Paramecium and Nyctotherus) possess only ultra-short introns, around 25 bp long. In Paramecium, ultra-short introns with length divisible by three (3n) are under strong evolutionary pressure and have a high frequency of in-frame stop codons, which, in the case of intron retention, cause premature termination of mRNA translation and consequent degradation of the mis-spliced mRNA by the nonsense-mediated decay mechanism. Here, we analyzed introns in five genera of Ciliates, Paramecium, Tetrahymena, Ichthyophthirius, Oxytricha, and Stylonychia. Introns can be classified into two length classes in Tetrahymena and Ichthyophthirius , but, surprisingly, comprise three distinct length classes in Oxytricha and Stylonychia . In most ranges of the intron lengths, 3n introns are underrepresented and have a high frequency of in-frame stop codons in all studied species. Introns of Paramecium, Tetrahymena, and Ichthyophthirius are preferentially located at the 5' and 3' ends of genes, whereas introns of Oxytricha and Stylonychia are strongly skewed towards the 5' end. Analysis of evolutionary conservation shows that, in each studied genome, a significant fraction of intron positions is conserved between the orthologs, but intron lengths are not correlated between the species. In summary, our study provides a detailed characterization of introns in several genera of Ciliates and highlights some of their distinctive properties, which, together, indicate that splicing spellchecking is a universal and evolutionarily conserved process in the biogenesis of short introns in various representatives of Ciliates.A typical eukaryotic gene is comprised of alternating stretches of regions, exons and introns, retained in and spliced out a mature mRNA, respectively. Although the length of introns may vary substantially among organisms, a large fraction of genes contains short introns in many species. Notably, some Ciliates ( Ciliates represent a unique and extremely heterogeneous group of protozoans containing many thousands of genetically diverse species . Named aThe micronuclear DNA consists of extremely long molecules typical of eukaryotic chromosomes, containing genes interrupted by short non-coding elements, called internal eliminated sequences (IESs), and large stretches of the spacer DNA . In contTetrahymena, Ichthyophthirius, Paramecium; class Spirotrichea, genera Oxytricha, Stylonychia, Urostyla, Paraurostyla, Laurentiella) [Euplotes crassus  had also been sequenced, but its annotation is not complete [Tetrahymena thermophila, 104 MB in length, is comprised of 225 chromosomes and encodes almost 27000 genes [Ichthyophthirius multifiliis, is approximately three-fold smaller and has three-fold fewer genes (around 8000), which are significantly shorter than those of Tetrahymena [Ichthyophthirius to the parasitic lifestyle. On the contrary, the MAC genome of Paramecium tetraurelia has undergone numerous whole-genome duplications resulting in about 40000 genes with dozens of copies [Oxytricha trifallax and Stylonychia lemnae, have a unique MAC genome organization comprised of thousands of so-called nanochromosomes, each typically encoding a single gene, and differently amplified up to ~2000 copies on average after extensive removal of ~96% of the MIC genome content [To date, macronuclear genomes of several ciliates have been completely sequenced and annotated  –10. The complete . The num00 genes . During f copies . Represes around 000, whics around 000, whic content .Paramecium and Oxytricha [Tetrahymena [The MIC-to-MAC genome reduction in Ciliates is also associated with fewer and smaller introns. In general, genomes with low intron densities also tend to have smaller introns and tend to be reduced in other ways as well, as seen for the yeast genome and more extremely compacted genomes such as those of microsporidia , 15. Thexytricha  to 4.8 irahymena . The alvrahymena . For spirahymena , 18. HowParamecium, the length of all introns lies in an extremely narrow range, 25–45 bp [Paramecium, 3n introns (with length divisible by three) are under strong evolutionary pressure and have a high frequency of in-frame stop codons [In 25–45 bp . The intp codons . In the Paramecium, Tetrahymena, Ichthyophthirius, Oxytricha, and Stylonychia , indicating that these organisms possess three distinct classes of introns.ibutions , which lIchthyophthirius, 45–62 bp introns of Tetrahymena, 27–69 bp introns of Stylonychia, and 27–38 bp introns of Oxytricha . Therefore, 3n introns depletion was intrinsic for short introns of all studied species, whereas no significant under-representation of 3n introns was identified for longer introns. Interestingly, in Tetrahymena, 3n introns in the range 22–44 bp were significantly more frequent, and, relative to 3n±1 introns, depleted in in-frame stop codons , 46–70 bp (short), and >70 bp (long) groups for Tetrahymena, and 35–65 bp (short) and >65 bp (long) groups for Ichthyophthirius. For Oxytricha and Stylonychia, the groups were <43 bp (ultra-short), 44–65 bp (short), and >66 bp (long), as defined based on the local minima of the fitted introns length distributions.In order to analyze densities of introns along the genes with respect to the introns length, we divided introns into several discrete groups by their length. For Oxytricha and Stylonychia were strongly skewed towards the 5' end of a gene, especially in the groups of short and long introns  [Like all eukaryotes, Ciliates have conserved dinucleotides at the intron termini –10, hencATAG–3') , 9, 24  tend to have strongest splice sites among other introns of these organisms. In Tetrahymena, the strongest 3' and 5' splice sites are found in introns with the length in the range 45–70 bp. These observations are in agreement with the intron-definition mechanism being predominantly used for splicing of short introns [Tetrahymena, that tend to have weaker splice sites. Similar to other introns of Tetrahymena, they possess the same conserved nucleotides at their 5' and 3' ends, (GT…AG), but are associated with gaps in protein alignments with orthologs from other studied species, indicating that these introns could be gene annotation artifacts .Paramecium, 50–70 bp introns of Tetrahymena, 30–40 bp introns of Oxytricha, and 30–70 bp introns of Stylonychia , and Oxytricha/Stylonychia (2266 introns) . As expected, given the branching order in the phylogenetic tree , the larintrons) . Of noteOxytricha and Stylonychia , and between Tetrahymena and Ichthyophthirius ; a weak correlation was observed between the numbers of introns in orthologs of Paramecium with either Tetrahymena or Ichthyophthirius .The numbers of introns in orthologous genes were moderately correlated between Ichthyophthirius, Oxytricha, and Stylonychia, but not in Tetrahymena  introns and a much flatter peak of longer introns, ranging up to thousands of base pairs are present in humans , 27. TheThe bimodality of intron lengths is also associated with two major modes of recognition and pairing between adjacent splice sites, so-called “intron definition” and “exon definition” models , 31. In Tetrahymena and Ichthyophthirius, bimodal intron length distributions are observed, suggesting that, similar to higher eukaryotes, they may possess two different mechanisms of the intron recognition and splicing, whereas, Paramecium has likely reduced or completely lost the exon definition mechanism. In general, the variability of the maxima of the intron length distributions and length classes across the studied oligohymenophoreans suggest that the evolutionary constrains imposed on intron length vary between the species possibly leading to the existence of several alternative mechanisms of introns recognition in these species. To our knowledge, spirotricheans are the only organisms with trimodal intron length distributions. Although the extent of this phenomenon in other species is not known, this could be evolutionarily associated with genome rearrangements and extensive IES removal during the MIC-to-MAC genome transition.Interestingly, in both Tetrahymena and Ichthyophthirius, ultra-short introns of Oxytricha, and both ultra-short and short introns of Stylonychia. The results indicate that 3n introns are under the strongest evolutionarily pressure in Paramecium [Tetrahymena, and Stylonychia, whereas in Oxytricha and Ichthyophthirius they are significantly underrepresented only among short introns. Then we analyzed frequencies of in-frame stop codons in introns of the studied species and found that 3n introns were enriched in stop codons in those intron length classes, where 3n introns were significantly underrepresented. Similar to the previous result, the pattern was most pronounced in three organisms, Paramecium, Tetrahymena, and Stylonychia. Further analysis of splice motifs at the introns termini revealed that short introns of Tetrahymena and ultra-short introns of Stylonychia and Oxytricha tended to have stronger splice sites than long introns of the respective species, suggesting a selection towards improved site-specific recognition and effective splicing of short introns.In most intron classes of all studied organisms, the intron length distributions had a deficit in introns whose length is divisible by three (3n introns). Here, we show that 3n introns are significantly underrepresented in short introns of ramecium , TetrahyTetrahymena. Unlike short and ultra-short introns of Tetrahymena and Oxytricha/Stylonychia, respectively, they are significantly overrepresented among 3n introns, and ultra-short 3n introns are depleted in in-frame stop-codons. These ultra-short introns also tend to have weaker splice sites. Hence, in the case of their retention, these introns would cause frequent read-through during the translation of a mis-spliced mRNA. However, we found that only a few ultra-short introns of Tetrahymena are conserved, and that ultra-short introns are