILs or explore any correlation that may exist between PD-1-positive TILs and expression of PD-L1 on cancer cells.D’Incecco et al.et al.et al. suggest that EGFR-mutant NSCLC is highly eligible for PD-1/PD-L1 immunotherapy, and PD-L1 may represent a favorable biomarker candidate for response to EGFR-TKIs. If this finding is reconfirmed in prospective studies, then immune checkpoint blockade combination with EGFR TKIs could be a major step forward in improving outcomes of EGFR-mutant NSCLC patients.A suitable test should be created to measure PD-L1 expression levels with established thresholds that can be used as a biomarker for anti-PD-1/PD-L1 therapies. A multitude of questions pertaining to companion predictive biomarkers to anti-PD-1/anti-PD-L1 therapies remain unanswered: Which PD-L1 antibody most accurately and reproducibly measures PD-L1 protein expression and predicts response to therapy? Which cutoff should be utilized to determine PD-L1 positivity/negativity? Should PD-L1 protein be measured in the tumor epithelium, stroma, or both? Should a different measure of PD-L1 expression, such as quantitative immunofluorescence or RNA, be used instead of standard immunohistochemistry protein methodology? Which additional components, such as TILs, PD-1, or PD-L2, play a role in predicting response? Currently, the various assays tend to be propriety to each of the groups developing the antibodies. Most assays examine PD-L1 staining on the tumor. Based on recent data from Herbst


In live Drosophila fat body tissue from third instar larvae, ReZolve-L1™ interacted mainly with lipid droplets, including the core region of these organelles. The presence of polar lipids in the core of these lipid droplets was confirmed by Raman mapping and while this was consistent with the distribution of ReZolve-L1™ it did not exclude that the molecular probe might be detecting other lipid species. In response to complete starvation conditions, ReZolve-L1™ was detected mainly in Atg8-GFP autophagic compartments, and showed reduced staining in the lipid droplets of fat body cells. The induction of autophagy by Tor inhibition also increased ReZolve-L1™ detection in autophagic compartments, whereas Atg9 knock down impaired autophagosome formation and altered the distribution of ReZolve-L1™. Finally, during Drosophila metamorphosis fat body tissues showed increased ReZolve-L1™ staining in autophagic compartments at two hours post puparium formation, when compared to earlier developmental time points. We concluded that ReZolve-L1™ is a new live cell imaging tool, which can be used as an imaging reagent for the detection of polar lipids in different intracellular compartments.Lipids have an important role in many aspects of cell biology, including membrane architecture/compartment formation, intracellular traffic, signalling, hormone regulation, inflammation, energy storage and metabolism. Lipid biology is therefore integrally involved in major human diseases, including metabolic disorders, neurodegenerative diseases, obesity, heart disease, immune disorders and cancers, which commonly display altered lipid transport and metabolism. However, the investigation of these important cellular processes has been limited by the availability of specific tools to visualise lipids in live cells. Here we describe the potential for ReZolve-L1 The. The™ lotructure . The loc675 cm-1 , as the 675 cm-1 , and spe1780cm-1 , which i-1 attributed to the ν(P-O), which is unique to phospholipids, such as phosphatidylcholine, phosphatidylethanolamine and sphingomyelin [ν(P-O) band (from the second derivative) with the ν(C = O) band, for the detection of triglycerides and sterol esters [™ in the lumen of Drosophila fat body lipid droplets.Further interrogation of the second derivative of the spectra taken from the lipid droplet revealed the presence of a band at ~ 1096cmgomyelin  Fig 3C)-1 attribl esters  it was pex vivo models  combined with Raman spectroscopy of both a 3T3 adipocyte cell line and Drosophila tissue provide evidence for the localisation of ReZolve-L1™ in cells with polar lipids. With the Raman data suggesting that lipid droplets are more heterogeneous in lipid composition, which may account for the dispersed staining of lipid droplets with ReZolve-L1™, rather than an interaction with non-polar lipids. However, it should be noted that this does not discount an interaction between ReZolve-L1™ and non-polar lipids (such as CEs and/or TAGs), but that the evidence suggests a preferential interaction with polar lipids. The authors suggest that the observed localisation patterns of ReZolve-L1™ is not due to an interaction with one ‘specific’ lipid, but more closely aligned