Group I catalytic introns have been found in bacterial, viral, organellar, and some eukaryotic genomes, but not in archaea. All known archaeal introns are bulge-helix-bulge (BHB) introns, with the exception of a few group II introns. It has been proposed that BHB introns arose from extinct group I intron ancestors, much like eukaryotic spliceosomal introns are thought to have descended from group II introns. However, group I introns have little sequence conservation, making them difficult to detect with standard sequence similarity searches. Taking advantage of recent improvements in a computational homology search method that accounts for both conserved sequence and RNA secondary structure, we have identified 39 group I introns in a wide range of archaeal phyla, including examples of group I introns and BHB introns in the same host gene. Group I introns are canonical examples of catalytic RNAs ,2. They et al.  which returned 591 443 sequences comprised of 6 710 959 751 total nucleotides.--anytrunc which can improve performance on truncated or interrupted sequences, such as group I introns interrupted by embedded HEGs. Hits with an E-value ≤0.01 were kept, and overlapping hits were removed, keeping the hit with the lowest E-value. Highly similar hits were removed such that no two remaining hits were more than 97% identical given their alignment to the CM.Infernal v1.1.2  was usedE-value threshold of ≤10−3.Homing endonuclease gene (HEG) homologs were detected by translating each of the 39 introns in all six frames and searching all ORFS ≥20aa against Pfam 31.0  using th--rfam, which accelerates searches by enforcing strict sequence-based filters.Large subunit (LSU) and small subunit (SSU) rRNA sequences were identified using cmsearch in all sequences with an IB4 or IA3 hit using the Rfam 13.0  archaealTo check that host LSU and SSU rRNA sequences were archaeal in origin, we scored each rRNA sequence with the domain-specific Rfam covariance models LSU_rRNA_archaea, LSU_rRNA_bacteria, and LSU_rRNA_eukarya  or the corresponding SSU models  and checked that the highest-scoring alignment was to the archaeal profile. This is only suitable as a coarse-grained check; similarity scores to multiple alignment profiles are a crude substitute for phylogenetic classification on trees.Taxonomic classification of 25 SSU rRNAs identified on the same contigs as the introns was done on the RDPClassifier web server  using tret al.  and further subdivided into fourteen subtypes  based on variation in conserved consensus secondary structure ,51. No set al.  and usedet al. , lightlyThe quantity and diversity of known archaeal genome sequence has grown rapidly recently, due to metagenomic studies of uncultivated archaea ,36–44, iE-value , the estimated number of false positives at that score threshold. After removing overlapping hits and keeping the hit with the lowest E-value, there were no significant hits at a threshold of E < 0.01 for ten subtypes and the RF00028 model. IA2 and IC3 searches found three and two significant hits, respectively, but upon examination, we were unsure whether these were truly in archaeal sequences, as opposed to misannotated or contaminating bacterial or phage DNA, and we did not consider these further.We searched this archaeal sequence data with each of the 14 group I subtype CMs and with the Rfam Intron_gp1 model. Most of the 15 searches take less than 30 minutes on four CPU cores. The IA1 and IC3 subtype alignments include large HEG insertions, and these larger queries take 1–4 h. The Infernal search program, cmsearch, assigns log-odds probability scores to RNA secondary structure alignments and ranks putative hits by a measure of the statistical significance, the E-value of 0.01 or less, after we removed overlapping, lower scoring hits. We examined these hits and removed 25 that corresponded to redundant identical sequences in the archaeal database; seven where the sequence record does not contain a complete intron; three where we were not confident that the sequence folded into a complete group I consensus structure; and four where we were not confident that the host gene is an archaeal sequence. Because introns may contain nonconserved insertions including HEGs, a single intron may be identified by Infernal in more than one local alignment piece. After assigning the remaining hits to single introns, we identified a total of 39 nonredundant, full length putative group I introns, 27 IB4s and 12 IA3s. Most were identified by at least one strongly significant hit; 30 of the 39 have at least one hit of E < 10−10. Table The IB4 and IA3 CM searches found a total of 95 significant hits with an We looked at three other lines of evidence, independent of Infernal similarity scores, that provide additional support for a conclusion that these are group I introns.E. coli coordinate numbering  and P7 is aligned only as primary sequence. Identifying a P7 helix, other secondary structure elements typical of group I introns, and additional base pairs that extend Infernal-predicted consensus helices provide more confidence in the predicted introns. We manually identified a P7 helix in all 39 predicted introns . We manuThe 39 introns are found in 31 different GenBank sequences annotated as archaeal, all of which were obtained by metagenomic environmental sampling. Because GenBank annotation can be unreliable, especially for the phylogenetic source of sequences obtained in metagenomic samples, we also checked that each host rRNA gene sequence scored higher for archaeal SSU and LSU rRNA Rfam consensus models than to bacterial or eukaryotic models. An example of a sequence that failed this check is an LSU rRNA in GenBank MBAA01000200.1, a 4.7 kb contig that was binned into a Lokiarchaeota genome assembly, but appears to be bacterial. We had already named two introns from this contig (IA3.13 and IB4.16) before removing them from our analysis, which is why they are missing in Table We asked whether group I introns are broadly distributed across the archaeal phylogeny, or if they only occur in some particular clade. Specific phylum assignments are annotated in 25 of the 31 GenBank records. Both because of the unreliability of these annotations, and also because the taxonomy of archaeal phyla has been expanding ,35,52, wet al. (Various sources say that group I introns have not been identified in archaea before ,12,55. Het al.  referredet al. .Infernal searches are convenient but not strictly necessary to find these introns. BLAST searches can also identify significant similarities between some of these archaeal intron sequences and some known group I introns in bacteria and eukarya. One reason they have been missed in previous archaeal genome annotations may be that a few informative similarities would be hard to spot amidst a long list of BLAST hits to rRNA and HEG sequences.The failure to identify group I introns in archaea has previously been attributed to their having evolved into BHB introns and gone extinct . It remaDevelopment of CM-based RNA similarity search methods was originally motivated by a desire to search for group I introns , but untSupplementary DataClick here for additional data file.


Post-traumatic stress disorder (PTSD) has a profound contextual component, and has been demonstrated to alter future contextual learning. However, the mechanism by which a single traumatic event affects subsequent contextual experiences has not been isolated. Acetylcholine (ACh) is an important modulator of hippocampus-dependent learning such as contextual memory strength. Using Stress-Enhanced Fear Learning (SEFL), which models aspects of PTSD in rats, we tested whether muscarinic acetylcholine receptors (mAChR) in dorsal hippocampus (DH) are required during trauma for the effect of trauma on subsequent contextual fear learning. We infused scopolamine or vehicle into DH immediately before stress, and tested fear in both the trauma context and a novel context after a mild stressor. The results show that during learning, ACh acting on mAChR within the DH is required for sensitization of future contextual fear learning. However, this effect is selective for contextual learning, as this blockade leaves discrete cue sensitization intact. Rather than simply sensitizing the BLA, as previous studies have suggested, SEFL requires cholinergic signaling in DH for contextual sensitization. For fear to be adaptive, it must be titrated to the level of threat and relatively specific for threat-related stimuli. Both aspects of this fear responding are dysregulated in anxiety disorders such as post-traumatic stress disorder (PTSD), disorders where fear responses are enhanced and disrupt an individual’s normal functioning . Though Though the amygdala is likely to be the core structure in the stress circuit leading to PTSD, the hippocampus has also been shown to play an important role. MRI studies of PTSD patients frequently report substantial loss of gray matter in the hippocampus, though there is debate as to whether this is caused by the trauma or due to an underlying predisposition for diagnosis; in rodent studies, both circumstances have been observed . FurtherProper hippocampal functioning, particularly during contextual memory acquisition, depends upon cholinergic inputs originating from the medial septum in the basal forebrain cholinergic system. These inputs are critical for the formation of normal contextual associations, as blockade of muscarinic receptors by scopolamine in the hippocampus prevents contextual fear learning while leaving tone fear learning intact , and enhAnimal models of aspects of PTSD such as stress-enhanced fear learning (SEFL) have recapitulated many aspects of human PTSD symptomology, such as resistance to extinction therapy , and senThe relationship between cholinergic signaling during the trauma and manifestation of the SEFL phenotype is currently unknown. In our first experiment, we infuse scopolamine or aCSF vehicle into the dorsal hippocampus (DH) 1 h prior to SEFL, and demonstrate that scopolamine disrupts both trauma context memory formation and later sensitization of fear in a novel context. In our second experiment, we administer a tone-shock pairing instead of a context-shock pairing as the novel mild stressor, and demonstrate that scopolamine in DH does not block this sensitization, suggesting that cholinergic signaling in DH during trauma sensitizes future contextual learning, but not future learning about discrete stimuli. Finally, we provide evidence that these effects are not due to state-dependent effects of scopolamine administration, nor by blockade of consolidation, but are rather a consequence of blocking cholinergic-dependent contextual sensitization during a traumatic experience.ad libitum. Animals were handled daily (one-two min per rat) for at least 1 week prior to the start of all experimental procedures. The procedures used in this study were in accordance with policy set and approved by the Institutional Animal Care and Use Committee of the University of California, Los Angeles.A total of 64 naïve male Long-Evans rats, weighing 270–300 g  were individually housed and maintained on a 12-hour light/dark cycle with access to food and water One week after housing, rats received surgical implantation of two guide cannulae aimed at the dorsal hippocampus. DH cannulae placements are shown in ® to provide the context with a scent. Chambers were individually lit from above by white lights and cleaned with 50% Windex in between squads. Fans mounted above each chamber provided background noise (60 dB). The experimental room was brightly lit with overhead white light. Animals were transported to the context in squads of eight in their home cages, which were slid onto hanging racks mounted to a portable cart and covered with a white sheet or black