The neural cell adhesion molecule L1  functions not only in cell migration, but also in cell survival, differentiation, myelination, neurite outgrowth, and signaling during nervous system development and in adults. The proteolytic cleavage of L1 in its extracellular domain generates soluble fragments which are shed into the extracellular space and transmembrane fragments that are internalized into the cell and transported to various organelles to regulate cellular functions. To identify novel intracellular interaction partners of L1, we searched for protein–protein interaction motifs and found two potential microtubule-associated protein 1 light-chain 3 (LC3)-interacting region (LIR) motifs within L1, one in its extracellular domain and one in its intracellular domain. By ELISA, immunoprecipitation, and proximity ligation assay using L1 mutant mice lacking the 70 kDa L1 fragment (L1-70), we showed that L1-70 interacts with LC3 via the extracellular LIR motif in the fourth fibronectin type III domain, but not by the motif in the intracellular domain. The disruption of the L1-LC3 interaction reduces L1-mediated neurite outgrowth and neuronal survival. Neural cell adhesion molecules are membrane proteins that play important roles in cell adhesion, migration, and signaling. One prominent member of this group is the surface glycoprotein L1, which is involved in cell migration, proliferation, survival and differentiation, axon fasciculation, and myelination during development of the nervous system, as well as in neurogenesis, neural plasticity, learning and memory, and regeneration in the adult nervous system ,4,5,6,7.Proteolysis of full-length L1 was shown to be important for L1’s functions, and extracellular and intracellular L1 fragments were found in different locations and organelles and regulate different cellular functions ,44,45,46To identify novel interaction partners of intracellular L1 fragments and to further determine the function of these L1 fragments, we searched for protein interaction motifs in L1 and intracellular proteins interacting with these motifs. We found two potential microtubule-associated protein 1 light-chain 3-interacting region (LIR) motifs in L1: one in the fourth FNIII domain and one in the intracellular domain. Proteins containing a LIR motif bind to the LIR docking site of proteins belonging to the autophagy-related protein 8 (ATG8) family. The microtubule-associated protein 1 light chain 3 alpha  is a mammalian homolog of ATG8E. The LC3 conjugation system is required for elongation and maturation of the autophagosome  and mitoHere, we show by ELISA and proximity ligation assay that L1 interacts directly with LC3 and that this interaction is mediated by the LIR motif within the fourth FNIII domain, but not by the LIR motif in the intracellular domain. LC3 interacts mainly with L1-70 generated by myelin basic protein, as seen by a reduction of L1/LC3 interactions in non-stimulated cerebellar and cortical neurons from L1-70-lacking mutant mice, and L1 antibody-enhanced L1/LC3 interactions were not observed in L1-70-lacking neurons in contrast to wild-type neurons. The treatment of cultured cortical neurons with a cell-penetrating peptide containing the LIR motif of the fourth FNIII domain reduces the interaction as well as L1-dependent neurite outgrowth and neuronal cell survival.Previous research from our group showed that fragments of L1 are present inside of the cell in the cytosol, in nuclei, and in mitochondria, and that L1 regulates mitochondrial and nuclear functions ,45,46,480-X1-X2-[LVI]3 [-2-[DEGMSTV]-1-[WFY]0-[DEILQTV]1-[ADEFHIKLMPST]2-[ILV]3, which is a redefined LIR motif [-2-[DEGMSTV]-1-[WFY]0-[HKRN]1-[ADEFHIKLMPST]2-[ILV]3 with the conservative glutamine (Q) substitutions histidine (H), lysine (K), arginine (R), and asparagine (N) at position X1 and identified one motif in the fourth FNIII domain (952LSYHPV957) and one in the in the intracellular domain (1177GEYRSL1182) of mouse L1. These motifs were also found in the fourth FNIII domain and in the intracellular domains of rat , human , and bovine  L1. These findings raise the question of whether L1 directly interacts with LC3 and if this interaction is mediated by the LIR motifs in L1.To search for putative LIR motifs in L1, we used the canonical LIR core motif [WFY]2-[LVI]3  to scan IR motif , was useTo investigate if L1 interacts with LC3 and if this binding is mediated by the LIR motif in the extracellular or intracellular L1 domain, an ELISA was performed. First, recombinant LC3 was used as a substrate-coat and was incubated with the recombinant His-tagged intracellular domain of L1 (L1-ICD). The intracellular domain of CHL1 (CHL1-ICD), the close homolog of L1, served as negative control. A concentration-dependent binding of the L1-ICD, but not of the CHL1-ICD to LC3, was observed a. Next, WT peptide containing the extracellular LIR motif LSYHPV or the LIRmut peptide containing the mutated LIR motif LSAHPA for competition ELISA with L1/Fc and fusion proteins containing human Fc and either FNIII domains 1–5 (FN1–5/Fc) or Ig-like domains 1–6 (Ig1–6/Fc). L1/Fc and FN1–5/Fc, but not Fc, bound to LC3, while Ig1–6/Fc showed only low binding to LC3. Moreover, the LIRWT peptide reduced the binding of L1/Fc and FN1–5/Fc by 60–65%, whereas the LIRmut peptide did not affect the binding of L1/Fc and FN1–5/Fc to LC3  were tested by ELISA for binding to LC3. L1/Fc, but not CHL1/Fc, showed a concentration-dependent binding to LC3 a. Next, c to LC3 b. These WT peptide in the absence or presence of the function-triggering L1 antibody 557 and then subjected the neurons to proximity ligation assay with a mouse L1 antibody and a rabbit LC3 antibody. L1 antibody 557 enhanced the number