with the polar lipid families and localisation pattern may in fact be the result of a particular combination of lipid(s).The accumulation of data from both the ™ could also detect autophagosomes, Drosophila fat bodies expressing the autophagic marker Atg8-GFP were stained with ReZolve-L1™ following exposure of the larvae to different autophagy inducing starvation conditions. Staining with Oil Red O was performed in parallel to allow for a comparison between a purely non-polar lipid dye and ReZolve-L1™; and to provide further evidence of the greater affinity for polar lipids.Autophagosomes are cellular compartments that are involved in cell degradation and like lipid droplets can accumulate lipids from a range of cellular components , 60. Pro™ was detected mainly in lipid droplets , these compartments had minimal to no Oil Red O staining  [TorTED reduced the fat body volume in larvae at later developmental time points, which prevented the analysis. TorTED expression increased the number of autophagic compartments detected by LysoTracker® Green in larval fat body tissue [® Green compartments detected in response to TorTED expression were co-stained by ReZolve-L1™, but there were some compartments that appeared to contain either only LysoTracker® Green or only ReZolve-L1™  , 63. Faty tissue , when coolve-L1™ . In contartments  and was artments . This ag™ to autophagic compartments, autophagosome formation was disrupted by RNAi silencing of the core autophagy protein Atg9 [Atg9RNAi, under the control of the fat body specific driver CG-GAL4, were either fed on standard media, or amino acid and sugar starved for four hours. In the control and Atg9RNAi fed larvae ReZolve-L1™ was detected in lipid droplets and was not detected in LysoTracker® Green compartments . At -4 h PF ReZolve-L1™ was detected primarily in lipid droplets and not in LysoTracker® Green compartments  and these compartments had some ReZolve-L1™ staining Drosophilysosomes . In addiysosomes . These fDrosophila fat body tissues. In these tissues ReZolve-L1™ was also localised to lipid droplets and was detected throughout the organelle. Although the mode of action of ReZolve-L1™ requires further analysis, lipid overlay experiments, liposome affinity experiments and Raman mapping of stained adipocytes showed positive correlations between ReZolve-L1™ and polar lipids to a much greater extent than neutral lipids. Although we cannot rule out that the lipophilic nature of ReZolve-L1™ drives the localisation of this molecular probe to the lipid droplet core, it is speculated that ReZolve-L1™ is localising with and hence detecting polar lipids in both the outer membrane and the core of lipid droplets. This hypothesis is supported by Fourier transform infrared microspectroscopy, which demonstrated a correlation between the location of polar lipid species such as phosphatidylethanolamine and sphingomyelin and ReZolve-L1™ in 3T3-L1 adipocytes [™ was most often detected in lipid droplets and was rarely associated with other cellular membrane structures where polar lipids are known to be abundant. This lack of other cell membrane staining may indicate that polar lipid packing in these membranes prevents ReZolve-L1™ interaction or that the interaction site is masked, or alternatively that ReZolve-L1™ only detects certain combinations of polar lipids.Lipid droplets are most often described as cytosolic organelles consisting of a neutral lipid core, surrounded by a monolayer of phospholipids , 70. Altipocytes . Interes™ was detected in autophagic compartments in response to certain starvation conditions, the inactivation of the target of Rapamycin (TorTED), or developmental cues. The ReZolve-L1™ staining of lipid droplets was also altered in response to these cues, and these changes in distribution may involve the trafficking of polar lipids during interactions between lipid droplets and autophagosomes.Autophagy has a pivotal role in lipid localisation, for example, to orchestrate the vesicular traffic and secretion of triglyceride rich lipoproteins, as well as to mediate the transient storage of lipids in lipid droplets , 76, 77.