sheet. All aspects of the context were altered to create a distinctive single shock context. This context was comprised of an alternating large small or height-staggered grid floor and a black plexiglass A-frame. The context light was off, the experimental room light was red, and chambers were cleaned and scented with a 7% acetic acid solution. Rats were transported to the context in groups of 4 in a black tub with individual dividers and bedding on the floor. Many aspects of the context were altered again for the cohort of rats that received tone conditioning and tone test in a separate, third context. This context consisted of a white plexiglass floor, white curving plexiglass rear wall in order to make the chamber shaped like a semicircle, context light off, red and white experimental chamber lights concurrently on, and cleaned with Pine Sol. Groups of four rats were transported to this context together in a large transparent plastic tub with blue pads on the floor. All chambers were cleaned with a 10% bleach solution following each day of behavioral testing.Behavioral training used a set of four identical fear conditioning chambers  equipped with a Med-Associates Video Freeze system. Individual boxes were enclosed in sound-attenuating chambers in an individual, dedicated experimental room. The SEFL context was comprised of chambers with aluminum sidewalls and a clear Plexiglas rear wall. The grid floor consisted of 16 stainless steel rods (4.8 mm thick) spaced 1.6 cm apart (center to center). Pans underlying each box were sprayed with a thin film of 50% WindexSEFL Context procedure is detailed in To assess cannulae placements, rats were anesthetized with isoflurane and decapitated. Brains were removed from the skull and placed in 10% formalin / 30% sucrose solution for 3 days prior to sectioning using a cryostat. Coronal sections (40 μm thick) were taken throughout the extent of the cannulae track and mounted on slides. Injection sites were reconstructed using bright field microscopy. Rats that had one or both cannulae or injector tracks outside the target structure were excluded from analysis .F = 22.53, p < 0.0001], Main effect of Shock Number , significant Interaction , Sidak’s multiple comparisons test, difference between vehicle and SCOP groups from PS1 (p < 0.01) through PS6 (p < 0.05), but no difference on subsequent trials (Naïve rats received an infusion of either scopolamine (SCOP) or vehicle (aCSF) into the dorsal hippocampus, and 1 h later experienced the 15-shock SEFL procedure or an equivalent context exposure with no stress . As ratst trials . These rF = 52.80, p < 0.0001), Drug main effect  = 17.68, p < 0.001), significant interaction between SEFL and Drug  = 14.71, p < 0.001); SCOP reduced fear in SEFL rats (p < 0.001) but not NS rats (p > 0.05)]. These findings suggest that cholinergic signaling in the DH, specifically at muscarinic receptors, is essential for the formation of a strong traumatic contextual memory.Though the effect of scopolamine infusion during trauma was mild, the effects on trauma memory recall were profound . While vWhile scopolamine is sufficient to disrupt the memory of a traumatic experience, it was unknown whether this temporary disruption would affect future fear learning to a novel contextual stimulus. Off drug, these rats were exposed to a novel context and given a single shock, and contextual freezing was measured the following day in the same context to test contextual sensitization.F = 32.37, p < 0.0001) and Drug  = 9.53, p < 0.01] and a significant interaction between SEFL and Drug ; SEFL rats who received scopolamine froze significantly less than those that received vehicle (p < 0.01).In vehicle rats, the prior stress enhanced subsequent fear learning to the new conditioning context, demonstrating the sensitizing nature of trauma to future fear learning . HoweverThis blockade of future contextual sensitization is not an obvious consequence of blocking traumatic memory formation, as many manipulations that disrupt this trauma memory have no effect on later contextual sensitization . Rather,Instead of a context-shock pairing, a cohort of rats received a single tone-shock pairing after the SEFL procedure. The following day, they were exposed to a novel context and the tone was played, in order to test sensitization to discrete stimuli .p > 0.05). Though some rats in the non-stressed scopolamine group had high initial freezing to the tone, which drove an interaction, the majority of non-stressed rats had very low responding to the tone . The disproportionate response of the SEFL groups continued in the period of time after the tone ended, while both non-stressed groups expressed normal levels of freezing . Though non-stressed vehicle rats did significantly increase freezing levels between the tone and post-tone periods (p < 0.05), freezing remained below both SEFL groups at that time point (p < 0.05). Together, these data suggest that scopolamine in DH does not block all consequences of the traumatic experience. Rather, it has a selective effect on future contextual sensitization.Despite the blockade of fear to the trauma context, scopolamine during trauma did not prevent future sensitization to discrete cues . Low basp > 0.05), suggesting that state-dependent effects are not the main mediator of the low levels of context fear seen in the scopolamine rats after SEFL.One potential mediator of the scopolamine disruption of freezing in the trauma context could be due to state-dependent effects, namely the incorporation of the mental attributes of experiencing scopolamine into the memory for the context. In order to test this, we subjected a subset of rats who had received scopolamine during SEFL to a second trauma test; this test occurred after an infusion of scopolamine . No signp > 0.05). Firstly, this test demonstrates that the reduced freezing levels seen during the sensitization test with pre-training scopolamine administration are not due to a prolonged duration of action of scopolamine, as post-training scopolamine did not disrupt freezing at that later time point. Secondly, this test is a conclusive demonstration that scopolamine in DH disrupts processes occurring during acquisition, but it does not exert its effects on trauma memory or later sensitization by blockade of memory consolidation.Scopolamine could exert its effects on both trauma memory formation and contextual sensitization by disrupting memory formation, by disrupting consolidation, or by a combination of the two. Indeed, scopolamine is known to disrupt both acquisition and consolidation of contextual memory . We testThe Stress-Enhanced Fear Learning (SEFL) procedure, in which an unpredictable 15-shock stressor is administered, generates a persistent sensitized state whereby novel mild stressors result in highly elevated levels of fear. This work extends the use of this model to study the role for cholinergic signaling during trauma on later sensitized responding, and demonstrates an important role for cholinergic signaling in DH for future contextual sensitization.Pre-training administration of scopolamine into DH prevented contextual fear memory formation for an intense, 15-shock stressor. Though cholinergic signaling in DH has long been known to facilitate contextual processing, this is the first demonstration that even intense trauma cannot overcome the blockade of memory formation by scopolamine.In addition to its role in contextual memory formation, this work has demonstrated a novel role for cholinergic signaling at muscarinic receptors in DH during a traumatic event for later contextual sensitization. Scopolamine administration into DH prior to the traumatic event blocked future contextual sensitization to a mild, single context-shock pairing in the absence of drug. The blockade of trauma memory formation is unlikely to be the mechanism for blocking this contextual sensitization for a number of reasons. The first is that blockade of trauma memory formation by blocking NMDA receptors, extinction of the trauma memory , and forThe mechanism by which scopolamine in DH disrupts this cholinergic-dependent sensitization was not a focus of this study, but some noted effects of scopolamine can be ruled out. For example, though scopolamine has been shown to affect consolidation processes, these effects are not sufficient to block contextual sensitization, as post-SEFL scopolamine infusion had no effect on later sensitization. Another noted effect of scopolamine, though after systemic administration, was an upregulation of M1 cholinergic receptors, alpha 7-containing nicotinic receptors, and NMDAR1 glutamate receptors in the DH , howeverMultiple mechanisms likely play a role in this cholinergic-dependent sensitization. During acute stress, ACh levels in DH become profoundly elevated  and co-vThough ACh has direct effects on hippocampal LTP, contextual sensitization may occur through other mechanisms, leading to a dissociation between the memory-impairing but sensitization-sparing effects of APV and the cholinergic effects on both processes. For example, muscarinic receptor activation on CA1 pyramidal cells altered spine morphology , while aThough scopolamine in DH blocks contextual sensitization, it does not block all consequences of the traumatic experience. With the known contribution of BLA GluR1 receptors and glucocorticoid signaling to the SEFL phenotype , it may In addition to the direct modulation of hippocampal processing, cholinergic blockade in DH may disrupt inter-regional communication necessary for future contextual sensitization after trauma. Scopolamine administered systemically disrupts resting state functional connectivity between mouse brain regions involved in memory , and thiAnxiety disorders such as PTSD may present with symptoms related to discrete stimulus sensitization, contextual sensitization, or both, however, the evidence that these symptoms may be produced by different patterns of neural activity was previously lacking. This study demonstrated for the first time a selective contribution of cholinergic signaling during trauma exposure to future contextual, rather than general, sensitized responding. For individuals who primarily overreact to unpleasant contexts, this dissociation could provide a basis for the development of specialized treatments. Even in the case where sensitized responding to contexts and discrete stimuli are both observed, pharmacological targeting of contextual circuitry could help to normalize responding to discrete stimuli, by facilitating improved discrimination between safe and unsafe contexts .This work highlights the important contribution of hippocampal signaling to the sensitization that occurs after an acute traumatic event and provides the first evidence that cholinergic signaling during the trauma may induce changes leading to enhanced contextual sensitization after trauma. Future work will endeavor to isolate the mechanism by which this signaling changes hippocampal processing and understand the contribution of cholinergic signaling during trauma to future anxiety states, hopefully leading to novel treatments for stress-induced sensitized states.All datasets generated for this study are included in the manuscript and/or the supplementary files.SH designed the experiments, performed surgeries, organized and ran behavioral experiments, analyzed results and was the primary writer of the manuscript. AH ran experiments and assisted with analysis and development of figures. LH and SS performed some surgeries and ran behavioral experiments. JL and AP ran behavioral experiments. MF assisted with design of experiments, analysis of data, and writing of the manuscript.MF is on the scientific advisory board of Neurovation, Inc. The remaining 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.