of red spots, which indicate L1/LC3 interactions, approximately four times, while the tat-LIRWT peptide reduced the L1/LC3 interactions in non-stimulated and stimulated neurons by 69 ± 10% and 63 ± 10%, respectively  and phenylalanine (F) at that position: out of 100 LIR motifs only 10 contain Y at the [WFY]0 position, while 42 and 48 contain W or F at this position, respectively [0 are mitochondrial LC3 receptors, which are involved in autophagy and/or mitophagy: FUN14 Domain Containing 1 (FUNDC1), Autophagy Related 4B (ATG4B), Mitogen-Activated Protein Kinase 15 (MAPK15) and Neighbor of BRCA1 Gene 1 Protein (NBR1). The mitochondrial outer membrane protein FUNDC1 binds to LC3 through its LIR motif DSYEVL and acts as an activator of hypoxia-induced mitophagy, ATG4B with its LIR motif LTYDTL is a cysteine peptidase which is required for autophagy as well as for mitophagy, MAPK15 carrying the LIR motif RVYQMI is an atypical MAPK protein that regulates basal and starvation-induced autophagy in a kinase activity-dependent manner, and NBR1 with the LIR motif EDYIII is an autophagy cargo receptor. The phosphorylation of tyrosine (Tyr-18) at position X0 in the LIR motif of FUNDC1 inhibits the activation of mitophagy, while hypoxia-induced dephosphorylation of this tyrosine and phosphorylation of the preceding serine at position X-1 (Ser-17) stabilizes the interaction of FUNDC1 with LC3 and triggers mitophagy [After its generation at the plasma membrane, L1-70 is transported from the cytoplasm into nuclei and mitochondria and is also present at the mitochondrial surface . L1-70 iectively . Among pitophagy .-1 in the LIR motif GSWVEL of the mitochondrial LC3 receptor Bcl2 interacting protein 3 (BNIP3) promotes the interaction between BNIP3 and LC3 and induces mitophagy under oxidative stress conditions [Interestingly, the phosphorylation of serine (Ser-17) at position Xnditions .-1 of L1’s extracellular LIR motif may become phosphorylated. We propose that L1 is a mitochondrial LC3 receptor like FUNDC1 and BNIP3 [-1 promotes mitophagy, as seen for the phosphorylation of serine at position X-1 in the LIR motifs of FUNDC1 and BNIP3. It is conceivable that the binding affinity of LIR motifs to LC3 in general is regulated through phosphorylation . Thus, serine at position Xnd BNIP3  and thatces, see ).L1 binds to dynamin-related protein 1 (DRP1) , which i953LSYHPL958. This mutation causes symptoms of the L1 syndrome, e.g., adducted thumbs, hydrocephalus, and spastic paraplegia [Since L1-stimulated neurite outgrowth and neuronal survival were abrogated in the presence of a peptide containing the LIR motif of the fourth FNIII domain and basal cell survival was also reduced in the presence of this peptide, it is plausible that L1-stimulated neurite outgrowth relies on the interaction of L1 with LC3 and that interference with the L1/LC3 interaction leads to enhanced neuronal cell death. Furthermore, one may conclude that neurite outgrowth and the protection of neurons against oxidative stress depends on mitophagy that is regulated by the interplay of LC3 with L1. The disturbance of this interaction may have severe functional consequences and may lead to the development of neurodegenerative diseases or L1-associated diseases, such as the L1 syndrome. Of note, T to G mutation in exon 21 of the human L1 gene (c.2858T > G) results in a leucine to arginine exchange at position 953 in the extracellular LIR motif raplegia .In summary, the extracellular LIR motif in the fourth FNIII domain of L1 is crucial for L1’s functions in the mouse brain.687 mutant) or with a mutation of the dibasic sequence RKHSKR to SKHSSS at positions 858–863 in the third FNIII domain (L1858–863 mutant) [687 mutant males, L1858–863 mutant males, L1-deficient males, and their wild-type male littermates, were used for all experiments. All animal experiments were conducted in accordance with the German and European Community laws on the protection of experimental animals and approved by the local authorities of the State of Hamburg . The manuscript was prepared following the ARRIVE guidelines for animal research [L1-deficient mice  and gene mutant)  have beeresearch .WT (SHNGVLTGYLLSYHPVEGESKEQ), and LIRmut (SHNGVLTGYLLSAHPAEGESKEQ), as well as tat-LIRWT (YGRKKRRQRRRSHNGVLTGYLLSYHPVEGESKEQ) and tat-LIRmut (YGRKKRRQRRRSHNGVLTGYLLSAHPAEGESKEQ), both containing the cell-penetrating HIV tat sequence YGRKKRRQRRR [Rabbit LC3 antibody  was from Proteintech Europe . Mouse L1 antibody C-2  against the L1-ICD, goat L1 antibody C-20 , and goat CHL1 antibody C-18  were from Santa Cruz Biotechnology . HRP-coupled goat anti-Fc antibody  was from Jackson ImmunoResearch . Rat monoclonal function-triggering L1 antibody 557 has been described . SecondaKKRRQRRR , were frhttps://prosite.expasy.org/scanprosite/; accessed on 10 August 2021) was used for scanning the sequences of mouse (L1CAM_mouse), rat (L1CAM_rat), human (L1CAM_human), and bovine (E1BBI3_BOVIN) L1 for putative LIR motifs. NetPhos prediction tool  was used to identify phosphorylation sites in L1.The Expasy ScanProsite tool  (PBS), treated with blocking solution  for 2  h, washed again with PBS containing 0.005% Tween 20 (PBST), and incubated with increasing concentrations of recombinant His-tagged L1-ICD and CHL1-ICD or L1/Fc and CHL1/Fc as ligands for 1 h under gentle agitation. For competition ELISA, 2.5 µM L1-ICD, L1/Fc, Ig1–6/Fc, or FN1–5/Fc were preincubated for 1 h without or with a 5-fold molar excess of peptide P1, P2, P3, P4, P5, Pa, Pb, Pc, Pd, Pe, LIRWT, or LIRmut. The mixtures were then incubated with substrate-coated recombinant LC3B. After washing two times with PBS and three times with PBST, L1 antibody C-2 (1:500), CHL1 antibody, or HRP-coupled anti-Fc