™ appeared to be directed to autophagosomes as a selective response to certain autophagic cues, rather than as a general response to autophagy induction. Under conditions of partial nutrient restriction by amino acid starvation, autophagosomes were formed, but ReZolve-L1™ had little interaction with these autophagic compartments. This would suggest that ReZolve-L1™ did not detect the polar lipids acquired from all cellular organelles that might be degraded under these conditions. ReZolve-L1™ did, however, co-locate with Atg8-GFP autophagic compartments following combined amino acid and sugar starvation, which indicated that it was recognised a specific event during autophagy. Amino acid and sugar starvation did induce lipid droplet degradation via autophagy, as shown by Oil Red O co-localisation with autophagosomes. While it is commonly accepted that neutral lipids are transferred to autophagic compartments in response to autophagy induction, by either combined amino acid and sugar starvation or the inhibition of Tor [™ detects polar lipid sequestration from lipid droplets by autophagosomes under these conditions. The down regulation of autophagy by genetic inhibition is known to reduce lipid transfer from lipid droplets to autophagosomes [™ detection in autophagosomes following autophagic reduction by Atg9RNAi knock down. Atg9RNAi knock down did not completely abrogate autophagic progression [™ positive structures near the periphery of the lipid droplets; which could be indicative of altered autophagosome formation and reduced incorporation of lipid into autophagosome membrane, a process in which Atg9 is suggested to be involved [Drosophila fat body, if lipid membrane content in autophagosomes is increased under certain conditions, however this may be an alternative explanation for the detection of ReZolve-L1™ in autophagic compartments.The lipids detected by ReZolveL1n of Tor , 62, 63,n of Tor , 80. Thuagosomes , 81, andgression , but cauinvolved , 83. Theinvolved . It is u™ had an altered staining pattern upon sugar starvation alone, but unlike exposure to combined amino acid and sugar starvation, similar changes were not observed in Oil Red O staining. This difference in staining patterns supports the interaction of ReZolve-L1™ with lipids that are distinct from the neutral lipids detected by Oil Red O. Furthermore, this suggested that the polar lipids detected by ReZolve-L1™ have increased association with autophagic compartments during the energy depravation induced by sugar starvation. The induction of autophagy by most signalling pathways converge at TOR but in contrast, energy depletion can lead to the direct activation of autophagy via interactions between energy sensing AMPK and the autophagy initiator Atg1/ULK1 [™ staining was detected throughout the cells with some brighter puncta seen localising with autophagic structures, unlike the staining observed following combined amino acid and sugar starvation, which was highly concentrated in autophagic compartments. This suggested a change in polar lipid distribution under these conditions, which may be associated with either an alternative signalling pathway being activated or a specific event in autophagosome maturation.Interestingly, ReZolve-L1tg1/ULK1 . FollowiDrosophila development, autophagy is important for the degradation and remodelling of larval tissue, including the fat body [Drosophila, and these lipids can be mobilised to provide energy or used as structural lipids for developing tissues during metamorphosis [Drosophila, the mobilisation of lipids during metamorphosis is in part facilitated by lipases, such as the lipid droplet associated Brummer and lysosomal acid lipase-1 [Drosophila metamorphosis [Drosophila fat body, at the later developmental stage of +2 h PF, ReZolve-L1™ staining was detected in autophagic compartments that were co-stained by LysoTracker®Green and Atg8a-GFP. Neutral lipids were also detected in these autophagic compartments at +2 h PF, which provided the first evidence of autophagy as a route for lipid delivery to the lysosome during metamorphosis. The detection of both neutral and polar lipids in the autophagosomes at the later developmental time point may also indicate that bulk degradation of lipid droplets is occurring at this stage.During fat body , 86. Theorphosis , 87. In lipase-1 . The invlipase-1  and the lipase-1  has led orphosis . In Dros™ appears to be able to identify the transfer of polar lipids from lipid droplets to autophagic compartments under conditions of high energy demand and during developmental autophagy. By reporting on this dynamic balance between lipid droplets and autophagic compartments during different energy requirements, ReZolve-L1™ has the potential to define new aspects of cellular lipid metabolism. ReZolve-L1™ overcomes many of the critical limitations for conventional molecular probes, including photobleaching, extended sample preparation, and the need for cell fixation, which could enable live cell imaging of specific metabolic events.There is a paucity of live cell imaging molecular probes available, to allow the specific investigation of lipids and to support the wider field of lipid metabolism. ReZolve-L1™ could be used to visualise the pathology in for example: the altered lipid metabolism associated with fatty liver disease; the increased autophagy and altered degradation of lipids observed in Alzheimer’s disease; the altered cholesterol deposition and plaque formation in cardiovascular disease; or the altered balance between lipid metabolism and autophagy in cancer. Therefore ReZolve-L1™ may provide a promising new tool for the live cell imaging of polar lipids and autophagy that has practical applications for both cell biology research and clinical diagnosis/prognosis.The bidirectional interaction of autophagy and lipid metabolism has important implications for disease pathophysiology and there is a defined need to develop new imaging technology for these disease processes . Consequiso-propanol for loading depending on solubility. Following lipid loading, the PVDF membranes were washed for 10 minutes in cold 10% ethanol, methanol or PBS, before they were incubated with 10μM of rhenium molecular probe in PBS for 1h. The neutral rhenium(I) species, ReZolve-L1™, was prepared according to a previously published procedure [To determine the binding properties of the rhenium molecular probe, a lipid overlay was performed. 50 μM of each lipid was loaded on to a PVDF membrane . Ten lipids were analysed including; sphingomyelin , sphingosine , ʟ-α-phosphatidylethanolamine , monosialodanglioside , oleoly-ʟ-α-lysophosphatidic acid , palmitic acid , cholesterol , ceramide , triacylglycerol mix C2-C10  and cholesteryl acetate . Lipids were dissolved in ethanol or sn-glycero-3-phosphocholine  and cholesterol , were dissolved in 2:1 chloroform:methanol at 20 mg/mL. Lipid mixtures were formed by evaporating liquid 666 μL DMPC alone or a mixture containing 443 μL of DMPC and 32 μL of cholesterol (8:1 molar ratio). The lipid mixture was then resuspended in 1 mL PBS and sonicated for 40 minutes. Following sonication liposomes were passed through an extruder with a 100 nm membrane 15 times. Liposome formation was confirmed using a Malvern Zetasizer DLS system. Mixtures were run through a Sephadex G-25 column to ensure no free lipid was present in solution. Fraction containing particles of approximately 100 nm (ascertained by DLS) were incubated with ReZolve-L1™ for 40 minutes. Solutions were then passed through a Sephadex G-25 column to remove unbound stain and fraction containing vesicles of approximately 100 nm were tested for fluorescence’s by flow cytometry and fluorometry.1,2-dimyristoyl-4 events were collected using 40x objective and camera with pixel size of 0.5 μm. Vesicles were identified by their low SSC signal and high fluorescence (405/560-595 nm) values; the accuracy of this separation was proven by viewing the images of single events.For analysis of vesicles by flow cytometry an Amnis ImageStreamX Flow Cytometer mkII (EMD Millipore) was used. Vesicles were excited with 405 nm laser (120 mW) and fluorescent data between 560–595 nm were collected. In parallel, data for forward scatter (FSC) and side scatter (SSC) light  were collected. Core diameter of the flow was 10 μm and flow speed was 66 mm/sec. Calibration beads (2 μm in diameter) were run in parallel to the samples. For each sample, images for 1.5–5.0x10™.For analysis of vesicles by fluorescence a Varian Cary Eclipse spectrophotometer was used. Vesicles were excited at 256 nm and emission was collected from 350–700 nm. Vesicles were tested for fluorescence before and after incubation with ReZolve-L1GAL4-UAS system was used for targeted gene expression [CG-GAL4 [UAS-Atg9RNAi stocks (#34901) and UAS-TorTED stocks (#7013) were obtained from the Bloomington Drosophila Stock Centre . Atg8a-GFP was driven by an endogenous promoter [Mammalian 3T3 adipocytes were obtained from culturing and differentiated the 3T3 pre-adipocyte fibroblast on calcium fluoride slides for Raman microscopy, as previously described  for a mipression . Fat-bod[CG-GAL4 . Transgepromoter  and was Bromophenol-blue was added to the fly media and used to define different developmental time points . Third iThird instar larvae at -8 h puparium formation (PF) were removed from standard media and placed on filter paper either soaked in 5% (w/v) sucrose (for amino acid deprivation), or 5% (w/v) tryptone pancreatic digest of casein , or soaked in water (for amino acid and sugar starvation). Larvae were incubated at 25°C for 4–5 hours prior to tissue isolation and imaging.