associated with insertions in alignments with orthologous proteins of other studied species. The latter indicates that these introns are probably annotation artifacts, which would also explain their low conservation and weak splice sites.Different properties have been observed for ultra-short introns of Paramecium, Tetrahymena, Ichthyophthirius) and spirotricheans (Oxytricha and Stylonychia). They possess different numbers of intron classes . Oligohymenophoreans have two conserved nucleotides at their intron termini (5'–GT…AG–3'), whereas spirotricheans have three additional conserved nucleotides at the introns boundaries (5'–GTAAG…TATAG–3') and have stronger splice sites. Introns of oligohymenophoreans have two peaks of the introns densities, at 5' and 3' ends of genes, whereas introns of Oxytricha and Stylonychia tend to occur at the 5' end of a gene.There are several distinctive features between the studied oligohymenophoreans  frequencies were generated as follows: for each length of introns, starting from the minimal intron length of the organism, through 100 bp, a random set of the same size, with the nucleotide frequencies as in the corresponding introns, of the length N–4 was generated 1000 times, where N is the intron length and 4 corresponds to two absolutely conserved dinucleotides at the intron termini. Next, the ratio between the numbers of the observed and randomly generated introns with in-frame stop-codons (TGA) was calculated. P-values were calculated as the number of times from 1000 replications when the expected ratio was larger or equal to the observed one.Paramecium, Tetrahymena, and Ichthyophthirius. Next, COGs (Clusters of Orthologous Groups) were constructed by the COGtriangles software  and Cognitor (for Oxytricha and Stylonychia) scores.First, two rounds of psi-BLAST protein search were performed, with and without low-complexity filtering and composition-based statistics (“psiblast -comp_based_stats F -seg no” and “psiblast -comp_based_stats T -seg yes”), between putative proteins of 20.04.12 ) and assCOGnitor , 45 aftea, the total number of gaps in the aligned proteins, and b, the distance between two introns positions in the alignment. Intron insertion was considered to be conserved if a ≤ 3 and b ≤ 2. The algorithm is schematically outlined in Conserved introns were identified by the following algorithm: (1) protein sequences of orthologous genes in the same COG cluster were aligned with the “muscle” R package ; (2) forLocations of introns and their sequences were obtained from the annotation and genome sequence data with custom scripts written in R. Sequence logos were constructed using the WebLogo on-line tool . DecompoS1 Fig(1) Protein sequences of orthologous genes in the same COG cluster were aligned with “muscle” R package ; (2) for(TIFF)Click here for additional data file.S2 FigFor comparison, two intron groups are shown: ultra-short (20–45 bp) and short (45–70 bp). In total, 254 ultra-short and 5199 short introns were considered. Orthologs were taken from COGs, which were constructed as described in Methods (“Construction of COGs”).(TIFF)Click here for additional data file.S3 FigThe percent of the total number of conserved introns in a pair of species is indicated in each cell.(TIFF)Click here for additional data file.S4 FigGenes containing at least three introns were selected and divided into three equal intervals; then numbers of introns in each interval were calculated.(TIFF)Click here for additional data file.S1 FileThe introns coordinates are given in numbers of base pairs and numbers of aminoacids for genes and proteins, respectively.(ZIP)Click here for additional data file.S2 FileTables include COG IDs, protein IDs, and corresponding intron IDs. Conserved introns were identified as described in Methods (“Identification of conserved introns”).(ZIP)Click here for additional data file.S3 File(TXT)Click here for additional data file.


The Long INterspersed Element-1  is the only autonomous mobile DNA element in humans and has generated as much as half of the genome. Due to increasing clinical interest in the roles of L1 in cancer, embryogenesis and neuronal development, it has become a priority to understand L1-host interactions and identify host factors required for its activity. Apropos to this, we recently reported that L1 retrotransposition in HeLa cells requires phosphorylation of the L1 protein ORF1p at motifs targeted by host cell proline-directed protein kinases (PDPKs), which include the family of mitogen-activated protein kinases (MAPKs). Using two engineered L1 reporter assays, we continued our investigation into the roles of MAPKs in L1 activity.We found that the MAPK p38δ phosphorylated ORF1p on three of its four PDPK motifs required for L1 activity. In addition, we found that a constitutively active p38δ mutant appeared to promote L1 retrotransposition in HeLa cells. However, despite the consistency of these findings with our earlier work, we identified some technical concerns regarding the experimental methodology. Specifically, we found that exogenous expression of p38δ appeared to affect at least one heterologous promoter in an engineered L1 reporter, as well as generate opposing effects on two different reporters. We also show that two commercially available non-targeting control (NTC) siRNAs elicit drastically different effects on the apparent retrotransposition reported by both L1 assays, which raises concerns about the use of NTCs as normalizing controls.Engineered L1 reporter assays have been invaluable for determining the functions and critical residues of L1 open reading frames, as well as elucidating many aspects of L1 replication. However, our results suggest that caution is required when interpreting data obtained from L1 reporters used in conjunction with exogenous gene expression or siRNA. The only active, autonomous mobile DNA element in humans is the Long INterspersed Element-1  retrotransposon, which is responsible for generating almost half of the human genome via insertion of its own DNA and that of non-autonomous Short-INterspersed repeat Elements (SINES) –5. L1 acPDPK target motifs consist of serines or threonines with a proline in the +1 position (S/T-P motifs), which in ORF1p are: S18/P19; S27/P28; T203/P204; and T213/P214. The PDPK family includes mitogen-activated protein kinases (MAPKs), cyclin dependent kinases (CDKs) and glycogen synthase kinase 3 (GSK3). Prior to our finding that the phosphorylation of ORF1p by PDPKs is necessary for L1 activity, several studies reported associations between L1 and the PDPK p38 –34, a MAGiven these associations between L1 and the PDPK p38, as well as our previous findings that host PDPKs are required for L1 retrotransposition, we decided to investigate the role of each p38 isoform on ORF1p phosphorylation and L1 activity. Although our studies are ongoing, we believe that dissemination of our present findings and their associated experimental pitfalls will be useful to the L1 research community. We report here that: 1) different populations of HeLa cells can result in different experimental outcomes; 2) two presumably complementary L1 retrotransposition reporter assays produced conflicting results when coupled with exogenously expressed p38δ; and 3) two different non-targeting control (NTC) small interfering RNA (siRNA) sequences differentially affected measured L1 activity.We first determined whether activated wild type p38δ  could phosphorylate ORF1p on its S/T-P motifs, which are required for robust L1 activity . In vitrIn order to compare the degree of phosphorylation at each motif, we constructed a series of mutants, each bearing only one intact S/T-P motif: SAGG (S27A/T203G/T213G); ASGG (S18A/T203G/T213G); AATG (S18A/S27A/T213G); and AAGT (S18A/S27A/T203G). S27 (ASGG) was phosphorylated by p38δ-WT to the greatest extent Fig.  top. T21E. coli, used for the in vitro kinase assays.Various p38δ mutants that retain some degree of constitutive activity independent of phosphorylation by their activating upstream kinases in the MAPK pathway have been described . In thosmneo cassette that encodes the neomycin-resistant gene (neo), driven by a Simian virus 40 (SV40) promoter located within the 3′ untranslated region (UTR) of L1 -II), phosphorylates and thereby inactivates G418. Selection with G418 is begun approximately three days following transfection of the reporter plasmid into retrotransposition-competent cells and is continued for 10–12 days. The arrangement of the neo gene in JM101 ensures that only cells that have undergone retrotransposition by the L1 reporter element will express APH (3′)-II. The coding sequence for neo and its promoter are located on the antisense strand in the 3′ untranslated region of L1. Within this sequence is the engineered artificial intron, but it can only be spliced from the L1 sense RNA driven by the L1 promoter due to the orientation of the splice donor (SD) and splice acceptor (SA) sequences. Once spliced, the L1 RNA is retrotransposed into cDNA and inserted into the genome. After synthesis of the complementary DNA strand, which contains the spliced neo template, the transcript for APH (3′)-II can be initiated from the antisense promoter.Given our findings that p38δ specifically phosphorylated ORF1p S/T-P motifs, we proceeded to determine the effect of p38δ on L1 retrotransposition. To assess this, we used two previously characterized L1 reporter assays. The original L1 retrotransposition reporter, JM101 (a kind gift from Dr. John Moran), relies on the splicing of an artificial intron from an L1-borne neomycin-resistant gene and its L1-mediated conversion into genomic DNA to produce cell foci resistant to the neomycin analog G418 -ATP (PerkinElmer). Reactions were incubated at 37 °C for 15 min and stopped with the addition of loading buffer supplemented with EDTA to a final concentration of 50 mM. Samples were heated