antibody (1:2000) in blocking solution was applied for 1 h. When samples were incubated with L1 and CHL1 antibody they were washed two times with PBS and three times with PBST and then incubated with HRP-coupled anti-mouse and anti-goat antibody (diluted 1:2000 in blocking solution) for 1 h. All samples were washed two times with PBS and three times with PBST, and wells were incubated with 1 mg/mL ortho-phenylenediamine dihydrochloride  to detect bound proteins. The reaction was terminated by the addition of 25 µL 2.5 M sulphuric acid. Absorbance was measured at 492  nm with an ELISA reader .For ELISA, 25 µL of 10 µg/mL recombinant LC3B were incubated overnight at 4 °C in 384-well microtiter plates with a high binding surface . All the following steps were performed at room temperature. Wells were washed with Dulbecco’s phosphate-buffered saline with MgCl4 cells per well of a 48-well plate coated with poly-L-lysine (Sigma-Aldrich). For proximity ligation assay, cells were seeded onto 12 mm glass coverslips poly-L-lysine-coated in a 24-well plate at a density of 1.25 × 105 cells per well. For cell death analysis, cells were seeded onto poly-L-lysine-coated 48-well plates at a density of 2.5 × 105 cells per well.Cortical neurons were prepared from cerebral cortices of 15.5- to 16.5-day-old embryos. Dissected cerebral cortices were incubated in Hanks’ balanced salt solution (HBSS) containing 0.025% trypsin (Sigma-Aldrich) at 37 °C for 30 min. The cortices were then incubated in HBSS containing 1% BSA (Sigma-Aldrich) and 1% trypsin inhibitor  at 37 °C for 2 min. After washing in HBSS, the tissue was mechanically dissociated, and the dissociated cells were cultured in Neurobasal medium (Thermo Fisher Scientific) supplemented with 1% B27, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin . For the determination of neurite outgrowth, cells were seeded at a density of 5 × 105 cells per well. For the determination of neurite outgrowth, cells were seeded at a density of 5 × 104 cells per well of a 48-well plate coated with poly-L-lysine (Sigma-Aldrich), and for neuronal survival analysis cells were seeded at a density 1.25 × 105 cells per well of a 48-well plate coated with poly-L-lysine.Cerebellar neurons were prepared from the cerebella of 6- to 8-day-old mice. Cerebella were incubated with 10 mg/mL trypsin and 0.5 mg/mL DNase I (Sigma-Aldrich) in HBSS for 15 min at 37 °C, washed with HBSS, dissociated, and centrifuged at 100 × g for 15 min. Cells were then diluted in Neurobasal A medium (Thermo Fisher Scientific) supplemented with 2 mM L-glutamine (Thermo Fisher Scientific), 4 nM L-thyroxine (Sigma-Aldrich), 0.1 mg/mL BSA (Sigma-Aldrich), 12.5 μg/mL insulin (Sigma-Aldrich), 30 nM sodium selenite (Sigma-Aldrich), 100 μg/mL transferrin , 0.1 mg/mL streptomycin, and 100 U/mL penicillin (Thermo Fisher Scientific). For the proximity ligation assay, cells were seeded onto poly-L-lysine-coated 12 mm glass coverslips in a 24-well plate at a density of 2.5 × 10WT peptide (100 µg/mL in vehicle solution) for 16–24 h. Following that, cells were treated with PBS (unstimulated) or L1 antibody 557  for 2 h. Cultured cells were fixed for 15–30 min at room temperature in 4% formaldehyde, washed with PBS, and subjected to proximity ligation assay using Duolink PLA products according to the manufacturer’s protocol  with minor modifications. Cells were incubated with 1% Triton X-100 in PBS for 30 min, washed once with PBS, and blocked with Duolink Blocking solution for 30 min and incubated for 24 h at 4 °C with mouse L1 antibody C-2 (1:10 dilution) and rabbit antibody against LC3 (1:30 dilution) in Duolink Antibody Diluent. Cells were washed two times using Duolink Wash Buffer A and incubated with a mixture of secondary antibodies conjugated with oligonucleotides (Duolink PLA Anti-Rabbit Probe MINUS and Duolink Anti-Mouse PLA Probe PLUS). The proximity ligation reaction was then performed according to the manufacturer’s protocol using the Duolink In Situ Detection Reagent RED. Thereafter, the coverslips were incubated in 5 µg DAPI/mL PBS for 15 min, washed twice with PBS and mounted in Immuno-Mount (Thermo Fisher Scientific). Ten images per condition were taken using an Olympus F1000 confocal microscope  and analyzed using ImageJ software . Numbers of red dots and numbers of DAPI-stained nuclei were determined using ImageJ and the number of red dots per image was divided by the number of nuclei per image. The average values of red dots per cell (=nucleus) were determined in 10 images per condition.After seeding, cells were treated with vehicle solution or tat-LIRWT or tat-LIRmut peptides (50 µg/mL) and 557 antibody (50 µg/mL) 30 min after seeding. After 24 h in culture, cells were washed gently with pre-warmed culture medium, fixed in 2.5% glutaraldehyde for 30 min at room temperature, and stained with 1% toluidine blue and 1% methylene blue in 1% sodium tetraborate for 30 min at room temperature. Neurite outgrowth was analyzed by measuring the total length of neurites with a Zeiss AxioObserver.A1 microscope with the AxioVision 4.6 imaging system . For neurite outgrowth analysis for each condition, at least 100 neurons were counted.To determine neurite outgrowth, cells were treated with cell-penetrating tat-LIRWT or tat-LIRmut peptides (50 µg/mL) and L1 antibody 557 (50 µg/mL) and exposed to oxidative stress by the addition of 10 µM H2O2 for 24 h. Live and dead cells were then stained with calcein-AM (Thermo Fisher Scientific) and propidium iodide (Sigma-Aldrich) and imaged with a Zeiss AxioObserver.A1 microscope (Carl Zeiss) with a 20× objective (aperture 0.4) and the AxioVision 4.6 software (Carl Zeiss). Live and dead cells were counted in five images (containing 350–400 cells