Moreover, the existence of tall graphene nanowalls was found not to be beneficial to the protection as a consequence of their layered orientation. These correlations among the morphology, structure, and corrosion properties of graphene nanostructures were investigated in this study. Therefore, the enhanced corrosion resistance in selected cases suggests that the low-temperature CVD graphene under appropriate conditions would be able to protect metal substrates against corrosion.Chemical vapor deposition (CVD) graphene is reported to effectively prevent the penetration of outer factors and insulate the underneath metals, hence achieving an anticorrosion purpose. However, there is little knowledge about their characteristics and corresponding corrosion properties, especially for those prepared under different parameters at low temperatures. Using electron cyclotron resonance chemical vapor deposition (ECR-CVD), we can successfully prepare graphene nanostructures on copper (Cu) at temperatures lower than 600 °C. Scanning electron microscopy (SEM), X-ray diffraction (XRD), Raman spectroscopy, and potentiodynamic polarization measurements were used to characterize these samples. In simulated seawater, i.e., 3.5 wt.% sodium chloride (NaCl) solution, the corrosion current density of one graphene-coated Cu fabricated at 400 °C can be 1.16 × 10 Their g in 2015 . It ment in 2015 . The rol in 2015 . Graphen2SO4 solution [On the other hand, graphene as an oxidation and corrosion resistive coating is of great interests recently ,28,29,30solution . Hsieh esolution . Miskovi2H2 at 600 °C for different times. On the other hand, electron cyclotron resonance chemical vapor deposition system , Hsinchu, Taiwan) was used for growing graphene films through flowing CH4 at different temperatures for 5 min. For film growth, the Cu foil (area: 2 cm × 2 cm) was used at the microwave power of 1200 W. The detailed rest process conditions and parameters were listed in Cu foils (30 cm × 150 cm × 0.00254 cm) were obtained from Alfa Aesar . Ag paste  and pipe sealant  were purchased for electrochemical measurements. A multi-sources electron cyclotron resonance chemical vapor deposition system , Hsinchu, Taiwan) was used for growing graphene films through flowing CFor material analyses, a field-emission scanning electron microscope  and an X-ray diffractometer  were adopted. In addition, the graphene films on the Cu substrate were checked by a micro-Raman spectrometer . Potentiodynamic polarization measurements were carried out by using a CHI potentiostat system  in a typical three-electrode setup with an Ag/AgCl reference electrode and a Pt wire counter electrode. For preparing the working electrode, the Cu wire was mounted on the backside of Cu substrates through silver paste, while the backside and surrounding sides were protected by pipe sealant. The drying conditions of the silver paste and pipe sealant are 80 °C for 3.5 h and 40 °C for 30 min, respectively. The electrolyte is 3.5 wt.% NaCl solution, and the sweeping potential range is between −0.8 and 0.6 V at the scan rate of 10 mV/s. All of the solutions were prepared with deionized water with a resistivity of more than 18 megohm/cm. We used CorrView software (version 2.6) to do data fitting in the Tafel plot in order to get the corrosion potential.2H2 flow, the surface texture seems not that clear, with few relatively large spots on gray surface. 4 flow, respectively. In the beginning, some rectangular stereostructures appear randomly at 350 °C, and gradually become larger through increasing temperatures, finally form the graphene nanowalls at 500 °C. In the later stages, even rose-like or flower-like structures start to stack and form big spheres under the influence of growth conditions. In general, graphene films grow for the most part in width, and not in height. While the film is thin, this coating appears flat. With a further increase in the amount of material in the film, it gets pinched and forms a texture. 2H2 flow. These large grain and good crystallinity would be attributed to the high growth temperature and better horizontal coverage. On the other hand, because the carbon ratio of C2H2 is higher than CH4, the C2H2 precursor could get more C2 radicals, which might be helpful for film deposition.−1 (D band), 1590 cm−1 (G band), 1620 cm−1 (D′ band), 2690 cm−1 (2D band), and 2950 cm−1 (D + G band). These peaks concur the existence of graphene films and nanowalls on Cu foil substrates that are observed in the above SEM and XRD analyses. First, the small 2D peaks imply multilayer graphene structure for all of the cases instead of a few-layer or even a single-layer structure. For the 2H2 flow, the growth time is the parameter that we controlled at the same temperature of 600 °C. The decreasing ID/IG ratios are 1.70, 1.60, and 1.47 as a result of the better crystallinity for long growth times. For the 4 flow, the growth temperatures are 350 °C, 400 °C, 450 °C, and 500 °C with the same time of 5 min. In D/IG ratios are 1.69, 0.87, 0.84 and 0.72 for the increasing growth temperatures. Here, the high G-band intensity at higher growth temperatures is due to the much superior sp2 crystallization of graphene nanostructures compared with that of 300 °C. Especially, there is a rapid transition from 350 °C to 400 °C, and the finally forming non-flat and porous graphene nanowalls at 500 °C.2H2, 5 min)-coated, and CVD graphene -coated Cu foils. The experiments were carried out ranging from −0.8 V to 0.6 V v.s. Ag/AgCl at the scan rate of 10 mV/s at room temperature. Originally, the corrosion potential for pure Cu foil is around −0.267 V. After adding good CVD graphene coatings, the corrosion potentials are shifted toward −0.2 V with several tens mV with relatively low corrosion activities. It depicts that the suitable CVD graphene coatings are able to improve the anticorrosion property under the appropriate growth conditions. corr) and corrosion potential (Ecorr) for the first scan. In addition, the scan numbers were raised 30 times in order to investigate the long-term stability. The corrosion current densities (jcorr) and corrosion potentials (Ecorr) for CVD graphene (C2H2)-coated Cu foils at different growth times and pure Cu foil were displayed in the left-hand side of corr of all of the CVD graphene (C2H2)-coated ones are lower than that of pure Cu foil. It is worthwhile to mention that the Ecorr for graphene  is closer to the positive side with lower jcorr, although it is relatively higher than those with shorter growth times. The right-hand side of corr and Ecorr for CVD graphene (CH4)-coated Cu foils at different growth temperatures and pure Cu foil, respectively. Despite the curve for 500 °C, all of the remaining jcorr are lower than that of pure Cu foil. However, the Ecorr for graphene -coated Cu foils are closed to the more negative potentials. This indicates that the appearance of vertical structures of graphene nanowalls might be fatal to the protection as a consequence of the fast diffusion channel against the horizontal surface. Among all of the growth conditions, the champion film is CVD graphene , and its jcorr and Ecorr are 1.16 × 10−5 A/cm2 and −0.219 V. Therefore, it is claimed that the protection graphene nanostructure can be successfully achieved at a relatively low temperature of 400 °C for 5 min in our system. 4, 500 °C)-coated Cu foils. Hence, basically the porous and high graphene nanowalls do not help the protection against corrosion in terms of morphology. In contrast, the surface with a low CVD growth temperature of 350 °C seems better. 2H2, 5 min) coating. This flatness is correlated with the relatively low jcorr and Ecorr on the more positive potential side. It indicates that the intermediate temperature CVD graphene with C2H2 as the carbon source can form the complete protection layer in a short growth time of five minutes, even suffering from multiple scans. Still, the corrosion will cause the surface oxidation confirmed by comparing the energy-dispersive x-ray spectroscopy (EDS) spectra  and graphene  coatings. However, after 10 scans, the Ecorr of pure Cu will shift to around −0.16 V, which could be a consequence of forming a passivation layer and thus be higher than those of all of the graphene-coated ones. spectra . Similarcorr and Ecorr closed to the more positive side of graphene-coated Cu can be achieved simultaneously for selected and optimal cases. More importantly, the lowest jcorr of graphene -coated Cu is one order of magnitude lower than that of the pure Cu. Meanwhile, its Ecorr of −0.219 V is 52 mV more positive than that of unmodified Cu. In contrast, the cases located outside the appropriate process window may suffer from non-uniform coverage, poor crystallinity, or porous nanowall structures, which also provide vertical diffusion pathway into the substrates. Our study suggests that the appropriate process and associated structures are essential to the development of graphene-based anticorrosion coatings through LTCVD.In conclusion, we have successfully demonstrated LTCVD graphene nanostructures with promising anticorrosion characteristics. The low j


This is achieved by engineering the asymmetric electronic environment of the generated hot carriers on chemical vapor deposition grown large area nanopatterned monolayer graphene, which leads to a temperature gradient of 4.7 K across the device terminals for an incident power of 155 nW, thereby enhancing the photo-thermoelectric voltage by manifold compared to previous reports. The results presented outline a strategy for uncooled, tunable, and multispectral infrared detection.Due to the low photon energy, detection of infrared photons is challenging at room temperature. Thermoelectric effect offers an alternative mechanism bypassing material bandgap restriction. In this article, we demonstrate an asymmetric plasmon-induced hot-carrier Seebeck photodetection scheme at room temperature that exhibits a remarkable responsivity of 2900 VW Designing electronically asymmetric environment across monolayer graphene enhances the photo-thermoelectric effect and enables efficient infrared photodetection. Here, the authors report a photodetector based on CVD grown nano-patterned graphene with high responsivity of ~2900 V/W within 8–12 µm wavelength regime by Dirac plasmon-assisted hot carrier generation at room-temperature. It is known that upon illumination, the intrinsic carrier temperature of graphene increases by means of hot carrier generation that manifests as a Seebeck voltage (ΔV)7. Despite the proof-of-concept demonstrations, so far, due to the modest Seebeck coefficient of graphene ~100 µVK−1, it has not been possible to harness this effect as an effective approach that can rival contemporary technologies4.Two-dimensional (2D) materials, especially graphene has shown a lot of potential as a candidate material for infrared detection. An ultrafast (~ps) infrared detection process is to excite hot-carriers in absence of carrier-phonon scatteringT) of the charge carriers can be engineered with minimal effect on the lattice temperature in order to enhance Seebeck voltage generation for highly sensitive, spectrally tunable, fast infrared detection in the LWIR band at room temperature. The carrier temperature of graphene at a specific spectral range can be manipulated by the plasmonic excitation of Dirac fermions which can be controlled by electrostatic tuning of the Fermi level. In our recent work, spectrally tunable infrared absorption of ~60% in the LWIR was demonstrated for nanopatterned monolayer graphene coupled to an optical cavity8. At resonance, due to the strong confinement of electric field at the discrete nanoresonator edges, the electronic system of graphene heats up by means of boundary-assisted intraband Landau damping to generate hot-carriers9. Although the hot-carrier generation develops a change in conductance of graphene, the resultant photoresponse arising from ΔT is limited by the theoretical Seebeck coefficient of graphene. The key to an efficient photo-thermoelectric device is the generation of high temperature gradient which we demonstrate in our asymmetric plasmon-induced hot-carrier Seebeck photodetector. In the asymmetric device  of 1.1 × 109 Jones along with a fast response of ~100 ns.The motivation of the present work is to identify avenues on how the temperature gradient , but also maintains