to 98 °C for 10 min then separated via denaturing gel electrophoresis. Gels were dried and exposed using Phosphorimaging.In vitro kinase reactions contained 85 nM p38δ or p38δ dialysis buffer and 200 μM ORF1p in 50 mM Tris–HCl (pH 7.4), 10 mM MgClHeLa-JVM cells (a kind gift from Dr. John Moran) were cultured in Dulbecco’s Modified Eagle Media (DMEM) with high glucose and pyruvate  supplemented with 10% Fetal Bovine Serum  and 100 Units/ml penicillin and 100 μg/ml streptomycin from a combined formulation . The cells were maintained at 37 °C in a standard incubator and passaged using 0.05% Trypsin-EDTA .2E or pcDNA-MKK62E. Lysates were harvested 4 days post transfection and processed in 96-well plates with the Dual-Luciferase Reporter Assay System (Promega) according to manufacturer’s protocol.Culture plates were seeded with HeLa-JVM cells in antibiotic-free DMEM with 10% FBS at a density to achieve approximately 50% confluency in 24 h, at which time cells were transfected using a ratio of 3 μl Fugene6 (Promega) per 1 μg DNA. For the G418-based assay, cells were seeded in 6-well plates and transfected with 500 ng JM101 and 500 ng pcDNA per well, allowed to grow for 72 h, then selected with media containing 400 μg/ml G418 sulfate  for 10–12 days. Cells were then washed with phosphate buffered saline (PBS) and fixed with 2% formaldehyde and 0.2% glutaraldehyde in PBS for at least 30 min at room temperature. Cells were then washed twice with PBS, stained with KaryoMAX Giemsa  for 1 h at room temperature, rinsed briefly twice with 50% ethanol and then water. For luciferase assays, cells were seeded in 24-well plates and transfected with 200 ng of reporter and 200 ng pcDNA-p38δ per well or 25 ng pcDNA-MKK3bHeLa-JVM cells were plated in 8-well glass bottom μ-Slides  in antibiotic-free DMEM with 10% FBS at a density to achieve approximately 60% confluency per well in 24 h. Wells with siRNA were reverse transfected as described in the following section. After a 24-h incubation, cells were transfected as described above with a pcDNA-EGFP expression plasmid (for siRNA wells) or cotransfected with pcDNA-EGFP and each pcDNA-p38δ expression plasmid. The ratio of DNA to surface area was identical to that used in the 6-well plates. After 24 h, cells were rinsed twice with PBS, then DMEM sans phenol red plus 10% FBS was added to each well. Cells were visualized with a Keyence BioRevo BZ-II 9000 digital microscope fitted with a Nikon PlanApo 4×/0.20 objective lens and 49002 ET-EGFP filter set from Chroma . Tiled images covering approximately 70% of each well were stitched with Keyence BZ-II Analyzer software, and total fluorescence in each stitched image was quantified in Fiji software using the Integrated Density function.HeLa-JVM cells were plated in antibiotic-free DMEM with 10% FBS at a density to achieve approximately 60% confluency in 24 h and reverse transfected per manufactures’ protocol using Lipofectamine RNAiMAX (ThermoFisher) at a ratio of 1 μl RNAiMAX per 8 pmols siRNA. All siRNAs were purchased from Dharmacon: NTC #3, NTC #5 and SMARTpool siRNA against p38δ . Following reverse transfection, cells were incubated for 24 h, then siRNA-containing media was removed and replaced with fresh antibiotic-free plating media with 10% FBS at the time of transfection with L1 reporters as described above.


In conversation, turn-taking is usually fluid, with next speakers taking their turn right after the end of the previous turn. Most, but not all, previous studies show that next speakers start to plan their turn early, if possible already during the incoming turn. The present study makes use of the list-completion paradigm (Barthel et al.,  Taking turns at talk in conversation is an essential feature of human interaction. When talking to one another in everyday encounters, interlocutors efficiently align their turns-of-talk, most of the time leaving only very short gaps of about 200 ms , planning of the content of a response, Barthel et al.  came to Substantial research on task (ii), turn end detection, suggests that, at least in participants overhearing a conversation, projection of the incoming turn's completion point is influenced by the presence or absence of turn-taking cues Kendon, . In partThe present study was designed to disentangle the relative contribution of early planning on the one hand and reaction to the upcoming turn-end on the other hand to the fast timing of turn-taking that is commonly observed in conversation. It makes use of the list-completion paradigm  use downsteps of successive pitch peaks on list items, which means that the rise in pitch in non-final list elements is lower and lower with every successive element Féry, . These dand that often precedes the final item of closed lists and indicates that the turn will end after the following noun phrase, such as in I have a key, a kite, anda ruby. Pitch contours, boundary tones, and lexical cues could therefore be monitored by listeners to identify turn-completion points and used to minimize gaps at turn transitions.A third cue to the end of a closed list, next to the two intonational cues, can be a conjunction like To disentangle the contributions of early planning and reaction to the upcoming turn-end, the present study applied two measures, namely gaze direction as a measure for the timing of planning, and voice onset time as a measure for the latency of launching the response turn. A combination of these two measures can be used to partly disentangle the processes of response preparation and response initiation. Assuming that next speakers aim for short gaps between turns, an earlier start in planning  should also lead to shorter response latencies (assuming that the head start in planning will not be canceled out by interference of the incoming speech with the planning process). If, however, no difference in planning can be observed in eye-movements, a difference in response latencies should reflect a difference in response initiation. If next speakers can take advantage of any of the cues tested in this experiment to start planning their response early, they should be able to move their gaze for planning earlier and respond faster in turns displaying the cue than in turns without the cue. If however a cue cannot be used to initiate response planning early , it could still be useful to detect the end of the incoming turn and to launch the articulation of a response. In that case, the presence of the cue should make no difference to the timing of gaze movements but lead to shorter response latencies compared to the absence of the cue. Early turn-taking cues, including pitch downsteps on non-final items of a list and a lexical cue before the final item of a list, are therefore hypothesized to lead to earlier response planning and consequently shorter gaps between turns. Late cues to turn-completion, however, such as the final boundary tone, were argued to not aid response planning I have a key, a kite, a ruby.und einen RubinIch habe einen Schlüssel, einen Lenkdrachen . (L%)I have a key, a kite and a ruby.Ich habe einen Schlüssel, einen Lenkdrachen, einen Rubin. (M%)I have a key, a kite, a ruby.Ich habe einen Schlüssel, einen Lenkdrachen, einen Rubin. I have a key, a kite, a ruby.and) to mark the final item of the list (−LEX) and ended in a low falling boundary tone (+BT). Non-final list items were produced with high rising intonation and pitch peaks of equal height around 400 Hz, i.e., without downsteps of pitch peaks on non-final list items (−DWNS). Sentences in condition (2)  were similar to sentences in condition (1), except that the lexical cue und (“and”) preceded the final list item to mark the item as being the last one of the list . Sentences in condition (3) (no boundary tone condition) were the same as in condition (1), except that their final intonation contour was manipulated to end in a flat mid tone instead of a low falling boundary tone (−BT). Sentences in condition (4) (downstepped condition) were similar to condition (1), except that non-final list items were produced with consecutive downsteps in pitch peaks in non-final list items (+DWNS). Figure Sentences in condition 1) (baseline condition) did not contain a lexical cue (like  (baselinSD = 2.9) were tested as paid participants at the MPI for Psycholinguistics. All participants reported normal or corrected-to-normal vision and normal hearing abilities. Data of three participants were not considered in the analyses due to technical failure during recording. Of the remaining participants, 10 answered “yes” to a post-experiment query whether pre-recorded materials were presented to them during the experiment. This factor was included as a binary control variable in the analyses (±recording_noticed). The experiment was approved by the Ethics Committee of the Faculty of Social Sciences, Radboud University Nijmegen. Written informed consent was obtained from all subjects.Thirty-eight German native speakers .Four-hundred and twenty-four pictures of concrete objects that were used in the study by Barthel et al.  were useOne-hundred and four pairs of displays (participant displays and corresponding confederate displays) that showed a differing number of objects drawn from the pool of object pictures were used as visual stimuli  were manipulations of the corresponding sentences in condition 1 (baseline condition). The final low falling boundary tone was flattened to a mid level with Praat , as in Ich habe nichts (“I have nothing”). Sentences accompanying the twelve practice trials were produced live. These sentences were produced to sound similar to the pre-recorded sentences, using the formats that were otherwise used in conditions 1, 2 and 4 in the pre-recorded sentences.Eight sentences did not contain any object nouns and were used as fillers. In these sentences, the object list was replaced by A participant display in combination with the