each) from each of the three wells per condition and experiment using ImageJ.To determine cell death, neurons were maintained overnight in serum-free medium and then treated with cell-penetrating tat-LIRFor the preparation of nuclear and non-nuclear fractions, the Subcellular Protein Fractionation Kit for Tissue (Thermo Fisher Scientific) was used. Fractions in cytoplasmic extraction buffer (CEB) and membrane extraction buffer (MEB) were pooled and taken as non-nuclear fractions. Fractions in nuclear extraction buffer (NEB) were taken as nuclear fractions. Nuclear and non-nuclear fractions were used for immunoprecipitation.For immunoprecipitation, Protein G magnetic beads (25 μL per sample) were washed twice in PBS, pH 7.4, and incubated in dilution buffer  for 10 min at 4 °C under rotation. The mouse L1 antibody C-2, rabbit LC3 antibody, or non-immune control rabbit or mouse antibodies (10 μg per sample) were diluted in dilution buffer. The diluted antibody solutions were incubated with the beads for 1 h at 4 °C under rotation. The beads were washed in dilution buffer for 5 min at 4 °C under rotation. A freshly prepared 13 mg/mL stock solution of dimethyl pimelimidate (DMP) was 1:1 diluted with wash buffer (0.2 M triethanolamine in PBS). After washing the beads in PBS, the diluted DMP solution (pH 8–9) was added to the beads, and beads were incubated for 30 min at room temperature (20–24 °C) under rotation. The beads were washed in wash buffer for 5 min at room temperature under rotation. The beads were then incubated in DMP solution and wash buffer for two further times. Finally, the beads were incubated twice in quenching buffer (50 mM ethanolamine in PBS) for 5 min at room temperature under rotation. After washing, the beads were incubated with brain fractions overnight at 4 °C under rotation. The beads were washed twice with lysis buffer and once with PBS. The beads were then boiled for 5 min in sample buffer .® TGX™ Precast Protein Gels . PageRuler™ Plus Prestained Protein Ladder (Thermo Fisher Scientific) was used as molecular weight marker. Proteins were then transferred to 0.45 μm Protran™ nitrocellulose membranes  and stained with Ponceau S to control protein loading. The membranes were then incubated for 1 h in blocking solution   with 0.05% Tween 20 (TBST) and then incubated overnight with goat L1 antibody C-20 or rabbit LC3 antibody (1:1000) in blocking solution at 4 °C with shaking. After washing five times for 5 min in TBST, the membranes were incubated for 1 h with HRP-conjugated secondary anti-goat or anti-rabbit antibodies . Bands were detected using enhanced chemiluminescent solution  and a CCD camera .For Western blot analysis, samples were run on 4–20% Mini-PROTEANHere, we show that L1-70 interacts with LC3 via the extracellular LIR motif in the fourth fibronectin type III domain and that this interaction is required for L1-mediated neurite outgrowth and neuronal survival. Phosphorylation at the serine residue in L1’s extracellular LIR motif might alter binding affinity to LC3. Since L1 also interacts with the mitochondrial fission protein DRP1 that is involved in mitophagy, we propose that L1, together with LC3, is involved in mitophagy. Altogether, we provide evidence that the extracellular LIR motif in the fourth FNIII domain of L1 contributes to L1’s functions in the nervous system and that impaired L1/LC3 interactions can lead to neurodevelopmental and neurodegenerative diseases.


What are the most important research priorities in concussion from the perspective of patients, caregivers, and clinicians?This survey study used 2 cross-sectional surveys and a consensus workshop with a nominal group technique to gather perspectives from patients, caregivers, and clinicians to identify the top 10 concussion research questions. The main question themes focused on accurate concussion diagnosis, effective symptom management, and prediction of poor outcomes.This research priority list provides a guide to the concussion research community and can help prioritize funding for research that matters most to those living with concussion and those who care for them. This cross-sectional survey study uses priority-setting partnership methods to identify the most important unanswered research questions in concussion from the perspectives of patients, caregivers, and clinicians. Identifying research priorities of patients with concussion, their caregivers, and their clinicians is important to ensure future concussion research reflects the needs of those who will benefit from the research.To prioritize concussion research questions from the perspectives of patients, caregivers, and clinicians.This cross-sectional survey study used the standardized James Lind Alliance priority-setting partnership methods . Data were collected between October 1, 2020, and May 26, 2022, from people with lived concussion experience (patients and caregivers) and clinicians who treat concussion throughout Canada.The first survey collected unanswered questions about concussion that were compiled into summary questions and checked against research evidence to ensure they were unanswered. A second priority-setting survey generated a short list of questions, and 24 participants attended a final priority-setting workshop to decide on the top 10 research questions.Top 10 concussion research questions.The first survey had 249 respondents , including 145 with lived experience and 104 clinicians. A total of 1761 concussion research questions and comments were collected and 1515 (86%) were considered in scope. These were combined into 88 summary questions, of which 5 were considered answered following evidence review, 14 were further combined to form new summary questions, and 10 were removed