a comparable high detectivity of ~109 Jones.The two important parameters that determine a detector’s performance are (a) detectivity and (b) response time. The uncooled detector devices or bolometers working at room temperature are very slow with the response time of 12 arising from localized Dirac plasmon excitations associated with intraband transitions. As a result, a gradient is introduced into the charge carrier density of the graphene channel across the source-drain electrodes. This creates a temperature difference across the graphene channel that as we discuss later in details plays the key role in infrared detection mechanism. Based on the applied source-drain voltage (VSD), henceforth called bias voltage, and the gate voltage (VG), multiple electronic processes work in tandem or against each other that influence the detector response. The graphene channel width is chosen to be 10 µm which is comparable to the diffusion length of the charge carriers3. However, in order to enhance carrier collection, the graphene channel width is elongated to maintain an effective active area of 2000 µm2 as shown in Fig. 8, of period P = 600 nm and hole diameter D = 400 nm was chosen in the detector fabrication  detector design and the fundamental electronic processes involved are schematically shown in Fig. 14 that arises due to the residual impurities on the graphene surface. In addition, the Al2O3 gate dielectric is known to enhance p-type doping in graphene16  of different graphene devices as a function of Fermi energy is shown in Fig. 18 on graphene doped away from the charge neutrality point. It can also be noticed that with decrease in temperature, S diminishes  of the fabricated cavity-coupled graphene nanohole array absorber were measured using a Microscope-coupled Fourier transform infrared spectrometer (FTIR) . The light reflection from the absorber stack without patterned graphene, i.e. Si++ (100 μm)/Al2O3 (15 nm)/SU-8 (1.3 μm)/Al2O3 (50 nm)/gold (200 nm) was taken as the reference and the light absorption spectra was calculated as A = 1-R.Upon illumination with infrared light the electronic properties across the half-patterned graphene channel exhibit contrasting electronic behavior. When light is incident on the unpatterned section of the graphene channel coupled to an optical cavity, the light absorption is a modest ~3%, however in the patterned section, owing to Dirac LSP excitations, ~60% light is absorbed by way of strong confinement of electric field near the nanohole edges  in our case occur at energies lower than the optical phonon energy of graphene (200 meV)20 and far from that of the substrate (~105 meV)21, plasmon damping through the emission of optical phonon has negligible effect23. Therefore, the plausible pathway for the plasmon damping is by generation of hot carriers via edge scattering-assisted Landau damping24 and resistive loss due to electron-impurity and electron-acoustic phonon scatterings25.Once excited, the LSPs dissipate energy through various damping pathways like phonon emissionS1)17. Second, the half-patterned graphene channel can be treated as a region consisting of two series connected thermoelectric materials with different Seebeck coefficients  and patterned (S2) sections which drives the system further into thermoelectric imbalance. The different Seebeck coefficients of the two sides induce a directional photo-thermoelectric current accompanied by the bias current. The resulting potential gradient can be expressed as a function of the channel width, XL − XRXL and XR are the positions of the left and right contacts, respectively and Tcr is the local carrier temperature. Finally, the different carrier mobilities of the patterned and unpatterned sections of the channel lead to differential Joule heating during carrier transport, which further enhances the thermal gradient in the system by increasing the temperature-dependent Seebeck coefficients in the patterned and unpatterned regions.Multiple factors now contribute to the asymmetric environment within the graphene channel that determine the effective thermoelectric response of this complex system when irradiated with infrared light. First, there exists the photo-thermoelectric effect originating from the intrinsic Seebeck coefficient of graphene  where the patterned section has elevated temperature as can be observed in Fig. T across the channel yielded a photo-thermoelectric voltage VPTE of ~0.44 mV. During the simulation, the Fermi level of graphene was maintained at −0.85 eV, and a bias voltage VSD = 0.9 V was applied , a measurement strategy was employed that eliminated bolometric effects from the signal  Fig.  was doneT and VPTE obtained across the graphene channel. It is to be stressed that generation of such a high position-dependent ΔT at room temperature exceeds previously reported values by an order of magnitude4. A series of FEM simulations were performed to investigate the role of Fermi energy and bias voltage on the photo-thermoelectric effect  ΔT) which monotonically decreases with EF. Considering these competing effects, there is a trade-off where above a threshold Fermi energy (~−0.8 eV), the effect of enhanced ΔT on VPTE is negatively impacted by the lower Seebeck coefficient that results in a decrease in VPTE. Therefore, there is an optimum range of gate (VG) and bias voltage (VSD) for the best performance of the detector associated with maximum VPTE at a desired spectral wavelength which can be observed from Fig. Figure ect Fig. . It can D.C = VPTEPinc−1) for different bias voltages (VSD) and substrate temperatures (TS), where Pinc is the band limited incident IR power. The highest responsivity measured in the present work is 2.9 × 103 VW−1  curves were recorded at regular temperature intervals within the range of 80–320 K. As the temperature is decreased, the responsivity reduces as shown in Fig. 27. In the proposed device, the absorption of light is almost independent of temperature, in other words, the hot-carrier generation and subsequent development of ΔT remains unaffected by the temperature of the sample. However, the Seebeck coefficient of graphene decreases as the temperature is lowered  and the responsivity (NEP = Sn ℛ−1) of the detector for different substrate temperatures  is more than one order of magnitude lower than thermal photodetectors26 which establishes its superiority as an uncooled, room-temperature mid-IR photodetector. The specific detectivity (D*) is derived from the NEP and the detector active area (A) as D* at EF = −0.8 eV and VSD = 0.9 V is measured to be 1.1 × 109 Jones  is calculated by measuring the noise spectral density  assists the drift of the hot-carriers (holes) generated on the patterned section towards the drain (ground), whereas in the negative bias condition (source voltage: 0 V to −0.5 V), the flow of holes towards the drain (ground) is inhibited leading to diminished responsivity, as shown in Fig. To further elucidate the role of LSPs in hot carrier generation and how the proposed asymmetric design excels in creating a high responsivity detector, we compare the A.C photoresponse of three detectors that were fabricated with (i) half-patterned, (ii) full-patterned and (iii) unpatterned graphene channels, respectively. We postulate that for the unpatterned and full-patterned detectors, the photoresponse primarily arises from the bolometric effect. Furthermore, owing to the symmetric design of the unpatterned and full-patterned devices, it is expected that the polarity of bias voltage should not affect the photoresponse. In contrast, due to the asymmetric architecture of the half-patterned detector, a bias voltage in the direction of Δ6. Since light modulation by mechanical chopping was not a feasible technique for high-speed measurements, we adopted an alternate method to study the A.C photoresponse by electronic modulation of the source-drain bias  and is limited by the capacitance of the external circuitry36.The time response of the half-patterned detector was measured to quantify the operational bandwidth. Due to the ultrafast plasmonic excitation and charge transport in graphene, a fast photoresponse is expected37 was used to image a Pegasus and UCF logo printed on a substrate. The tunable response of the detector is evident from greyscale images shown in Fig. The proposed asymmetric graphene device is a multispectral gate tunable infrared detector. This opens up the possibility for making an uncooled multi-pixel infrared camera with performance comparable to the commercial cooled cameras. To demonstrate the real performance of the photodetector, a single-pixel imaging method  which is critical to the remarkable photoresponse. We identify various processes contributing to the photoresponse and present conclusive evidence that the fast (τres ~ 100 ns), high responsivity (2900 VW−1) and high D* ~ 1.1 × 109 Jones can be attributed to the photo-thermoelectric effect. The proposed frequency-tunable graphene detectors not only offer spectroscopic detection but also pave the path towards dynamic multispectral imaging in the IR domain, which is lacking in the present IR imaging technologies.In conclusion, this work demonstrates outstanding room temperature photodetection using 2D monolayer graphene that is possible by the interplay between multiple physical phenomena: (i) tunable enhanced infrared absorption induced by localized Dirac plasmonic excitations, (ii) graphene mobility engineering, and (iii) excitation of asymmetric hot carriers and consequent electronic photo-thermoelectric effect. The asymmetric graphene channel design facilitates generation of high-temperature gradient (Δ++ (100 μm)/Al2O3 (8 nm) substrate. The source and drain contacts were fabricated by UV-lithography following by Ti/Au (3 nm/60 nm) deposition. The electron beam lithography (EBL) following by oxygen-plasma etching techniques was used to nanopattern the half side of transferred graphene with period P = 600 nm and diameter D = 400 nm.The large-scale monolayer graphene grown by chemical vapor deposition (CVD) method on copper foil was transferred on the SiVSD) was applied across the source-drain (SD) terminals and the resulting current I1 = I + ITE and I2 = –I + ITE were measured for applied voltage ± VSD, where I is the current generated by the bias voltage and ITE is the thermoelectric current (ITE = 0.5 × (I1 + I2)). This thermoelectric current was measured in dark (ITE-D) and in the presence of mid-IR light (ITE-L). Any contribution due to the photoconductive effect is expected to be independent of the polarity of applied bias voltage, which was thereby eliminated in the ITE-L calculation. Therefore, the photo-thermoelectric current and voltage can be calculated as IPTE = ITE-L − ITE-D and, VPTE = RGIPTE respectively (Supplementary Methods). The D.C responsivity (ℛD.C = VPTEPinc−1) was calculated by using the measured incident light power (Pinc), the gate-tunable graphene resistance (RG)  and the responsivity R = VPTEPinc−1. The built-in modules Electric Currents and Heat Transfer in Solids coupled with the multiphysics module Thermoelectric Effect were applied to predict the behavior of the detector.The COMSOL Multiphysics 5.3a software was used to simulate the performance of the detector. The overall goal of simulations was to find the time-dependent solution for the bias-dependent photo-thermoelectric current, which was further used to calculate the photo-thermoelectric voltage  and 20 µm long. The channel width of detector was 10 µm wide and 20 µm long, where half of the width of the graphene sheet was patterned, and the other half kept unpatterned. The gold terminals were 5 µm by 20 µm, and the thicknesses of graphene, gold contacts, aluminum oxide, and silicon were 0.5 nm, 50 nm, 8 nm, and 3 µm, respectively. Gold, Silicon, and Aluminum oxide materials were directly imported from COMSOL material library, while the experimentally measured parameters were used for graphene. The electrical conductivity and Seebeck coefficient were gate-dependent for graphene, measured experimentally for the patterned and unpatterned graphene, separately. The temperature-independent electrical conductivity was used for all materials to neglect the bolometric effects.V0 = 0 V. In order to add the contact resistance, the electrical contacts were introduced between gold and graphene. The heat flux was applied in the form