accompanying sentence constituted an experimental item. In sixty-one of the items in which the confederate named at least one object, the objects were arranged in clockwise order as they were named, starting at the top of the display. In twenty-three of the items, other arrangements were used, so that the participants had to listen attentively and search for the items mentioned by the participant, rather than scanning the objects in the same order on all trials.Four lists were constructed, with each sentence and the accompanying display appearing once per list. Since sentences with less than three objects could not appear in condition 4 (downstepped condition), and sentences with less than two objects could not appear in condition 3 (no boundary tone condition), the number of items per condition was not balanced throughout the experiment. In each list, twenty-eight items appeared in condition 1, twenty-eight items in condition 2, sixteen items in condition 3, and twelve items in condition 4. Each participant was assigned to one of the lists.The procedure followed Barthel et al. . Particifurther objects (s)he has got. Participants were not instructed to use any particular utterance format.After the familiarization phase, the confederate arrived and was introduced as a second participant. Participant and confederate were informed that they would be seated in separate cabins and talk to each other via headphones and microphones to play the following game. They would see a number of displays on their respective screens, showing a number of objects. All objects that were displayed on the confederate display were also displayed on the participant display. The confederate was to tell the participant which things she has got on her display, so that the participant could tell the confederate what The confederate was instructed to try to remember which objects she had seen and which names she had heard. This served as a cover task to distract participants from the aim of the study. Participants were told that their eye-movements would be recorded in order to study looking behavior when searching for objects on a screen whose names were heard. After instructions were given, the eye-tracker was calibrated and calibration was repeated three times during the experiment.Before the test phase, participants completed twelve practice trials. During the test phase, all communication between the participants and the confederate was live, except for the critical pre-recorded sentences. The confederate started the trials and the corresponding pre-recorded utterances so as to make them fit naturally into the conversation.Participants were asked to look at a fixation cross that was presented in the center of the display at the beginning of each trial, which triggered the presentation of the item displays. After a preview that varied randomly between items between 600 and 1,000 ms, the stimulus sentence began.After the experiment, participants were asked in a computerized questionnaire whether they had noticed the presence of pre-recorded speech. The answers were used as a control variable (±recording_noticed).The experiment took about 25 min. The entire test session took about 1 h, including familiarization, test, and questionnaire.F-tests with Kenward-Roger approximations of degrees of freedom . The present latencies are relatively long compared to averages observed in natural conversation, probably due to task demands. They are comparable to the latencies obtained by Barthel et al.  = 21.04, p < 0.001], i.e., participants responded slower when no lexical cue to the turn end was present. Furthermore, response latencies were significantly longer in −BT items (condition 3) than in the baseline condition , i.e., participants responded slower when no final intonational cue to the turn end was present. ±DWNS did not significantly influence response latencies, meaning that the apparent difference in the descriptive statistics is merely an artifact of sentence durationSE = 7, F = 42.95, p < 0.001], meaning that participants responded faster, the longer the incoming turn, presumably because participants' level of preparedness to speak increases as the likelihood that the incoming turn will come to an end increases , presence of low falling boundary tone (±BT), and presence of downstepped pitch peaks (±DWNS) as predictors and the duration of the confederate turns in seconds, as well as ±recording_noticed as control variables. Response latencies were significantly longer in −LEX items (condition 2) than in the baseline condition [β = 90, In order to investigate the time course of participants' planning of their response to critical confederate turns, fixations to the first-mentioned objects in the participants' responses (target objects) were analyzed. Fixations toward an area of interest covering the target objects and approximately 0.25 degrees of visual angle around them were categorized as target fixations. Figure Participants' eye-movements were analyzed in a time window from 0 ms until 2,600 ms, corresponding to the beginning of the last noun in the confederate's turn (0 ms) and the grand mean duration from the time-lock point until the beginning of the first object noun in the participant turn , respectively. Fixations to the target object were aggregated to empirical logits in 100 ms time bins over the course of the analysis window by subjects and by items, respectively Barr, . The emp2) models the steepness of the curve, i.e., how “U-shaped” it is. The cubic time term (Time3) describes at what point in time fixations increase (“S-shaped” curve). An interaction of the linear time term with a factor of interest would signify a difference in the slope of the increase of proportions over time in one level of the factor vs. another level. An interaction of the quadratic time term with a factor of interest would signify a difference in the speed with which proportions increase in one level of the factor vs. another level, thereby describing the pointedness of a U-shaped curve. An interaction of the cubic time term with a factor of interest would signify a difference in latency, i.e., a difference in when proportions start to increase in one level of the factor vs. another level. This interaction is most interesting to us, since it models the predictions about when participants shift their gaze toward the target objects in the different conditions. Table Eye-movement patterns were analyzed using hierarchical quasi-logistic growth curve modeling Mirman, . Growth term Time describeVisual inspection of the proportions of fixations indicates that proportions of target looks are generally at a low level during the incoming turn, increase suddenly after the onset of the last noun of the confederate turn and start to decrease again after about two seconds Figure . In cond2× LEX and Time3× LEX in the direction of earlier and steeper increases in trials with a lexical cue than in trials without a lexical cue.Conditions 1 and 2 were compared to test for effects of the lexical cue to turn end (±LEX). Both by-subject and by-item comparisons showed interaction effects of Time2× BT or Time3× BT was found to be significant, indicating that proportions of target looks were not modulated by the presence or absence of a final low boundary tone.Conditions 1 and 3 were compared to test for effects of a boundary tone cue (±BT). No interaction of Time2 × DWNS or Time3 × DWNS was found to be significant, indicating that the presence or absence of downstepped pitch peaks on non-final list items had no influence on the growth of proportions of target looks.Conditions 1 and 4 were compared to test for effects of downsteps in pitch peaks on non-final list items (±DWNS). No interaction of TimeThe present study set out to investigate whether, in a conversation, next speakers make use of cues to turn ends to align the beginning of their next turns to the end of the incoming turn. Three types of cues were tested: a lexical cue that indicated that the turn would end after the following noun phrase, a final boundary tone that prosodically marked the turn as complete, and a pitch contour that allowed for an early estimation of the length of the unfolding turn. To test the use of these different cues in turn-taking, an experiment using the list-completion paradigm was designed, in which a naive subject and a confederate took turns in naming objects  was present before the last item of the list of the incoming turn, participants knew the following list item to be the last item before the turn would be complete. In sentences containing this lexical cue, participants started planning their response earlier than in sentences not containing this cue, showing that they started planning their response as soon as possible. Dependent on when the turn-final boundary tone becomes recognizable  the lexical cue gave participants a head-start in response planning of at least the length of one syllableParticipants were found to start planning their turn as soon as they knew which objects they had to name, replicating the results of Barthel et al. . When thContrary to the lexical cue, which was located before the last noun phrase of the turn, the final boundary tone was located right before the end of the turn. It was argued before that turn-taking cues which are located at the end of a turn could not be used to time the planning of the content of the response (de Ruiter et al., No evidence was found that next speakers make use of the early prosodic downstep cue to turn length. Participants were not found to use downsteps on pitch peaks in list items to plan their response earlier or respond faster than in turns without a downstepped pitch contour. This early prosodic cue could have been used by participants as much as the lexical cue to the last list item, since the number of list items might have been guessed from the size of the downsteps. However, it is less discrete than the lexical cue, which might be the reason why participants relied on this cue less than on the lexical cue. Both findings on the use of prosodic cues are in line with the conclusions drawn by Bögels and Torreira , who fouIn conclusion, the results suggest that next speakers plan the content of a response as early as the incoming turn's message becomes recognizable and that turn-final cues can function as “go-signals” to initiate response in a timely fashion. Given that lists are a natural kind of conversational turn that is frequently encountered in everyday situations, the present results can be assumed to be generalizable to casual conversation Selting, . Turn-fiMB designed and ran the experiment and analyzed the data. MB wrote the paper and AM and SL commented on it.This research was financed by the ERC Advanced grant # 269484 INTERACT awarded to SL and by the Max Planck Institute for Psycholinguistics.