for being submitted by only 1 or 2 respondents. The 59 unanswered questions were circulated in a second survey, which had 989 respondents , including 654 people who identified as having lived experience and 327 who identified as clinicians (excluding 8 who did not record type of participant). This resulted in 17 questions short-listed for the final workshop. The top 10 concussion research questions were decided by consensus at the workshop. The main research question themes focused on early and accurate concussion diagnosis, effective symptom management, and prediction of poor outcomes.This priority-setting partnership identified the top 10 patient-oriented research questions in concussion. These questions can be used to provide direction to the concussion research community and help prioritize funding for research that matters most to patients living with concussion and those who care for them. To address this, multidisciplinary research networks, including the Canadian Concussion Network, are being established to generate high-quality research.Concussion is a mild traumatic brain injury affecting 2% of the population each year.18 and the Patient-Centered Outcomes Research Institute in the US19 have encouraged researchers to seek out the perspectives of those with lived experience to determine important and unanswered questions. This approach leads to priority setting that is relevant to patients and more readily implemented in clinical practice.19In this context, an important first step is to establish research priorities. In the past, research priorities were typically set by academic researchers, pharmaceutical companies, medical device industries, or funding bodies. More recently, the Strategy for Patient-Oriented Research in Canada20 The JLA process is grounded in principles such as equal involvement of patients, caregivers, and clinicians; transparency; mandatory sharing of an audit trail; and exclusion of researchers and groups with competing interests from the prioritization process.20 The JLA-PSP method has engaged patients in setting research priorities for more than 150 discrete health conditions.21 Our aim was to conduct a research prioritization study using JLA-PSP methods to identify the most important unanswered questions in concussion from the perspectives of patients, caregivers, and clinicians.The James Lind Alliance (JLA) brings together patients, caregivers, and clinicians to form priority-setting partnerships (PSPs) to identify and prioritize important unanswered questions in health care.22 Steps included establishing a steering group for the partnership; identifying research uncertainties through a national cross-sectional survey; summarizing top-ranked questions and determining their inclusion via evidence verification; interim priority setting through a second national cross-sectional survey; and a priority-setting workshop to rank the questions in order of priority. The study followed the Reporting Guideline for Priority Setting of Health Research (REPRISE) and the American Association of Public Opinion Research (AAPOR) Standards Best Practices. The project was conducted from October 1, 2020, to May 26, 2022. Research ethics board approval was obtained from the Children’s Hospital of Eastern Ontario. Potential respondents were informed of the risks and benefits of the study in the survey introduction and informed that by completing the survey they were agreeing to participate in the study.In this cross-sectional survey study, we used standardized JLA-PSP methods, which draw on modified Delphi and nominal group techniques.17 Patients of all ages, any mechanism of injury, any symptom burden, and any duration of postinjury symptoms were included. The scope of questions included those relating to diagnosis, treatment, management, prognosis, and prevention of concussion. We excluded questions related to moderate or severe brain injury, brain injury not caused by physical trauma, and health care resourcing.This pan-Canadian PSP sought individuals diagnosed with or suspected of having a concussion or mild traumatic brain injury, as well as caregivers of people with concussion and clinicians of all disciplines who treat concussion. We used the 5th Consensus Statement on Concussion in Sport to define concussion as “a traumatic brain injury induced by biomechanical forces” that results in 1 or more symptoms in the clinical domains of somatic, cognitive, emotional and/or behavioral, or sleep.23 The PSP lead (M.H.O.) and senior JLA advisor (K.C.) were coleaders of the steering group. The steering group met monthly and was responsible for (1) finalizing the scope; (2) designing and piloting the surveys; (3) overseeing a plan to publicize the online surveys; (4) assisting with classification and verification of summary questions; and (5) identifying the short list of questions for the final workshop.The steering group, established to oversee the operations of the PSP, included 6 patients with concussion, 1 caregiver, 6 clinicians who treat concussion, and 1 concussion stakeholder organization member.24 and stored on a password-protected laptop in a locked room. The survey , anonymous, web-based survey. Study data were encrypted and securely collected on REDCapThe survey was open from February 16 to May 17, 2021, to patients of all ages with concussion, caregivers, and clinicians across Canada. The survey was distributed broadly through websites, emails, newsletters, and social media by strategic partners . Participant characteristics were analyzed using descriptive statistics.Questions from survey 1 were categorized into themes by the steering group through an iterative process. Out-of-scope questions were removed. Patient-clinician dyads formulated