of rectangular pulse of period 4 ms, which means for the first two milliseconds the heat flux was zero, corresponding to the dark state in the experiment. For the next two milliseconds, nonzero heat flux was applied on the patterned side of graphene using laser heating. A Gaussian beam with the spot size Rspot = 2 mm and the incident power Pinc = 153 nW was used. The absorbed heat flux depended on the absorption at different Fermi levels. The gate dependence of the light absorption was calculated by using the Lumerical FDTD software, which ranged from A = 34% at EF = −0.55 eV to A = 60% at EF = −1.0 eV for the patterned graphene (Supplementary Methods).The bias voltage was applied across the gold terminals; one side was set to ground, and the other at high potential. Except gold terminals and graphene, everything was considered electrically insulated. The current conservation boundary condition was applied for the whole geometry, and the initial values were set to T0 = 293.15 K, and the boundary condition open boundary was used across all the sides of the detector, except top and bottom surfaces which means the heat can flow inside or outside across the cross-sectional boundary depending on the ambient temperature. Thermal contacts were used between graphene, aluminum oxide, and silicon to control heat transfer in the vertical direction. The free tetrahedral mesh for gold and the free triangular mesh at the graphene surface were used, which were swept in vertical direction for the remaining geometry.The bottom side of the detector was kept at fixed temperature using the boundary condition in the software. The initial value of the temperature was set to −5 was used to measure the time-dependent thermoelectric voltage across the terminal for different Fermi energies. The dark and light thermoelectric voltages VTE,D and VTE,L were measured in absence and presence of the incident heat flux, respectively. The photo-thermoelectric voltage VPTE was then calculated by subtracting the dark from the light voltage, i.e. VPTE = VTE, L − VTE, D.The time-dependent solver with very low relative tolerance of 10Supplementary InformationDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2


Drosophila model mimicking key features of the TDP-43 autoregulatory feedback loop, we identified CG42724 as a genetic modulator of TDP-43 production in vivo. We found that CG42724 protein influences qualitatively and quantitatively the TDP-43 mRNA transcript pattern. CG42724 overexpression promotes the production of transcripts that can be efficiently released into the cytoplasm for protein translation. Importantly, we showed that TCERG1, the human homolog of the Drosophila CG42724 protein, also caused an increase of TDP-43 protein steady-state levels in mammalian cells. Therefore, our data suggest the possibility that targeting TCERG1 could be therapeutic in TDP-43 proteinopathies.TAR DNA-binding protein-43 (TDP-43) is a ubiquitously expressed DNA-/RNA-binding protein that has been linked to numerous aspects of the mRNA life cycle. Similar to many RNA-binding proteins, TDP-43 expression is tightly regulated through an autoregulatory negative feedback loop. Cell function and survival depend on the strict control of TDP-43 protein levels. TDP-43 has been identified as the major constituent of ubiquitin-positive inclusions in patients with Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD). Several observations argue for a pathogenic role of elevated TDP-43 levels in these disorders. Modulation of the cycle of TDP-43 production might therefore provide a new therapeutic strategy. Using a The online version of this article (10.1186/s40478-018-0639-5) contains supplementary material, which is available to authorized users. TARDBP/TDP-43 gene in patients with FTLD/ALS -insertion line (UY5237) that significantly increased TDP-43 production , indicating that the human TDP-43 antibody did not recognize Drosophila proteins, including the Drosophila homolog TBPH. Normalization of the amount of TDP-43 proteins using the stain-free technology [P(UY)5237 element caused a drastic increase (~ 18-fold) of TDP-43 protein steady-state levels (p = 0.0001). In contrast, the P(UY)5237 element did not modify significantly FUS (p = 0.524)  . This assay is based on simultaneous PCR amplification of short fluorescent fragments and allows the comparative quantitative analysis of mRNA. We compared the levels of CG42724 transcripts in GMR-Gal4 > + and GMR-Gal4 > UY5237 Drosophila heads. We indeed observed a significant upregulation of the CG42724 transcripts  in GMR-Gal4 > UY5237 flies, compared to control flies    5237 element displayed no obvious external phenotype, compared to control flies  species  to prevent dissociation of putative HMW forms. Only a faint signal was detected in the insoluble urea fraction, indicating that TDP-43 species were mainly recovered as soluble forms in Drosophila. As expected, the expression of CG42724 increased the TDP-43 protein steady-state levels. TDP-43 proteins were detected as either full-length monomeric forms or HMW species, with TDP-43 proteins being the most prone to form aggregates in the presence of the UY5237 transgene. When samples were analysed in the presence of the reducing agent (+DTT), we observed a decrease of the HMW forms and, concomitantly, an increase of TDP-43 monomeric species, indicating that these complexes were indeed DTT-sensitive. Altogether, these data showed that CG42724-mediated increased expression of TDP-43 results in the appearance of TDP-43 HMW species and is associated with cellular toxicity in Drosophila retina.Interestingly, we also observed that TDPBR region contributes to the CG42724-mediated regulation of TDP-43 protein production using the UAS-TDP-43 construct (no TDPBR region) previously described in [UAS-TDP-43_TDPBR construct , overexpression of CG42724 caused only a 2-fold increase in TDP-43 protein steady-state levels (p = 0.0002)  gene .We first evaluated whether changes in CG42724 expression could affect the nucleocytoplasmic export of TDP-43 mRNAs. We performed cell fractionation   experiments. PCR products were amplified using a human TDP-43-specific primer  and a smaller one of ~ 800 bp (short isoform). The sequencing of these two major bands revealed that they corresponded to alternative transcripts of different sizes resulting from differential usage of polyadenylation sites. Note that we failed to characterize the intermediate band by sequencing , with a concomitant decrease of the long isoform   isoforms ratios of TDP-43 mRNA levels, whatever the experiments were performed using total mRNA or mRNA extracted from the cytoplasmic compartment. Quantification of the relative abundance of splice isoforms showed that the S/L ratio in the cytoplasmic compartment was significantly higher compared to that quantified in total mRNA   or not (GFP::TDP-43) to the TDPBR region, and compared their expression in HEK293 cells. As expected, introduction of the TDPBR sequence into the reporter construct resulted in a significant ~ 50% decrease (p = 0.0053) in the GFP::TDP-43 protein expression levels  . Together these results showed that the human TCERG1 protein can regulate TDP-43 protein production in mammalian cells, and that TCERG1-mediated regulation of TDP-43 production is also predominantly mediated by the TDPBR region.To validate these findings identified in els Fig. a, b. Co-78) Fig. a, c. In TDPBR [TDPBR sensor region includes low-affinity binding sites for TDP-43 and the polyadenylation site pA1, the most efficient polyadenylation site of the TDP-43 gene. In steady-state conditions, most TDP-43 production within cells comes from the transcript that uses the polyadenylation site pA1. Increase in TDP-43 nuclear levels results in an increased occupancy of the TDPBR that in turn suppresses usage of the pA1 site, resulting in elongation of transcripts beyond pA1. The elongated transcripts present an acceptor site for normally silent intron that contains the TDPBR region and the pA1 sequence. The exclusion of this intron forces the system to use suboptimal polyadenylation sites. The mRNAs using these alternative polyadenylation sites show an increased incidence of alternative splicing, and are partially retained in the nucleus and/or degraded.TDP-43 is a critical RNA-binding factor that has been shown to play a central role in RNA metabolism. Cell functions and survival depend on the strict control of TDP-43 protein levels. TDP-43 expression is tightly regulated through an autoregulatory negative feedback loop mediated by the binding of TDP-43 protein in a specific region of its mRNA 3’UTR called TDPBR , 42, 54.Drosophila model previously developed and characterized by our group [Drosophila transgenic model is based on the expression of the human TDP-43 cDNA under the control of the TDPBR sensor region. This TDP-43_TDPBR Drosophila model recapitulates key features of the self-regulatory process of the steady-state levels of TDP-43 proteins described previously in mammalian and cellular models, namely alternative splicing events, differential usage of polyadenylation sites, nuclear retention of the transcripts, and a decrease in steady-state mRNA levels.To identify genetic modulators of TDP-43 production in vivo, we used an autoregulatory TDP-43 ur group . This DrCG42724 Drosophila gene as a genetic modulator of TDP-43 production in vivo. We showed that CG42724 overexpression caused a drastic increase of TDP-43 protein steady-state levels, whereas CG42724 down-regulation resulted in a decrease of TDP-43 accumulation. The study of the underlying molecular mechanisms allowed us to highlight that the CG42724 protein influences both qualitatively and quantitatively the TDP-43_TDPBR mRNA transcripts pattern. We found that CG42724 overexpression promotes the inclusion of the TDPBR sensor region as well as the production of transcripts ending at the pA1 polyadenylation, isoforms that can be efficiently released into the cytoplasm for protein translation. Of course, additional mechanisms could also be involved. Notably, we observed that TCERG1 expression resulted in a slight increase of TDP-43 mRNA steady-state level, which could also contribute to the increase in TDP-43 protein accumulation. Importantly, this effect predominantly depends on the presence of the TDPBR region.In this study, we report the identification of the Drosophila CG42724 protein. The protein is composed of 1123 residues and contains three WW domains at its N-terminus followed by six FF domains at its C-terminus. The CG42724 protein was detected in affinity-purified Drosophila spliceosome [To date, very little is known about the iceosome . On the iceosome . InteresCG42724 gene encodes a homolog of the human TCERG1 (Transcription elongation regulator 1) gene. The CG42724 and TCERG1 proteins share 35% sequence identity and 48% sequence similarity. The highest homology is observed in the WW and FF domains. Importantly, our data suggest a significant degree of functional conservation between flies and mammals regarding the regulation of TDP-43 production. Indeed, we showed that similarly to CG42724, human TCERG1 overexpression also caused an increase of TDP-43 protein steady-state levels in mammalian cells.The TCERG1 knockdown [Bclx gene through the modulation the RNAPII transcription rate [TCERG1, previously named CA150, is a highly conserved human nuclear protein, localized at the interface of nuclear speckles and presumed nearby transcription sites , 60. TCEnockdown , 53. Basion rate .Interestingly, TCERG1 and TDP-43 proteins have been linked to common aspects of mRNA life cycle, namely transcription, pre-mRNA alternative splicing and polyadenylation site selection. Uncovering the mechanism of action of TCERG1 on TDP-43 production is complicated by their multifaceted functions, but also by the fact that RNAPII transcription, alternative splicing and alternative polyadenylation can be influenced reciprocally. All these processes are tightly coupled and coordinated.TDPBR region. We demonstrated previously that the negative regulatory activity of the TDPBR region is specifically dependent on TDP-43 expression [TDPBR region, possibly by competition through binding the same mRNA site. Indeed, even if TCERG1 has not been described as an RNA-binding protein, it has been shown that the protein associates in vitro with the Bcl-x pre-mRNA [We showed in