™ was prepared according to a previously published procedure [™ was dissolved in DMSO to prepare a 10 mM stock solution, which was diluted in PBS to 10 μM for tissue staining. For Raman confocal microscopy, 3T3 cells were fixed using cold methanol and air dried [™ overnight. Fat body tissue was dissected from Drosophila larvae or pupae into PBS. For Raman confocal microscopy, the tissue was fixed in 4% paraformaldehyde  ready for mapping. For live confocal and two photon microscopy, the fat body tissue was incubated with ReZolve-L1™ for 15 minutes at room temperature and washed in PBS for 30 seconds. Tissues were counterstained for acidic compartments with LysoTracker® Green  for 2 minutes at room temperature. Tissues were then mounted in carbomer-940  based optical coupling gel to prevent dehydration prior to imaging; see protocol in [The neutral rhenium(I) molecular probe ReZolve-L1rocedure . ReZolveir dried  before btocol in . For Oil™ HR mapping. The spectrometer was fitted with holographic notch filters and 2400 mm/line (NIR) grating. The attached microscope was a Leica DMLM and was equipped with three objectives  and a trinocular viewer that accommodates a video camera allowing direct viewing of the sample. Sample excitation was achieved using a Modu-Laser  emitting at 785 nm. Calibration of the wavenumber axis is achieved by recording the Raman spectrum of silicon  for both static and extended modes. If necessary an offset correction is performed to ensure that the position of the silicon band is at 520.50 ± 0.10 cm-1. Spectra were not corrected for instrument response. The spectrometer was controlled by PC with instrument control software . The Raman maps were acquired using a step size of 2 μm for both x and y axes over the spectral range of 700–1850 cm-1.Raman spectra were collected using a Renishaw Raman inVia Reflex Microscope , equipped with an air-cooled charge-coupled device (CCD) cameras for StreamLineCosmic rays were removed from the spectral data using a nearest neighbour cosmic ray removal method and noise filtering was applied using the Renishaw WiRE 4.2 software. The false colour maps shown generated from the data using the direct analysis approach by calculating the area under bands of interest using the signal-to-baseline mode.®  was used for live imaging. The images were acquired using a Plan- APOCHROMAT 63X/ NA1.4 oil immersion objective. ReZolve-L1™ was excited at 830 nm using the two-photon pulse laser and detect at 600–654 nm. LysoTracker® Green was imaged by excitation at 488 nm and detected at 497–558 nm. Atg8a-GFP was imaged with ReZolve-L1™ by excitation at 830 nm (to excite both fluorophores) and resolved using spectral un-mixing using the Zen software package . A spectral profile for ReZolve-L1™ was acquired in lambda stack mode .A Ziess LSM710 META NLO confocal microscope , supplemented with a two-photon Mai-Tai™ images and co-location analysis involved at least five separate images from five independent biological replicates, and one representative image from each treatment was selected for presentation. For image quantification, two regions of interest (ROI) were selected at random, with an area of 2500 μm2 and the number of compartments stained with ReZolve-L1™ and LysoTracker®Green were counted to give a minimum of ten counts per treatment group. Comparison of means was performed by ANOVA with a Tukey post hoc tested in GraphPad Prism V.6.01 (USA).The ReZolve-L1S1 FigA) and DMPC and cholesterol vesicles (DMPC:cholesterol 8:1 molar ratio) (B) incubated with ReZolve-L1™ when excited at 405nm and representative fluorescence and transmitted lights images of vesicles by flow cytometry. (C) Fluoroscence spectra of liposomes alone (dashed grey line), ReZolve-L1™ alone (dotted grey line) and liposomes incubated with ReZolve-L1™  when excited at 256nm.Histogram showing fluorescence intensity 560-595nm of DMPC vesicles ((TIF)Click here for additional data file.S2 FigA) Structure and Raman spectrum of ReZolve-L1™ and (B) the second derivative of this spectrum. (C) Second derivatives of Raman spectra taken from a ReZolve-L1™ stained 3T3 adipocytes from a region of high (a) medium (b) and low (c) ReZolve-L1™ presence.((TIF)Click here for additional data file.S3 FigRepresentative Raman spectra from the lipid droplet core of -4 h PF larval fat body tissue. Corresponding second derivatives are presented above each of the five spectra. Important lipid regions are shaded and assigned above.(TIF)Click here for additional data file.S4 FigA) Confocal micrographs of Drosophila fat body cells explanted from +2 h PF from pupae expressing Atg8a-GFP (green) and stained with ReZolve-L1™ (red). (B) Confocal micrographs of Drosophila fat body cells explanted from +2 h PF from pupae expressing Atg8a-GFP (green) and stained with Oil Red O (red). LD indicates lipid droplets arrows indicate co-location between ReZolve-L1 or Oil Red O and Atg8a-GFP autophagic compartments. Scale = 10 μm.((TIF)Click here for additional data file.