summary questions. The steering group reviewed summary questions, and the final wording of the questions was settled by consensus.17 or national concussion evidence-based practice guidelines.16 Unanswered questions were included for the interim priority-setting survey. Questions considered answered by previous research were compiled separately.Evidence checking was completed by a team convened by one of us (L.M.L.) who provided perspectives from specialized fields, including knowledge translation, research, public policy, and clinical practice. These information specialists examined recent research, databases, and current guidelines. For pragmatic reasons, focused searches to identify systematic reviews, primary research, clinical guidelines, and consensus statements were conducted. We searched the MEDLINE, Embase, CINAHL, Epistemonikos, and Cochrane Central Register of Controlled Trials databases from 2010 onward for English language publications. Questions were classified as unanswered if the search found no systematic review, if recent systematic reviews found the question unanswered or yielded contradictory results, if there was a lack of primary research, or if there was insufficient evidence in the 5th International Consensus Statement on Concussion in SportA second anonymous online survey (available in English and French) shared the long list of unanswered summary questions  and requested that participants choose the 10 most important questions from their perspective. The order in which the list of questions was presented was randomized weekly to minimize bias. The survey was pilot tested on 5 people for comprehension, clarity, and time for completion. Demographic data were also collected in survey 2, which was open from January 10 to April 10, 2022; promotion, distribution, and data storage paralleled that of survey 1. A scoring system was used to analyze the data. One point was assigned for each question in a respondent’s top 10 list. Points were tallied separately for 2 distinct groups: (1) those with lived experience  and (2) clinicians who treat concussion. To ensure equal influence for each group, questions were ranked from highest to lowest for each group and the 2 scores were summed to give a total score which determined the combined rank order for each question. The steering group reviewed the final rankings and reached consensus on the short list of questions for the priority-setting workshop.26 Three additional JLA advisors facilitated breakout sessions to ensure balanced participation and respectful discussion across participants. To enable a wide exchange of knowledge and consider all perspectives, participants rotated through different combinations of breakout groups. Consensus on the top 10 questions was reached using established JLA methods.22 Degree of participant engagement was assessed through a postworkshop survey.A workshop consisting of 2 half-day sessions  and conducted through video teleconference (Zoom Video Communications) was used to rank the final list of unanswered research questions by order of importance and generate a final top 10 list. Twenty-four participants were invited to attend . The workshop sought equal representation from those with lived experience and clinicians and sought diversity in age, sex, geography, concussion mechanism, and clinician expertise. Two weeks prior to the workshop, participants were sent the short list of 17 final questions via email and post. The workshop was led by a senior JLA advisor (K.C.) and used a modified nominal group technique.Two hundred forty-nine participants completed survey 1 . Of thesA total of 1761 concussion questions and comments were collected from the 249 participants . Of thesOf the 88 summary questions, 5 (6%) were determined to be sufficiently answered by existing research evidence . This leBox. What is the effect of a delay in concussion diagnosis on symptom severity and outcomes?What relationship exists between neck injuries  and concussions?Are impact sensors  effective tools to help diagnose and assess concussions?What criteria are required for a health care provider to request a CT scan for a child with a head injury and possible concussion?What type of family support, education, and resources result in the best outcomes for families coping with concussion?Abbreviation: CT, computed tomography.A total of 989 participants completed survey 2 . Of thesTwenty-four participants (13 with lived experience and 11 clinicians) attended the workshop. Of the 13 with lived experience, 8 (62%) identified as female and 5 (38%) as male. One participant was younger than 18 years, and the mechanism of concussion injury included sports (6 [46%]), non–sport-related falls (4 [31%]), motor vehicle–related injuries (2 [15%]), and assault (1 [8%]). Clinicians included 2 physiotherapists, 2 neuropsychologists, 1 nurse, 1 athletic therapist, and 5 physicians . Consensus was reached on the final ranking of the top 10 unanswered research questions in concussion .A postworkshop anonymous survey was completed by all 24 attendees. Overall, all attendees (100%) strongly agreed or agreed that they were able to share thoughts and opinions effectively, 23 (96%) strongly agreed or agreed that the facilitators were fair and impartial, and 22 (92%) strongly agreed or agreed that the process of determining the top 10 questions was fair and robust.This JLA PSP gathered opinions from more than 1000 patients, caregivers, and clinicians and, through a multistep process, identified the top 10 unanswered research questions that matter most to those affected by concussion. The project brought together a broad and diverse group of patients, caregivers, and clinicians from across Canada and, to our