this study that these processes of regulation predominantly depend on the presence of the pression , suggestpre-mRNA . Alternapre-mRNA , 63, 75.As a component of the splicing machinery, TCERG1 could also modulate the spliceosome assembly and activity. Consistent with this possibility, it has been shown that spliceosome assembly across the 3’UTR region induced by TDP-43 is a key event in the reduction of the amount of TDP-43 . The 3’UTDPBR sequence to the downstream region [TDPBR region could interfere with the recognition of pA1, forcing the use of suboptimal polyadenylation sites. As mentioned above, TCERG1 modulates the rate of RNAPII transcription by increasing its elongation rate [Otherwise, it has been shown that TDP-43 overexpression causes a rise in RNAPII density from the m region . Such a m region  and the m region , 40. Thuion rate . Therefoion rate . Thus, aTranscriptomic studies showed that TCERG1 is widely and highly expressed in the brain . Interestingly, TCERG1 has already been implicated in the pathogenesis of the neurodegenerative disorder Huntington’s disease (HD). TCERG1 interacts with the huntingtin (HTT) protein and has been associated with the morphological deposits related to the disease . TCERG1 Drosophila model that recapitulates key features of the TDP-43 auto-regulatory feedback loop, we have identified TCERG1 as a modulator of TDP-43 production in vivo. Further studies will be necessary to unravel the exact mechanisms through which TCERG1 modulates TDP-43 production. Nevertheless, regardless of underlying mechanisms, our data suggest the possibility that targeting TCERG1 could be therapeutic in TDP-43 proteinopathies.To conclude, using a Additional file 1:Figure S1. Schematic representation of the organization of the human TDP-43 gene. (TIF 282 kb)Additional file 2:The table lists the detailed genotypes of the flies used in this study. (DOC 64 kb)Additional file 3:The table lists the primers used in this study. (TIF 573 kb)Additional file 4:Figure S2. Quantification of CG42724 steady-state mRNA levels by RT-QMPSF. (A) Schematic representation of the CG42724 transcription unit, the relative location of RNAi target sites (green boxes) and the RT-QMPSF amplicon (red box). (B) Expression analyses of CG42724 mRNA transcript by RT-QMPSF. The single-stranded cDNA was PCR-amplified using one pair of primers spanning CG42724, yielding a 173 bp product, and a pair of primers spanning the reference gene Cyp1 (162 bp). The diagrams shown were obtained from GMR > + (control), GMR > UY5237 flies. The y-axis displays fluorescence in arbitrary units, and the x-axis indicates the size in bp. The electropherogram of the GMR > + (blue) and GMR > UY5237 (green) flies were superimposed by adjusting the peaks obtained for the control amplicon to the same level. (TIF 456 kb)Additional file 5:Figure S3. Light micrographs of new-born Drosophila adult eyes. Compared to control flies , expression of CG42724  or TDP-43_TDPBR  alone triggered no structural defects. Similarly, flies co-expressing CG42724 and TDP-43_TDPBR have no external phenotype. (TIF 25334 kb)Additional file 6:Figure S4. Homology TCERG1 and CG42724. Alignment of the human TCERG1 and Drosophila CG42724 proteins. TCERG1 and CG42724 share 35% sequence identity and 48% sequence similarity. The highest homology is observed in the WW domain (blue) and the FF domain (red). Alignment was performed using DRSC Integrative Ortholog Prediction Tool. (TIF 338 kb)Additional file 7:Figure S5. Quantification of TDP-43 steady-state mRNA levels by RT-QMPSF. (A) Schematic representation of the TDP-43 transcription unit and the relative location of the RT-QMPSF amplicons. (B) Expression analyses of TDP-43 mRNA transcripts by RT-QMPSF. This assay is based on simultaneous PCR amplification of short fluorescent fragments in a single tube. The single-stranded cDNA was PCR-amplified using: TDP-43F1/R1 yielding a 115 bp product, TDP-43F2/R2 that yielded fragments of 132 bp and CG42724 that produced an amplicon of 173 bp  and Cyp1 (162 bp) cDNAs were amplified as internal references. The number of cycles of amplification was determined by testing a range of cycle numbers in order to remain in the linear phase of the PCR. Fluorescent amplicons were separated on a genetic analyzer and the resulting fluorescent profiles were analyzed. The diagrams shown were obtained from GMR > + , GMR > TDP-43_TDPBR (red) or GMR > TDP-43_TDPBR, UY5237 (green) flies. The y-axis displays fluorescence in arbitrary units, and the x-axis indicates the size in bp. The electropherograms were superimposed by adjusting the peaks obtained for the control amplicons to the same level. (C) TDP-43 amplicons were amplified using a TDP-43-specific primer (F3) and an oligo-dT adapter primer (AUAP). The diagrams shown were obtained from GMR > + , GMR > TDP-43_TDPBR (red) or GMR > TDP-43_TDPBR, UY5237 (green) flies. Cyp1 cDNAs was amplified as internal reference. The electropherograms were superimposed by adjusting the peaks obtained for the control amplicons to the same level. Note that the “mis-alignement » of the longest pics is due to the imprecise sizing of the fragment > 1 kb. (TIF 1151 kb)Additional file 8:Figure S6.. Purity of subcellular fractions. Cytoplasmic/nuclear fractionation was performed on GMR > + (control), GMR-Gal4 > UAS-TDP-43_TDPBR or GMR-Gal4 > UAS-TDP-43_TDPBR, UY5237 transgenic flies. Nuclear (N) and cytoplasmic (C) fractions were qualified by performing Western blot experiments. Results shown are representative of 3 independent biological replicates. β-tubulin was used as a cytosolic marker, while histone H3 was used as a nuclear marker. Total protein was used as the loading control by Stain-free technology. (TIF 683 kb)


Whispering gallery mode (WGM) resonators are versatile high sensitivity sensors, but applications regularly suffer from elaborate and expensive manufacturing and read-out. We have realized a simple and inexpensive concept for an all-polymer WGM sensor. Here, we evaluate its performance for relative humidity measurements demonstrating a sensitivity of 47 pm/% RH. Our results show the sensor concepts’ promising potential for use in real-life applications and environments. Monitoring and measuring the relative humidity (RH) is an important factor in many chemical processes and industrial applications, for example in air quality management, food manufacturing or in metal processing and polymer synthesis . There iOptical techniques have the advantage of immunity against electrical and vibrational influences and offer interesting multiplexing capabilities, among others . Opticalpm/% RH) .R. Hence, changing one of these parameters will cause a resonance wavelength shift WGM-based sensors enable very long photon life times or high quality factors, respectively. This makes them ideal sensors for many applications, especially for biological and chemical sensing . Typicalshift Δλ :(1)Δλλ=Δ pm/% RH . The manAs a first step to overcome these issues, we developed an inexpensive, easy to handle, easy to manufacture and completely polymer-based sensor. The general setup of the WGM sensor concept was introduced earlier for the application of wavelength measurement . Using pAs outlined in the Introduction, the sensor concept was developed to rely on a simple scheme and to be easy to manufacture. The design is shown in The plate acts as an optical waveguide due to total internal reflection. Thus, an evanescent field is present at the surface of the plate. In this field, commercially available PMMA spheres  were placed with a random distribution and positions. Two different mean diameters, Germany) . At a paIn order to evaluate the sensor performance, the whole sensor was enclosed in an acrylic box, against environmental influences. To adjust the relative humidity, a Petri dish with a saturated salt solution was positioned inside the box together with the sensor . By usinh shifts .Before the sensor can be used for relative humidity measurements ,16, it nmera see . AfterwaIn each sphere, a variety of different modes is excited, so specific modes cannot be distinguished by the intensity measurements recorded with the sensor sketched in r(λ) ,16,17: for RH measurement showed promising results. For unfixed spheres, we achieved sensitivities of 47 esonator ). RatherEven though the sensor was not able to reliably measure humidity in the fixed sphere configuration, more favorable results may be achieved with an improved fixation layer, e.g., reduced coverage of the sphere surface by the fixation material and/or using a more elastic material. In view of the transfer of the sensor concept to real-life applications, it needs to be mentioned that in the experiments presented here, the temperature was constant over the whole trial period. In a real environment, this will, rather, not be the case. As temperature changes induce diameter changes, as well, the resonance pattern of the spheres will shift accordingly. Consequently, the two effects need to be separated in a humidity measurement. This can be realized in the future by making one part of the sensor array insensitive to humidity, for example by application of a protective layer. With this protected part, it would be possible to measure temperature variations simultaneously with the same sensor array in order to correct the evaluated humidity response for the actual temperature.


First-generation epidermal growth factor receptor (EGFR) targeted kinase inhibitors (TKIs) are still used in selected non-small cell lung cancer (NSCLC) patients despite the resistance. Based on the correlation of programmed cell death receptor ligand 1 (PD-L1) and EGFR signaling pathway, whether continuous TKIs treatment will affect PD-L1 expression after disease progression remains unclear. To investigate the potential change of PD-L1 expression in TKI-resistant NSCLC after continuous TKIs treatment, we treated H1975 and HCC827 for more than one month and explored the possible effect on immune cells as well as underlying biological mechanisms. We found that continuous exposure to TKIs induced upregulation of PD-L1 in H1975 and HCC827. Moreover, PD-L1 upregulation significantly inhibited proliferation and slightly promoted apoptosis of T cells. We observed the activation of STAT3 and ERK1/2 along with the PD-L1 upregulation. With the pathway inhibitors, we found ERK1/2 pathway involved in inducing PD-L1 in resistant lung cancer. This study provides preclinical evidence that continuous TKIs treatment may induce PD-L1 expression in resistant NSCLC, resulting in the suppression of T cell function and immune escape. ERK1/2 pathway inhibitors, PD-L1/PD-1 inhibitors or combination strategies should be considered to reverse the resistance to TKIs in NSCLC patients. It is still urgent to optimize therapy strategies for patients with advanced disease. Currently, 83% of lung cancers are classified as non-small cell lung cancer (NSCLC), most of which are at an advanced stage when the first diagnosis is performed. Chemotherapy with or without radiation therapy used to be the standard resolution, but in recent decade targeted kinase inhibitors (TKIs) are proved to be superior, especially in the oncogene-driven tumors, such as epidermal growth factor receptor (EGFR) or anaplastic lymphoma kinase (ALK)5. EGFR, also named HER1/erbB1, is a critical member of the HER/erbB family of receptor tyrosine kinases (RTKs). About 85–90% mutations in the TK domain of EGFR are exon 19 deletions and exon 21 L858R mutations, resulting in constitutive phosphorylation of important tyrosine residues and activation of downstream signaling pathways (such as mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), signal transducer and activator of transcription(STAT))7. Tumors bearing these EGFR mutations are particular sensitive to EGFR TKIs compared to those with wild-type EGFR8. EGFR TKIs reversibly inhibit EGFR activity through competing with adenosine triphosphate (ATP) for binding to the receptor’s kinase pocket, thus blocking EGFR auto-phosphorylation. Unfortunately, widespread acquired resistance to TKIs always takes place within 6 to 12 months, which greatly restricts the long-term efficacy of these drugs. The most common mechanism of acquired resistance is a second EGFR mutation on threonine 790 in the ATP binding pocket, named T790M9. The T790M mutation increases the ATP affinity of the oncogenic L858R