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Low-temperature electronics operating in below zero temperatures or even below the lower limit of the common −65 to 125 °C temperature range are essential in medical diagnostics, in space exploration and aviation, in processing and storage of food and mainly in scientific research, like superconducting materials engineering and their applications—superconducting magnets, superconducting energy storage, and magnetic levitation systems. Such electronic devices demand special approach to the materials used in passive elements and sensors. The main goal of this work was the implementation of a fully transparent, flexible cryogenic temperature sensor with graphene structures as sensing element. Electrodes were made of transparent ITO (Indium Tin Oxide) or ITO/Ag/ITO conductive layers by laser ablation and finally encapsulated in a polymer coating. A helium closed-cycle cryostat has been used in measurements of the electrical properties of these graphene-based temperature sensors under cryogenic conditions. The sensors were repeatedly cooled from room temperature to cryogenic temperature. Graphene structures were characterized using Raman spectroscopy. The observation of the resistance changes as a function of temperature indicates the potential use of graphene layers in the construction of temperature sensors. The temperature characteristics of the analyzed graphene sensors exhibit no clear anomalies or strong non-linearity in the entire studied temperature range . The sensors’ sensitivity in the range of cryogenic temperatures (particularly below 50 K) is much higher than the sensitivity of comparable resistive  temperature sensors (RTD). Temperature sensors made of the graphene film transferred on the polymeric substrate have significantly better flexibility and resistance to thermal stresses in comparison to bulk graphite sensors. Application of thin conductive polymeric films (ITO—Indium Tin Oxide and AgHTTM) allowed us to obtain the sensors with electrodes characterized by high optical transparency and therefore may be applicable in low-temperature optical studies (UV—Ultraviolet).The application of transparent conductive films and multilayer films for resistance temperature detectors in cryogenic systems was discussed for the first time in . GraphenThe crucial features for flexible, transparent temperature sensors are their electrical conductivity, thermal properties, and the optical transmittance of the materials used. These properties of graphene and transparent conducting oxides films are briefly discussed below.min ≈ 4 e2/h; resistivity ρ ≈ 6.5 kΩ/sq) is close to the theoretically predicted quantum conductivity for Dirac fermions [The electrical conductivity of large-surface graphene sheets is a very complex issue, resulting from the characteristics of their carrier transport. The electronic properties of graphene are mostly considered and investigated from the viewpoint of its use as a material that could replace silicon in microelectronic devices controlled by electric fields (MOSFETs). The experimentally measured minimum conductivity in graphene . The phonons’ mean free path in supported graphene, predicted theoretically and estimated from measurements, is about 100 nm [The thermal conductivity perature . These operature ,21,22. Bperature ,24. The t 100 nm . An optical transmittance of single layer graphene equal to 97.7% was theoretically derived, assuming a constant high-frequency conductivity for Dirac fermions through the visible range of the spectrum, and proved experimentally ,26. ThisThe above brief considerations suggest that, especially in applications that use graphene sheets of large size (of the order of millimeters), the dependence of the electrical, thermal and optical properties on methods for graphene synthesis, sample size, grain structure, purity, homogeneity and substrate type must be taken into account.Although graphene has attracted greatest interest as a material for active microelectronic devices (graphene FETs), its exceptionally high electrical conductivity and furthermore its transparency, flexibility and mechanical strength, make it also suitable for passive electronic elements. Recently the requirements of modern optoelectronic technologies, such as photovoltaic technology, flat panel displays, OLEDs (Organic Light-Emitting Diodes) and optoelectronic devices, have caused the rapid development of new conductive transparent materials in the form of thin layers ,30,31,32−1 for the bi-layer graphene, about −0.003 K−1 for the monolayer graphene, and about −0.0015 K−1 for the few-layer graphene, respectively. The bilayer graphene had the highest negative TCR, measured as the temperature changed between RT and 80 °C. Kong et al. [Recently, several innovative temperature sensors based on graphene have been developed, thus providing an alternative to conventional rigid ceramics. Mono- or bi-layer graphene nanofabricated on a silicon substrate has been used as a thermally sensitive element, however no flexibility has been achieved in such a system. Al-Mumen et al.  studied g et al.  fabricatg et al.  proposedg et al.  inventedg et al.  demonstrg et al.  reportedThese excellent properties indicate graphene is a candidate for the preparation of the transparent, flexible electrodes, e.g., instead of ITO . Many in™) with surface resistance of 15 Ω/sq and 4 Ω/sq, respectively. AgHT™ is a highly conductive film on a polyester substrate which is significant in shielding against EMI/RFI (ElectroMagnetic Interference/ Radio-Frequency Interference) and also infrared heat rejection. Its good electrical conductivity, high optical transparency and flexibility determine applications of this material in membrane switches, photovoltaic structures, displays, and passive elements of flexible and transparent electronics. The ITO film on PEN  substrate had a thickness of 125 nm and an ITO/Ag/ITO film thickness of 150 nm.To create electrodes for cryogenic temperature sensors two different conductive films were used, namely commonly used ITO and ITO/Ag/ITO film (AgHTLaser Direct Writing (LDW) methods for nanometer films are used in the manufacture of flexible electronic circuits and sensors on the sub-millimeter scale. It is well known that laser processing of thin layers of nanometer thickness can be performed using laser beams of short wavelength and short pulse duration. Thin functional layers  on transparent substrate materials  have similar ablation threshold fluences, therefore damage of the substrate layers during laser processing should be avoided. Processing of most materials demands applying of UV laser beams with femto- or picosecond pulses to ensure the best effects. Patterning of ITO thin films was performed using laser beam pulses of ultraviolet to infrared wavelength of picosecond, femtosecond or nanosecond duration ,58,59,60The main goal of our former research was to establish the possibility of using a single mode fiber laser in micromachining considering the quality of the obtained structures and their smallest dimensions while maintaining acceptable quality . We haveThe optical transparency was not changed, although some thermal effects were observed after laser treatment. In case of the ITO/Ag/ITO layer even improvement of the optical transmission after laser micromachining was noted. Previous studies have shown that structures patterned in AgHT conductive film can be useful for sensors in cryogenic systems and passive elements in flexible electronics . A redENThe characteristics electrode resistance changes with temperature in the range of 12–300 K are shown in ®) sheets were synthesized in an industrially scaled thermochemical facility based on the process described in previous papers [The high strength metallurgical graphene —PMMA foil, which is described in details in our previous paper [The modified transfer procedure of HSMGus paper . Methodsus paper .