knowledge, is the first patient-oriented research priority-setting project in concussion. The top 10 questions identify priority areas on which researchers and research networks can focus to improve outcomes for people living with concussion.Taken as a whole, the list of research priorities is broad in scope and reflects the diverse concerns of patients, caregivers, and clinicians. Priorities include early and accurate diagnosis after a head injury, specific symptom management, approaches to ensure a safe return to physical and cognitive activities after concussion, predicting and preventing prolonged symptoms and poor outcomes, and understanding the pathophysiology of concussion.6 early and accurate identification of risk factors for prolonged symptoms was deemed the highest priority. Risk factors associated with prolonged recovery have been identified, and a clinical risk score has been validated for pediatric patients14 and is now in development for adult patients.27 However, despite considerable research, there are no validated physiological, serological, or radiological biomarkers that can accurately diagnose concussion and predict recovery.31 Therefore, more research is needed to implement existing validated tools into clinical pathways and to develop additional biomarkers and strategies to refine diagnosis and personalize therapies to improve outcomes.The top-ranked question focuses on identifying patients at high risk for prolonged symptoms at the time of diagnosis and developing a personalized management strategy. With up to 30% of people with concussion experiencing symptoms longer than 1 month,Three questions focused on improving early diagnosis and management of concussion. The first concerns the role of interdisciplinary teams of health care professionals  in assessing and managing concussion and asks which patients should be referred early to these teams to achieve the best outcomes. A second question focused on the need to develop approaches to differentiate symptoms due to concussion from those of nonconcussion medical problems that may have similar symptoms but require different therapies. The third question concerns the variation in abilities of clinicians to recognize and manage concussion. With concussion diagnosis and management recommendations changing frequently, educating clinicians on concussion was considered important to improve recognition and standardize management.32 Some patients have difficulty increasing mental and physical activity without exacerbating postconcussion symptoms. Effective strategies to reintroduce cognitive and physical activities in those with refractory symptoms, to reduce time away from school, work, and/or physical activity, and to improve recovery time are needed. Two additional management-related questions focused on the effectiveness of interventions to manage concussion-related chronic headache and sleep disturbances.Two highly ranked questions focused on identifying the best approach for a safe and effective return to activities. Recent evidence has shown that after a short period of initial rest (24-48 hours), mild to moderate physical and cognitive activity is beneficial for recovery.Five questions were deemed to be sufficiently answered by literature review. These answered questions highlight gaps in knowledge translation and should be a priority for dissemination to stakeholders and the public to improve awareness and enhance implementation of existing research evidence.22 Results were analyzed transparently and are shared openly on the project website. Other strengths include a mix of ages of survey participants across Canada, including speakers of both official languages and those in urban and rural areas. The steering group, survey respondents, and workshop participants included a diverse group of clinicians who treat people with concussion, allowing for different perspectives across professions.Strengths of this study include the methodological rigor and transparent nature of the JLA-PSP methods. The JLA PSP is a widely used and highly cited approach for facilitating collaboration between patients and clinicians to prioritize research questions.33 Second, we may have had difficulty reaching groups who do not have internet access. However, both a paper version of the survey and telephone completion were offered. Third, greater than 50% of survey participants had concussion symptoms lasting longer than 1 year, and this may have resulted in research questions reflecting more chronic concerns. Despite this limitation, many questions in the top 10 focused on acute concussion management. Fourth, as the surveys were completely anonymous, we were not able to determine whether people completed them more than once. Fifth, although many questions on concussion prevention came from survey 1, they were not highly ranked by participants in survey 2 and therefore not selected as priorities to include in the final workshop. We believe that this omission does not reflect a lack of importance of research on concussion prevention. Rather, we believe it reflects the overwhelming importance of diagnosis, treatment, and prognosis as the major concerns of individuals living with concussion and clinicians managing concussion. Finally, this is a Canadian study, and the research priorities may not reflect people from countries with different health care expertise, access, and resourcing.This study has some limitations. First, it was challenging to recruit people from some racial, ethnic, and cultural backgrounds, particularly those who identify as Black, Latino, and Middle Eastern. Our study was able to recruit 4% of individuals with concussion who self-identified as Indigenous (5% of Canadians identify as being Indigenous).This PSP of patients, caregivers, and clinicians worked together in an equal partnership to identify the top 10 patient-oriented research questions in concussion. These priority questions help inform the concussion research agenda and may be useful to stakeholders, researchers, clinicians, funders, hospital leaders, and policy makers. The goal is that these questions will stimulate funding for concussion research that will answer questions that matter most to those living with concussion and those who care for them.