mutant and sterically interferences the binding of TKIs10.Lung cancer remains the leading health challenge to humanity worldwide, with the second highest incidence and the highest mortality in both males and females12. However, whether initial TKI therapy should be continued in resistant NSCLC has been debated. The IMPRESS trial indicated the continuation of Gefitinib failed to prolong progression-free survival in resistant NSCLC when combined with platinum-based doublet chemotherapy13. On the contrary, a retrospective study showed survival benefit from EGFR-TKIs beyond progressive disease compared to cytotoxic chemotherapy14. The ASPIRATION trial suggested Erlotinib was feasible for selected patients after progression15. As a result, Gefitinib and Erlotinib are still utilized in some TKI-resistant NSCLC in spite of possible limited benefit.Currently, new generation TKIs (such as Afatinib and Osimertinib), toxic therapy, immunotherapy or combination strategies are advocated to deal with this intricate situation16. The PD-1/PD-L1 pathway transfers inhibitory immune signals, which can limit tumor-infiltrating CD4+ and CD8+ T cells and contribute to immune evasion17. Accordingly, Gefitinib and Erlotinib may have a notable influence on the PD-L1 expression through changing the downstream signal pathways of EGFR, such as MAPK, PI3K, Janus kinase (JAK)/STAT). However, despite the initial inhibition of PD-L1 in EGFR-driven tumor, very limited information is known about the effect of continuous TKIs treatment on PD-L1 expression when NSCLC become resistant to TKIs. Based on STAT3 activation after continuous TKI treatment in our previous research18, we hypothesized that PD-L1 expression will increase in resistant NSCLC with the continuation of TKIs. To test the hypothesis, we treated H1975 and HCC827 for more than one month and tended to explore the possible effect on immune cells and the underlying biological mechanisms. Identifying the possible change of immune checkpoint will provide important information before clinical treatment strategies are made.As reported recently, the expression of programmed cell death receptor ligand 1 (PD-L1) could be induced by the oncogenic EGFR mutation and reduced apparently by EGFR TKIs in EGFR-driven tumorTo examine the influence of continuous exposure to TKIs on the PD-L1 expression in TKI-resistant NSCLC, H1975 cells which harbor the EGFR L858R/T790M double mutations were treated with Gefitinib or Erlotinib for 5 weeks. PD-L1 expression gradually upregulated with Gefitinib and Erlotinib treatment as showed by western blot Fig. . Though 19. To investigate the effect of acquired resistance on PD-L1 expression in NSCLC, HCC827 (EGFR Del 19), which is sensitive to TKIs therapy, was exposed to Gefitinib (2 μM) for more than 6 months. Finally, HCC827 became resistant to Gefitinib with increased IC50 values of 15.22 μM  were isolated from healthy donor and expanded 827 Fig. . When T 827 Fig. . Given t827 Fig. , the par20. To elucidate the mechanism involved in PD-L1 upregulation, we assessed the STAT3 signaling pathway. The level of total STAT3 and p-STAT3 (Tyr705) exhibited increase trend during the TKIs treatment  was another critical pathway for PD-L1 regulationime Fig. . Likewisime Fig. . The res22. Along with the development of new effective agents targeting specific resistance mechanisms, a correlation between immune suppression and TKIs resistance is gradually being understood, indicating that immunotherapy can be a promising way to deal with the situation. Here, our study showed that PD-L1 expression was upregulated by continuous TKI treatment in resistant NSCLC resulting in persistent immune suppression.In the past decade, EGFR-TKIs yield significant success in the treatment of lung cancer. Routine test for EGFR mutation status characterizes NSCLC patients into different subgroups, resulting in encouraging efficacy and survival benefit in selected ones. However, primary or secondary drug resistance usually inevitably emerge over time, limiting the duration of response25. However, Jiang, X.et al. demonstrated that resistance to selective BRAF inhibition increased PD-L1 expression in melanoma19, which means there is a dynamic change of PD-L1 expression during the TKIs treatment. In our study, enduring use of EGFR TKIs in NSCLC induced not only drug resistance but also the increase of PD-L1. Notably a new correlation was revealed between the resistance of EGFR TKIs and PD-L1 expression in the long-term treatment setting.An increasing body of evidence indicates that the activation of common oncogenic drivers in NSCLC  is correlated with the induction of PD-L1 and corresponding inhibitors will reduce PD-L1 expression in initial treatment28. So far, immune checkpoint inhibitor therapy has been considered a revolutionary treatment in lung cancer. After the success of CTLA-4 inhibitors in metastatic melanoma30, PD-L1 inhibitors show unprecedented responses in numerous clinical trials. PD-L1 expresses on a wide range of hematopoietic and non-hematopoietic cells including human cancer cells. High expression of PD-L1 is now regarded as a poor prognostic factor, contributing to immune escape and disease progression31. We found that PD-L1 upregulation mainly inhibited proliferation rather than promoted apoptosis of T cell, which would greatly restrain the antitumor immunity. The result suggested upregulation of PD-L1 is able to induce the immune suppress, indicating optional immune targeted therapy.Early studies reported that PD-L1 on tumor cells was sufficient for tumor immune evasion through directly inhibiting CD8+ T cell responses. Therapeutically blocking the PD-1 pathway leaded to immune reinvigoration and tumor regression33. Our findings indicated that the increased phosphorylation of ERK1/2 pathway was engaged in inducing PD-L1 in resistant lung cancer. ERK1/2 pathway participates in cell proliferation, differentiation, and development and is critical for tumor survival. Meanwhile, previous study showed that activation of ERK1/2 signaling implicated in the resistance to EGFR kinase inhibitors34, suggesting an overlap in signaling mechanisms between TKI resistance and PD-L1 modulation. Moreover, clinical trials combining BRAF and MEK inhibitors with immune checkpoint blockades have been shown manageable safety profile and enhanced antitumor activity in melanoma36. As patients benefit temporarily from all the TKIs because of the acquired resistance, therapeutic synergy between EGFR-TKIs and PD-L1/PD-1 blockades or targeting downstream ERK1/2 signaling may prolong responses to these agents and delay the salvage treatment38.A number of preclinical studies have explored the possible mechanisms of PD-L1 modulation. The reported molecular mechanisms include MAPK pathway, PI3K-mTOR-S6 pathway, PI3K/PKB pathway, and JAK/STAT pathway, most of which have been extensively investigated in melanoma39. It was reported previously that STAT3 could bind to the PD-L1 promoter and modulated PD-L1 expression at the transcriptional level, and its inhibitors could decrease PD-L1 level in both parental and resistant melanoma19. Interestingly, the STAT3 inhibitor had no suppression effect on PD-L1 expression according to our study, conversely, strongly induced it, while ERK1/2 inhibitor suppressed STAT3 signaling at the same time but decreased PD-L1 expression successfully. Given that STAT3 siRNAs decreased PD-L1 expression by only 10–32% in two KRAS-mutant lung adenocarcinoma cell lines23, it is possible that STAT3 only partially involved. Considering the complexity of transcription and signaling networks, further investigation is needed to confirm the function of STAT3 in the regulation of PD-L1 in resistant lung cancer.STAT3 belongs to the STAT protein family, which involves various biological processesIn conclusion, continuous TKIs treatment upregulates PD-L1 expression in resistant NSCLC, contributing to the suppression of T cell function and immune escape. ERK1/2 pathway plays a vital role in the regulation of PD-L1. Thus, reassessment of genetic alterations and signaling pathways after clinical progression is necessary for subsequent therapy. Based on our preclinical study, ERK pathway inhibitors, PD-L1/PD-1 inhibitors or combination strategies should be considered to overcome the TKI resistance and improve outcomes in NSCLC patients.2 at 37 °C. Gefitinib was bought from AstraZeneca ; Erlotinib was kindly provided by Roche Pharmaceuticals , while inhibitors including S3I-201 and FR180204 were bought from Selleck Chemicals. These drugs were dissolved in dimethyl sulfoxide (DMSO) and diluted in culture medium before use. Besides, primary rabbit antibodies against PD-L1 (CST#13684), ERK1/2 (CST#4695) and p-ERK1/2 (CST#4376) were from Cell Signaling Technology; rabbit antibodies to STAT3 (ab68153), p-STAT3 (Tyr705) (ab76315) were from Abcam while mouse antibody to β-actin was from Santa Cruz biotechnology.Human NSCLC cell lines including H1975 (EGFR L858R/T790M) and HCC827 (EGFR Del 19) were obtained from State Key Laboratory of Biotherapy, Sichuan University, and maintained with RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin  under a moist atmosphere of 5% CO5 cells/ml) continuously to 5 μM Gefitinib or 3 μM Erlotinib for 5 weeks. Total proteins were collected every week. 5 weeks later, the expression of PD-L1 was tested by flow cytometry while the total RNA was extracted as well. Meanwhile, HCC827 was constantly incubated (1 × 105 cells/ml) with 2 μM Gefitinib for 6 months.H1975 was exposed  containing protease inhibitor cocktail and 1 mM phenylmethylsulfonyl fluoride. And the protein concentration was detected by bicinchoninic acid (BCA) protein assay. After cell lysates were separated in the SDS-PAGE Gels, they were transferred to the polyvinylidene difluoride (PVDF) membrane , which was then blocked with 5% non-fat milk. After the membrane was cut into small pieces, they were incubated in different primary antibodies at a 1:1000 dilution overnight at 4 °C. Then the membranes reacted with the anti-rabbit or mouse IgG conjugated with horseradish peroxidase (HRP) . Finally, visible bands were obtained through the enhanced chemiluminescence (ECL) detection.TM RT reagent Kit . Quantitative real-time PCR was conducted with Bio-rad CFX manager and SsoAdvanced SYBR Green Supermix  as the detection reagent. All samples were analyzed in triplicate for each primer set. The primer sequences of PCR were as follows: hPD-L1, sense 5-GGAATTGTCTCAGAATGGTC-3 and antisense 5-GTAGTTGCTTCTAGGAAGGAG-3; hGAPDH, sense 5-GAAGGTGAAGGTCGGAGT-3 and antisense 5-GAAGATGGTGATGGGATTTC-3. RT-PCR amplification was performed 40 cycles with DNA denaturation at 95 °C for 5 s and annealing/extension at 60 °C for 20 s. Target gene expression was quantified with the relative quantification method.Total RNA was extracted and purified using RNA simple Total RNA Kit  according to the manufacturer’s instructions. The RNA samples were reverse-transcribed into cDNA with the PrimeScriptCells were collected and dissociated as single cells, then washed with 0.1% bovine serum albumin in PBS (PBSA) and stained with APC-conjugated anti-human CD274 antibody  or isotype-matched control antibody . After 30 minutes of incubation, cells were washed and resuspended in PBS, then performed on a BD Biosciences FACSCalibur cytometer. Data were analyzed using FlowJo software program.7 beads/mL) (bead-to-cell ratio 1:1). H1975, which were already treated with Gefitinib or Erlotinib for 5 weeks, and Gefitinib-resistant HCC827 cells were seeded in 24-well plates (5 × 104 cells/well) with parental cells as control and cultured in plain medium (with serum) overnight. Then isolated T cells were added to each well at a ratio of 2:1 (T cells: tumor cells) and co-cultured for 24 hours. Parental tumor cells were set as control.Human peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy volunteer donors by Ficoll Paque density centrifugation. T cells were selected by magnet  and stimulated with recombinant human IL-2  and Dynabeads Human T-activator CD3/CD28 beads  for 30 minutes on ice. Then washed T cells were counterstained with FITC Annexin V and PI for 15 min at room temperature (25 °C) according to the manufacturer’s instructions of FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen). Finally the FITC Annexin V and PI signal of T cells were analyzed by flow cytometry.Expanded T cells were labeled with carboxyfluorescein diacetate succinimidyl ester  prior to co-culture with tumor cells (described above). After 48 hours, suspended cells were harvested and counterstained with anti-human CD3-APC antibody. The CFSE signal of T cells was analyzed by flow cytometry.3 cells per well. Cells were allowed to grow for 24 hours and then treated with Gefitinib at various concentrations , in six replicates. After 72 hours of treatment, 20 μL MTT -2,5 diphenyltetrazolium bromide) (Sigma-Aldrich) (5 mg/mL) was added to each well, incubated at 37 °C for 4 hours and terminated with 150 μL of dimethyl sulfoxide (Sigma-Aldrich). Cell viability was finally determined with an ELISA reader (Bio-Rad) at 570 nm. The 50% inhibitory concentration (IC50) values were calculated using a proper analysis (nonlinear regression-curve fit).HCC827 were plated in 96-well at a concentration of 2 × 10t test for two groups and one-way ANOVA Tukey test followed by Dunnett’s multiple comparison test for multiple groups were performed using GraphPad Prism 7. All data were summarized and presented as means ± SD. Statistical significance was defined as: “ns”p > 0.05, “*”p < 0.05, “**”p < 0.01, “***”p < 0.001 and “****”p < 0.0001.Experiments were all repeated in triplicate. For significance analysis of PD-L1 expression , western blot analysis and proliferation and apoptosis of T cells, All experiments involving human tissue samples were performed in accordance with relevant guidelines and regulations approved by medical ethics committee of Sichuan University. Informed consent was obtained from all healthy donors.supplementary figure S1–6