® allowed manufacturing of a single layer of graphene. The intentional and controlled modifications of the synthesis process lead to a graphene-like material (G-LM) with a slightly lower value of resistance/square. The production method of HSMG−1 wavenumber range. The acquisition settings listed above did not cause any changes on the surface, like damage by local heating. Data processing was performed using the PeakFit software . Gauss–Lorentz curves were used for spectra deconvolution. Data obtained from the deconvolution for example: peak positions, intensities, half-widths were used for the calculation of characteristic peak ratios of graphene structures which were used in further analysis are provided in following tables . All acquisitions of Raman spectra for graphene structure were performed with using an Ar+ laser at a laser excitation wavelength λ = 532 nm, exposure time was 10 s; signal was averaged from three times repeated exposition per spot. The maximum laser output power was 29.3 mW but the test was carried out at only 10% of the output power. Raman scattering was observed for the 1200–3100 cmg tables .The construction of the graphene cryogenic temperature sensor should eliminate or reduce the sensitivity of the sensor to stimuli other than the temperature. Particularly important is the ability to control the influence of gases on the electrical properties of graphene. This means that the graphene should be insulated from the influence of gas (vacuum inside) or the sensor should be filled with gas in a controlled way (gas inside). Moreover, the structure should provide a simple method of implementing the electrical connections. The reduction of heat exchange with the room temperature environment and heat capacity of the cooled part are just as important in cryogenic systems. The presented transfer of graphene film on a polyester substrate with highly conductive electrodes provided for a small sensor thermal capacity and reduced the heat transfer through the electrodes. The LDW method enabled patterning of electrodes in various shapes, while maintaining the transparency of the electrodes and the substrate. The transparency of the sensor is a unique feature of the presented encapsulation technology. In addition, the small cross section of the thin film electrodes effectively reduces the heat transfer to the cryogenic. Features of the applied substrate and the method of electrode patterning favored the use of a protective thin transparent polymer foil (about 10 μm). The protective film coated on graphene and partially on the electrodes isolates these elements electrically and chemically from the environment . Integration of the polymer film onto the substrate is the result of a thermal activation adhesive. Proper preparation of the protective layer made it connect only to the substrate and does not affect the graphene film. The encapsulation process did not adversely affect the electrical and optical properties of the sensors. The encapsulation process may be performed either under vacuum or in a gas. This is important because thus can be used to change the parameters of the temperature sensor by doping the gas. The sensors retain the transparency and flexibility of conventional polymers after encapsulation process. The proposed encapsulation of the samples provides various options for electrical connections between the sensor and measurement systems. Two types of electrical joints were applied: 1st—a pressed contact as in butt joints, where a thin silver foil (35 μm) has been used and 2nd—an adhesive contact, where the electrically conductive silver-epoxy Elpox AX 15s  has been applied. No effects of the various types of contacts on sensor properties were observed. The proposed technology of encapsulation ensures the possibility of forming fully transparent sensors with different size and shapes . ®) and graphene-like material (G-LM) were used in the construction of sensors. The total resistance of the manufactured sensor is the sum of the resistance of electrodes eR, the resistance of graphene-electrode interfaces iR and the resistance of the graphene layer gR (HSMG® or G-LM) . A graphene monolayer (HSMGor G-LM) . The temperature affects the all listed resistances to a different extent. Therefore, proper selection of materials for electrode construction is particularly important. In the proposed solution, the substrates with electrodes were made of ITO/Ag/ITO AgHT (4 Ω/sq) and ITO (15–100 Ω/sq). The resistance value of the graphene layer is relatively high (>20 kΩ/sq). A significant difference of the resistance of the graphene layer and the resistance of the substrate, resulting from the different thickness of graphene monolayer and electrodes (~120 nm), minimizes the participation of the electrode resistance and the interface between the graphene and electrodes in the total resistance of the sensor.All tests and measurements of electrical properties of graphene cryogenic temperature sensors were performed in a helium closed-cycle DE-210 cryostat . The sensors were cyclically cooled from room temperature (295 K) to cryogenic temperature (20 K) at a rate of about 0.1 K/s. The tested graphene sensors and reference temperature probes were placed directly on the surface of the massive copper heat exchanger (a copper block 80 mm × 80 mm × 10 mm). The encapsulation of the sensor provided the electrical and chemical isolation of the sensor from the environment and other elements of the cryogenic system. In order to eliminate the temperature gradient along the sensor the entire surface of the sensor was fixed to the heat exchanger (including electrodes). The exchanger was mounted directly on the cold finger of the cryocooler (second stage of the cryocooler) . The temperature of heat exchanger was measured and controlled by a precise Lakeshore 331 temperature controller and the reference sensors . The glass-epoxy laminate G10  was used for preparing the fastening and support elements. The observation of the temperature effect on resistivity of the graphene sensors was the main research work. Resistivity measurements were performed using a Keysight 34420A Micro-Ohm Meter . The resistivity of sensors before and after encapsulation has been measured in wide range of temperature (20–295 K). This allowed observing any influence of the encapsulation process on electrical parameters of graphene. Spectra for the studied graphene structures are presented on ® graphene consists of characteristic G and 2D peaks. The intensity ratios IG/I2D or I2D/IG are 0.2 and 4, respectively. According to the studies of Das and Ferrari it can be noted that these values correspond to a single layer of graphene [® is defected, single-layer graphene.The spectrum of HSMGgraphene ,72. The graphene . Taking ® graphene and post-processing procedures preclude the formation of graphene oxide. In G-LM graphene spectra a 2D symmetrical peak can be observed. The 2D peak for G-LM graphene is broadened in comparison to the peak for HSMG® graphene. On analysis of the Raman spectrum an additional D’ peak can be also observed. The D’ peak, like the D peak, indicates a defect and a low ordered carbon structure [G/I2D equal to 3.1 [The spectrum shape of G-LM graphene looks like a spectrum of graphene oxide (GO) or reduced graphene oxide (rGO) with a distinctive arrangement and shape of the G and D peaks ,74,75. Itructure . Das in l to 3.1 . The menl to 3.1 . Taking ® and G-LM showed a significant dependence of resistance on temperature in the range of 20–195 K characterized by a continuous, nearly linear decrease of the resistance (negative temperature coefficient of resistance). The process of encapsulation for samples with HSMG® and G-LM caused the relatively small, permanent increase in the resistance of the sensor  is much higher than the sensitivity of comparable resistive  temperature sensors (RTD). Measurements carried out at temperatures below approximately 20 K showed the increase of dynamics of the sensors’ resistance changes with temperature. It should be noted that the measured changes of electrical resistance as a temperature function in the case of the sensor based on a monolayer graphene are significantly less than for a sensor prepared using a defected graphene-like material. The explanation of this phenomenon requires further study. Temperature sensors made of the graphene film transferred on a polymeric substrate have significantly better flexibility and resistance to thermal stresses in comparison to bulk graphite sensors. The application of thin conductive polymeric films (ITO and AgHTTM) allowed obtaining sensors with electrodes characterized by high optical transparency. These sensors may be applicable in low-temperature optical studies (UV). In cases where transparency is not particularly required, Kapton tape with Au electrodes can be used because of its good electrical and thermal properties in cryogenic temperatures. Future studies of sensors placed in a controlled atmosphere (gas/vacuum in the capsule) are planned.The observation of the effect of temperature on the electrical characteristics of graphene indicates the potential use of graphene layers in the construction of temperature sensors. The temperature characteristics of the analyzed graphene sensors exhibit no clear anomalies or strong non-linearity in the entire studied temperature range . The as-fabricated sensors have a low thermal capacity due to their construction. Changes in resistance of sensors using HSMG


This study aimed to evaluate the correlation between positive PD-L1 expression and driver gene mutations in NSCLC and to seek preliminary evidence in favor of the strategy of PD-L1 inhibitors plus targeted agents.P = 0.010), but no correlation with clinical characteristics , driver gene status  and overall survival (OS): male versus female , never smoking versus former/current smoking , adenocarcinoma versus non-adenocarcinoma , EGFR mutation versus EGFR wild type , ALK positive versus ALK negative , OS of positive PD-L1 expression versus that of negative PD-L1 expression , respectively. Noteworthily, subgroup analyses exhibited that in Chinese cohort studies, positive PD-L1 expression was significantly correlated with OS ; and in the studies using PD-L1 monoclonal antibodies (McAbs), positive PD-L1 expression was significantly correlated with KRAS mutation  and EGFR mutation .The overall analyses revealed that positive PD-L1 expression had a significant relationship with KRAS status (RR = 1.26; 95% CI, 1.06−1.50, After thoroughly searching PubMed, EMBASE and Cochrane Library databases, 11 relevant studies incorporating 3128 cases were identified. The pooled data were analyzed via Review manager 5.3 software.PD-L1 inhibitors probably was a potential promising option to manage advanced NSCLC harboring KRAS mutation. Worldwide, lung cancer, including non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), is one of the leading causes of cancer-related deaths , and itsRecently, immune checkpoints including programmed death-ligand 1 (PD-L1), programmed death 1 (PD-1), have made waves in anticancer immunotherapy. Some studies exhibited that PD-L1 expressed on the surface of tumor cells, including in NSCLC, melanoma, breast cancer , 9. PD-1After initial comprehensively searching from PubMed, EMBASE and Cochrane Library databases, 622 potential relevant articles were found, whereas 595 (235 duplications and 360 irrelevant articles) were excluded. Then, another 16 articles were further removed after screening full-text for the following reasons: 8 for insufficient data, 4 for driver genes unavailable and 4 for the same population. Eventually, 11 articles incorporating 3128 cases were identified. The flow chart of literature searching and the clinical characteristics of included studies were listed in Figure P = 0.15, Figure P = 0.17, Figure P = 0.77, Figure Overall analyses revealed no significant correlation between positive PD-L1 expression and gender , subgroup analyses were conducted based on the studies using PD-L1 McAbs or PoAbs, and on Chinese cohort studies. The outcomes demonstrated that in the studies using PD-L1 McAbs, positive PD-L1 expression more frequently occurred in EGFR mutation group than in wild type group , thus a fixed-effect model was employed. The pooled result indicated that positive PD-L1 expression was not associated with ALK status  [ cases 1, 21, 22 I2 = 7%, P = 0.38), and the outcome revealed a significant correlation between PD-L1 expression and KRAS status . The result indicated that positive PD-L1 expression tended to be associated with poor OS, despite no statistical significance .Ten studies –22  is the first meta-analysis to focus on the correlation between positive PD-L1 expression and driver genes in NSCLC. Our study revealed that positive PD-L1 expression tended to occur more frequently in female, never smoking, ADC, EGFR mutation and ALK positive, but no significant association between them. Interestingly, there was a significant correlation between positive PD-L1 expression and KRAS status. Meanwhile, concerning the diversity of PD-L1 antibodies, further subgroup analyses based on the studies using PD-L1 McAbs or PoAbs were implemented. And the results showed that positive PD-L1 expression was significantly associated with KRAS status and EGFR status in the studies using PD-L1 McAbs, but not in the studies using PD-L1 PoAbs. Furthermore, we also conducted another subgroup analyses on Chinese cohort studies, which revealed that positive PD-L1 expression was associated with only OS, but not with other parameters.KRAS mutation is one of the most common driver genes in NSCLC, but the aim of designing therapeutic regimen for lung ADCs harboring KRAS mutations has far proven elusive. Our study demonstrated that positive PD-L1 expression had a significant correlation with KRAS status. The result mentioned above was obtained in the studies using PD-L1 McAbs, while it did not occur in the studies using PD-L1 PoAbs and in Chinese cohort studies. One reason for the inconsistent results between overall and subgroup analyses might be the diversity of races , because KRAS mutation was low expression in Chinese population . The othWith respect to the relationship between PD-L1 expression and EGFR status, some studies showed that the activation of EGFR pathway might up-regulate PD-L1 expression . Our stuAdditionally, this study revealed that PD-L1 expression had no significant correlation with ALK status in both overall and subgroup analyses. However, Ota et al showed that EML4-ALK rearrangements and downstream signaling pathways could induced PD-L1 expression in NSCLC models . Thus, tFurthermore, subgroup analyses demonstrated that positive PD-L1 expression was significantly associated with OS in Chinese cohort studies but not in the studies using PD-L1 McAbs or PoAbs. This differences may attribute to the diversity of races, the definition of positive/negative PD-L1 expression, the various therapeutic regimen, the duration of follow-up, the baseline characteristics as well as the potential bias of several HRs which were extracted from Kaplan-Meier curves.Nevertheless, we also encountered some limitations: firstly, the sample size of participants was not substantial, only 11 studies containing 3128 cases, thus, more large scales studies including various races were required to further determine the correlation between PD-L1 expression and drive genes; subsequently, all eligible articles were published in English, and other languages were not included, which may be a potential bias; finally, the obvious heterogeneity among these eligible studies would affect the stability of statistical analyses to some extent, despite the fact that subgroup analyses had lowered the heterogeneity.Accordingly, PD-L1 inhibitors probably was a potential promising option to manage advanced NSCLC harboring KRAS mutation.Relevant articles were thoroughly searched from PubMed, EMBASE and Cochrane Library databases using the following terms: programmed cell death-ligand 1, PD-L1, B7-H1, lung cancer, NSCLC, EGFR, ALK, KRAS and driver genes, from their inception till September 2016. The search was performed with language limitation to English. If the same population was found in different publications, the latest or the most complete articles were included. Once the articles were identified, the references were also searched for extending the search. All potential relevant articles were scanned by two researchers (Yang HT and Chen HJ) independently via the following process: initially excluding duplication by Endnote X7 software, then selecting pertinent articles via screening titles or abstracts and eventually identifying eligible articles through scrutinizing full-texts. Disagreement was resolved by consensus or the third researcher (Xie XH).The eligible articles should meet the following criteria: i) all patients had histologically or cytologically confirmed NSCLC; ii) PD-L1 expression as well as one or more driver genes  were available; iii) all studies investigated the correlation between positive PD-L1 expression and driver genes mutations in NSCLC and acquired sufficient data; iv) all articles were published in English. Reviews, letters, ongoing studies and insufficient data were excluded.Two researchers (Yang HT and Chen HJ) extracted the following data independently from all eligible studies: the first author's name, year of publication, country, gender, smoking status, histological type, PD-L1 expression (positive/negative), driver genes  and OS. When the extracted data existed discrepancy, consultation was held to settle the problem.P < 0.1 and I2 > 50%, indicating significant heterogeneity, a random effects model was utilized, otherwise, a fixed-effect model was employed [The pooled data were calculated via Review manager 5.3 software. Analyses were conducted according to gender, smoking status, histological type, driver genes  and OS. The effective value, RR with 95% confidence intervals (95% CIs), was employed to evaluate the correlation between positive PD-L1 expression and driver gene mutations, clinical characteristics in NSCLC. Hazard ratios (HRs) with 95% CIs were extracted from the articles or calculated from survival curves according to Zhou et al method if these data were unavailable . HR>1 inemployed . With re


The photodetectors exhibit a selective photo responsivity for the UV light with the wavelength of 405 nm, confirming the main light absorption from QDs. The photo-generated charges have been found to transfer from QDs to graphene channel, leading to a gate-tunable photo responsivity with the maximum value obtained at VG about 15V. A recirculate 100 times behavior with a good stability of 21 days is demonstrated for our devices and another flexible graphene/QDs based photoconductors have been found to be functional after 1000 bending cycles. Such UV photodetectors based on graphene decorated with cadmium-free ZnSe/ZnS quantum dots offer a new way to build environmental friendly optoelectronics.Graphene-based optoelectronic devices have attracted much attention due to their broadband photon responsivity and fast response time. However, the performance of such graphene-based photodetectors is greatly limited by weak light absorption and low responsivity induced by the gapless nature of graphene. Here, we achieved a high responsivity above 10 However, a low absorption coefficient, which causes a barrier from photon to generated charge carriers, has limited the responsivity of graphene-based photodetectors to ~10−2 AW−1 9. So far, great efforts on the achievement of high-performance graphene-based photodetectors have been focused on the development of graphene hybrid structure such as graphene/semiconductors11, graphene/polymer12 or graphene/quantum dots13. In such structures, the photoelectric conversion usually occurs in the as-metioned introduced parts working as light harvesting layers or the junction generated between graphene and other semiconductors. The photo-generated charge carriers could shift into the graphene under the build-in field and change the conductance of graphene to achieve high responsivity. Among these hybrid structures, quantum dots stand out because of high-quality crystalline structures, solution processability, low cost, novel size-tunable wavelength and high responsivity15. Especially, colloidal semiconducting quantum dots (QDs) synthesized by chemical methods, which are tiny crystals of semiconductors with particle size as small as a few or several tens of nanometers, have attracted much attention for the light-emitting diodes and photosensitive devices17. For example, an infared photodetector based on CVD-grown graphene and PbS quantum dots has been proposed with ultrahigh responsivity of 107 AW−1 at an