The primary outcome was 3-year all-cause mortality and the secondary outcome was 3-year major adverse cardiovascular events (MACEs). Low L1 SMI was present in 141 (29.7%) of 475 patients. The incidence of all-cause mortality  and MACEs  was significantly higher in patients with low L1 SMI than in those with high L1 SMI. In multivariate analysis, low L1 SMI was an independent predictor of higher risk of all-cause mortality (hazard ratio (HR): 4.07; 95% confidence interval (CI): 1.95–8.45; p  < 0.001) and MACEs . These findings remained consistent after propensity score-matched analysis with 91 patient pairs (C-statistic = 0.848). CT-diagnosed low skeletal muscle mass is a powerful predictor of adverse outcomes in patients with CAD undergoing PCI.The impact of sarcopenia on atherosclerotic cardiovascular disease remains unclear. We aimed to investigate the prognostic impact of sarcopenia on coronary artery disease (CAD). A total of 475 patients with CAD who underwent successful percutaneous coronary intervention (PCI) and computed tomography (CT) were enrolled. The cross-sectional area of skeletal muscle at the first lumbar (L1) vertebral level was measured, and sex-specific cut-off values of L1 skeletal muscle index (L1 SMI; male <31.00 cm Sarcopenia is characterized by age-related generalized loss of skeletal muscle mass (SMM) and muscle strength, and frequently accompanies metabolic, physical, and functional disabilities ,2,3,4. APrevious studies have reported sarcopenia as a potential predictor of the prevalence and prognosis of atherosclerotic cardiovascular disease (ASCVD) ,15,16,17This study investigated the long-term prognostic impact of CT-diagnosed low SMM in patients with CAD who underwent successful percutaneous coronary intervention (PCI). We identified the sex-specific cut-off values of SMM at the first lumbar (L1) vertebral level to predict all-cause mortality and validated its prognostic significance.The PCI registry of Korea University Guro Hospital  is a single-center, prospective, all-comer cohort that consecutively enrolls patients undergoing PCI for CAD. The study population was enrolled from the KUGH PCI registry between January 2004 and April 2014, and was analyzed retrospectively. The study protocol complied with the Declaration of Helsinki and was approved by the Ethics Committee and Institutional Review Board at KUGH (2018GR0352). From the KUGH PCI registry, 788 patients who underwent PCI for CAD with stent implantation and performed CT scans within 180 days of PCI were selected. The following were excluded sequentially: (1) Patients without successful PCI; (2) patients who did not undergo CT within 30 days of PCI; and (3) CT exams without cross-sectional images at the L1 level. Finally, a total of 475 patients were included in the study .All participants in the present study underwent successful PCI. PCI was considered successful if the final angiography showed residual stenosis of <30% with achievement of Thrombolysis in Myocardial Infarction grade 3 flow. The morphology of the coronary artery lesion type was determined according to the American College of Cardiology and American Heart Association classification . MultiveAt initial admission for PCI, demographic features and underlying cardiovascular risk factors were recorded through detailed patient interviews, and baseline laboratory results including lipid profiles were obtained. The Canadian Study of Health and Aging (CSHA) Clinical Frailty Scale (CFS)  was usedAfter discharge, a regular outpatient clinic visit was scheduled at the end of the first month and every 3 to 6 months thereafter. If a patient did not show up for a scheduled visit, a telephone interview was conducted as an alternative approach for assessment of adverse outcomes. The follow-up duration was commenced with the date of successful PCI.The primary outcome was 3-year all-cause mortality, and the secondary outcome was 3-year major adverse cardiovascular events (MACEs), a composite of all-cause mortality, non-fatal myocardial infarction (MI), and repeat revascularization. Mortality was classified as cardiovascular or non-cardiovascular. MI was defined as a significant elevation of cardiac biomarkers accompanied by concomitant symptoms or electrocardiographic findings indicative of ischemia . Repeat CT measurement of the skeletal muscle area (SMA) of a specific body part accurately reflects the muscle mass of that region , and the2) of skeletal muscles on CT images were measured manually by utilizing the freehand module of the PACS workstation  by two independent observers (one radiologist and one cardiologist). Both observers were blinded to the clinical information during the measurement period. Repeat measurements were performed at least 4 weeks after the initial measurement to assess intra- and inter-observer variability with 50 randomly selected CT images. The cross-sectional areas were normalized for height (cm2/m2) and presented as the L1 skeletal muscle index (L1 SMI). Due to the absence of established reference values for defining sarcopenia with the L1 SMI in an Asian population, sex-specific cut-off point analysis was performed with the L1 SMI to determine the best reference value for predicting the primary outcome episodes. Based on the determined reference values, patients were divided into two groups by the presence of low SMM. Transverse CT images obtained at the level of the L1 transverse process were assessed in each patient. The CT Hounsfield unit ranges of −29 to 150 and −190 to –30 were considered to represent skeletal muscle and adipose tissue, respectively. The total SMA at the L1 level was composed of the following four components: Paraspinal muscle, extracostal abdominal wall muscle , intercostal muscle (including transversus abdominis muscle), and psoas muscle and diaphragm. Cross-sectional areas  curve. The cumulative incidence rate in each group was analyzed using the Kaplan–Meier method with the log-rank test. Multiple stepwise Cox-proportional hazards regression analysis was conducted to test the prognostic impact of low L1 SMI and frailty, and four models were established using subsequent adjustment of confounding factors. To adjust for additional confounding factors, propensity score-matched analysis was performed using a logistic regression model. The following factors, which showed significant differences at baseline or had proper relevance, were included in the model: Age, sex, body mass index (BMI), presentation as MI, hypertension, diabetes, dyslipidemia, previous malignancy, clopidogrel and statins at discharge, multivessel disease, creatinine clearance <60 mL/min, LVEF <50%, total cholesterol, number of treated lesions and treated vessels, number of implanted stents, average stent diameter, total stent length, and implantation of second generation drug-eluting stents. The logistic model estimating the propensity score revealed a good predictive value (C-statistic = 0.848). Intra- and inter-observer variability was quantified using the intra-class correlation coefficient (ICC) and visualized with Bland–Altman plots. Significant differences were defined at a p-value of <0.05, and all analyses were two-tailed. All statistical analyses were performed using Statistical Package for the Social Sciences (SPSS) software, version 20.0 .Data are expressed as mean ± standard deviation (SD) or median with interquartile range (IQR) for continuous variables, and frequency (percent) for categorical variables. Differences between groups were analyzed using Student’s p  < 0.001; p = 0.045; 2/m2  in males and 25.00 cm2/m2  in females.The average duration from the date of PCI to CT scan was −3.3 ± 11.7 days (median −2.0 days). 2/m2 in males, respectively, and 21.77 ± 2.64 and 31.36 ± 4.02 cm2/m2 in females, respectively. The L1 SMI showed a weak correlation with BMI  of the 475 study patients. The measured L1 SMI values of the low L1 SMI and high L1 SMI groups were 26.52 ± 3.74 and 38.36 ± 5.24 cmwith BMI . Both inwith BMI . DetailsMean follow-up duration was 4.11 ± 3.02 years (median 4.04 years), and 82.3% of the study participants completed a 3-year follow-up. Baseline clinical characteristics, procedural characteristics, and treatment strategies are displayed in p < 0.001) and MACEs . Other patient-oriented outcome measures such as MI and repeat revascularization also showed significantly higher incidence in the low L1 SMI group than in the high L1 SMI group. Detailed information about the cause of non-cardiovascular death is provided in A comparison of 3-year clinical outcomes between the groups is displayed in p  < 0.001 and HR: 3.76; 95% CI: 2.27 to 6.23; p  < 0.001, respectively). The complete dataset for univariate analysis and stepwise multivariate analysis of the study population is displayed in p = 0.009; and 46.1% vs. 18.1%, p  < 0.001, respectively)   . Low L1 ctively) .p  < 0.001) and MACEs  than non-frail patients. In fully adjusted multivariate analysis, the presence of frailty was an independent predictor of 3-year all-cause mortality . However, the impact of frailty on 3-year MACEs was markedly attenuated .Detailed study results based on the presence of frailty are provided in n = 313) of patients were excluded from the initial enrollment, mostly due to the absence of CT scans within 30 days of PCI and unavailability of SMA data at the L1 level. Details of study participants and those excluded at initial enrollment are displayed in A modest proportion  of patients were excluded from the initial enrollment, mostly due to the absence of CT scans within 30 days of PCI and unavailability of SMA data at the L1 level. For balanced interpretation of the study results, detailed features of the excluded study population were provided and compared to those of the enrolled study participants in the Online First, the CT measurement of L1 SMI was not a primary goal. However, the image quality was optimal in most cases and L1 SMI measurement was highly reproducible. Under the widespread use of coronary CT in CAD, CT images to assess SMM at the L1 level could be easily acquired without additional radiation burden or extra payment in this population. Second, because of the intrinsic limitation of non-randomized registry data, the baseline differences between groups could have affected the outcome. To minimize such biases, multiple stepwise Cox regression and propensity score-matched analyses were performed. However, remaining confounders could have influenced the analyses. Third, a modest proportion n = 3; 39.7% oLow SMM is a powerful predictor of adverse clinical outcomes in patients with CAD who undergo successful PCI. CT-diagnosed low SMM may further aid in risk stratification and decision-making in patients with established ASCVD.