incident power of about 30 pW18.Graphene is a promosing 2D material for optoelectronics19. However, their efficient emissions are mostly limited in the range from the green to near-infrared spectral window due to a narrow band gap, which also limits the applications in short-wave band20. Another important factor, which hinders the further development of these QDs, is the toxicity of the Cd element. In contrast, the ZnSe QDs may be an excellent candidate for short-wave band appliactions due to their wide band gaps and low toxicity. Especially, when the surface of ZnSe is passivated by ZnS, a ZnSe/ZnS core shell quantum dot is formed and brings a remarkable enhancement of the optical properties compared with pristine ZnSe QDs22. The ZnS shell with higher energy can fill the detects on the surface of ZnSe QDs, resulting in a more perfect particle surface and an improved quantum yield. In addition, this core/shell structure also reduces the recombination of semiconductor nanocrystals and improves the stablity of QDs24. The synthesis methods for such ZnSe/ZnS core/shell quantum dots have been widely discussed and this class of materials has been applied as emitting materials in blue QD-LEDs due to their narrow emission peak and wavelength tunability27. However, their optoelectrical properties and potential applications in photodectors are rarely reported.Recently, great progress has been made in the synthesis of QDs, and a large number of high quality QDs, such as CdSe, CdTe and CdS QDs, have been successfully synthesized by organometallic and aqueous approaches3 AW−1. The sensing mechanism is attributed to the photo-generated charge carriers in the QDs and the tranfer process from QDs to graphene, which may directly modulate the Fermi Level of graphene and the conductance of graphene channel. Besides, the ligand capping the surface of QDs, which is used to gurantee the stablity of QDs, was found to have an impact on the photo responsivity of QDs, especially the response/recovery time, due to its role in charger transfer process between graphene and QDs.In this work, ultraviolet (UV) photoconductors based on CVD-grown monolayer graphene decorated with ZnSe/ZnS core/shell QDs by a simple solution method. The devices were fabricated on the bottom-gated Si substrates and exhibited a high reponsivity up to 2 × 102 substrate also indicates a dispersed arrangement . In order to furtherly characterize the size of ZnSe/ZnS QDs, the transmission electron microscope (TEM) image of the QDs is shown in Fig. The scanning electron micrograph (SEM) image of this device is shown in Fig. IDS- VG curves) of GFET was measured in the absence of light and is shown in Fig. 29, W-shaped curves in transfer characteristics of GFETs have been widely observed and the origin of such abnormal behaviors is due to the non-uniform doping in graphene channel induced by the adsorbate from ambient environment or the charge defects during the device fabrication process. Recent studies on II–VI group semiconductor nanostructures, such as ZnS and ZnSe NWs/NR, demonstrated that complementary doping, i.e., both n- and p-type doping, could be realized by carefully controlling the experimental conditions31. As shown in the SEM image , the QDs are scatterred across the graphene channel and the holes in p-type ZnSe/ZnS QDs will transfer to the graphene film and induce a local p-doping in graphene channel as shown in Fig. Vg to get the charge neutral point and achieves at least two Dirac points. Then, the optoelectrical properties of our device was measured under the illumination with a wavelength of 405 nm. The photocurrent (IP) defined by IP = ILight − IDark, where ILight is the drain current under the illumination and IDark is the drain current in the dark, and the responsivity to UV light was measured and calculated as a function of source-drain voltgate (VDS) in Fig. VDS and the light irradiation. The responsivity increases with the increase of VDS but decreases with the increase of the light irradiation, which is consistent with the reported UV-detectors32. The maximum responsivity is about 2 × 103 AW−1 at VDS = 5V and an incident power of about 5.54 mW/cm2, which is a higher value compared with other previously reported graphene-based UV-detectors , the conductance of graphene channel increases for VG < VDirac, where the hole carriers are dominant, but decreases for VG > VDirac, where the electron carriers are dominant. In our work, ZnSe/ZnS QDs play an importance role in the photosensitive behaviors of our device, where a maximum photo responsivity is achieved at the wavelength of 405 nm compared with the results at the wavelength of 470 nm and 590 nm , which is consistent with results of absorption spectrum and PL spectrum in Fig. ΔVG) is plotted as a function of light irradiation is shown in Fig. 34. From Fig. et al.18 Fig. 36.To get insight into the sensing mechanism, the transfer characteristics of our phototransistor were characterized under light illumination at the wavelength of 405 nm with different irradiation. The transfer curves shift to higher positive gate voltage with the increase of light irradiation as shown in Fig. 38. Acordding to the conversion mechanism from photo to charges talked above, the photo-generated charges need tranfer from QDs to graphene and collected to drain electrodes through the graphene channel. So, the response time includes the charges generation time, charges tranfer time between QDs/graphene and the charges collection time. It is notable that the charger generation occurs in QDs, which is a fast process due to a direct bandgap structure for ZnSe and the charge collection is achieved through the graphene channel, which offers a high carrier mobility. In this case, the slower response time may be due to the delay for the charge transfer from the QDs to graphene. The ligand capping the surface of QDs may be the origin because the oleic acid, ligand used here, is a long chain polymer, leading to an inefficient charger transmission. This issue may be solved by a ligand replacement technology by other short chain polymers, which may minimize the interparticle spacing to promote carrier transport39. Considering the complexity of ligand replacement technology, this improvement will be discussed in our future works. Figure 3 AW−1 after being put in ambient for 21 days.The transient behaviors with the illumination on and off and the reliability of graphene/QDs hybrid structure was investigated as shown in Fig. 2. The photocurrent is promoted with the increase of irradiation, indicating a strengthed absorption of UV light when the bias voltage increases. The responsivity of flexible photoconductors was measured after a 1000 times bending test as shown in Fig. −1 after the bending test. In this case, our device based on graphene/QDs can be easily fabricated on the flexible substrate and compatible with conventional CMOS technology to be pattern to smaller size, indicating a great potential in future wearable electronics.Considering the flexibility of graphene and QDs, such photoconductors could be fabricated on the flexible substrate as shown in the inset of Fig. 3 AW−1 at a light irradiation of 5.54 mW/cm2 and VG of 15V. Such photodetectors are found to dynamically response to periodic light switching on/off as many as 100 cycles and the response time of our devices is calculated to be 0.52s, which is limited by the ligand covering the surface of QDs. The optimization of ligand for QDs will be a crucial issue to further improve the response time of photodetectors. The responsivity of such photodetectors remains to be above 103 AW−1 after being placed in air for 21 days. Moreover, flexible graphene/QDs hybrid photoconductors are realized and show an excellent flexible stability after 1000 times bending test, which confirms great potential of such low dimensional materials in future wearable and stretchable electronics.In conclusion, high responsivity UV photodetectors based on CVD-grown graphene decorated with ZnSe/ZnS core/shell quantum dots were realized by facile solution processing method. The ZnSe/ZnS QDs afford the conversion from photon to charges, which could be transferred from the QDs to graphene under the electric field of heterojunction between graphene and QDs. The responsivity of such photodetectors could be effectively tuned by the gate voltage and increases with the decrease of light irradiation. The maximum value reaches as high as 2 × 102 gate dielectric by thermal oxidation process as bottom gate dielectrics. Monolayer graphene grown by CVD method was transferred to the SiO2 substrate by a PMMA-assist transfer process to form the conducting channel. Two-step standard photolithography process was used to pattern the graphene channel and the S/D electrodes deposited by Cr/Au (10 nm/50 nm) through electron beam evaporation process. The ZnSe/ZnS quantum dots solution diluted to be 1mg/ml was spin-coated on the graphene surface and dried on the hot plate. Flexible photoconductors were fabricated on the flexible PET substrate and fabrication process was similar to that on SiO2 substrate.Figure SEM images were carried out on a Sirion-200 field-emission scanning electron microscope (FEI). TEM images were collected on a Hitachi H-7650 electron microscope operated at 80 kV. Absorption spectra were recorded on a Lambda 35 UV-vis spectrometer (Perkin Elmer). PL spectra were performed on a FLSP920 fluorescence spectrometer (Edinburgh). The electrical performance of the phototransistors was studied by using a B1500A Semiconductor Device Analyzer (Agilent Technologies) and Summit 11000 AP probe station (CASCADE microtech) at room temperature. The monochromatic lights with different wavelengths were provided by CEL-LEDS35 LED illuminant (CEAULIGHT). In the bending test, the flexible samples were bended to a radius of 7mm and bended up for 1000 times.Supporting Information