However, it is problematic for other ceramic materials , especially porous ones. In this case, it is mainly due to poor adhesion to the substrate, resulting in strong degradation of the graphene. For these reasons, the research topic of this study was undertaken. This article presents research on the development of the methodology of graphene transfer onto ceramic Al2O3 surfaces. Polycrystalline graphene chemical vapour deposition (CVD) monolayer and quasimonocrystalline high-strength metallurgical graphene (HSMG®) synthesised on liquid copper were used. When developing the transfer methodology, the focus was on solving the problem of graphene adhesion to the surface of this type of ceramic, and thus reducing the degree of graphene deterioration at the stage of producing a ceramic–graphene composite, which stands in the way of its practical use. Plasma and chemical ceramic surface modification were applied to change its hydrophobicity, and thus to improve the adhesion between the graphene and ceramic. The modification included the use of dielectric barrier discharge (DBD) plasma, oxygen plasma , and hydrofluoric acid treatment. Changes in surface properties caused by the modifications were determined by measuring the contact angle and  measuring the degree of surface development. The effectiveness of the applied surface preparation methodology was evaluated based on the damage degree of CVD and HSMG® graphene layer transferred onto modified Al2O3 using optical microscopy and Raman spectroscopy. The best average ID/IG ratio for the transferred HSMG® graphene was obtained after oxygen plasma modification (0.63 ± 0.18) and for CVD, graphene DBD plasma was the most appropriate method (0.17 ± 0.09). The total area of graphene defects after transfer to Al2O3 was the smallest for HSMG® graphene after modification with O2 plasma (0.251 mm2/cm2), and for CVD graphene after surface modification with DBD plasma (0.083 mm2/cm2).Graphene transfer onto ceramics, like Si/SiO Graphene, thanks to its unique mechanical, thermal, and electrical properties, is widely known as an innovative material of the future ,2,3,4,5.®) [The methods of production of large-area graphene include synthesis on solid metal by chemical vapour deposition (CVD) , synthes®) ,12,13. AMany literature reports concern the use of graphene in filtration, but these are solutions based mainly on polymer membranes ,21,22,232) is used as the target ceramic substrate. Silicon itself or its oxide are widely used substrate materials for studying the effectiveness of improved transfer methods. The lack of use of other ceramic materials for this purpose is explained by their surface morphology and physicochemical properties, by which graphene shows poor adhesion to ceramics and is damaged during the removal of the supporting polymer [Most of the graphene transfer methods presented in the literature refer to procedures where quartz  groups on the surface. Commercial membranes are modified by hydrophobization for use in separation processes. This effect would also be possible by covering them with a layer of graphene ,36,37. O2 plasma  were pro2 plasma .® methods was used for the study. The surface of Al2O3 ceramics was modified by means of barrier plasma, oxygen plasma, and etching with hydrofluoric acid. The surface changes were assessed based on FTIR (Fourier Transform Infrared Spectroscopy) tests and the wetting angle measurement. The quality of the transferred graphene layers was evaluated based on optical microscopy and Raman spectroscopy. On this basis, the relationship between the state of the ceramic surface and the obtained quality of graphene transferred to its surface was demonstrated. We also determined which of the surface modification methods is optimal for obtaining the best quality transferred graphene layer.Bearing in mind, on the one hand, the potential benefits of using graphene–ceramic composite membranes, and on the other hand, the problems with transferring the graphene layer to ceramics, the purpose of this work were focused on this basis. The work aimed to determine the effect of chemical and physical modifications of the surface of the ceramic porous membrane on the quality of the transferred graphene. Large-area graphene produced by the CVD and HSMG2O3 discs with a diameter of 47 mm and pore size <0.1 µm  were used for surface modification and graphene transfer. CVD graphene synthesized on solid copper and HSMG® graphene synthesized on liquid copper were used for transfer onto ceramics.Porous oxide ceramic Al® synthesis was conducted according to the patent [1.2.Preparation of nickel foils with a thickness of 0.1 mm and dimensions of 220 × 120 mm2.4·5H2O), at a current density of 0.02 A/cm2 for 6 h.Galvanic coating of the Ni foil with a layer of copper with a thickness of 100 µm in a solution of copper sulphate pentahydrate (CuSO3.a.Heating in vacuum (10 Pa pressure) up to 1060 °C.b.Carburizing in an atmosphere of acetylene, ethylene, and hydrogen (2:2:1), at a flow rate of 4.0 L/min acetylene and ethylene, and 2.0 L/min hydrogen, consisting of four alternating stages of carbonation and annealing, 5 s of gas dosing/15 min diffusion.c.Heating in an atmosphere of argon and hydrogen to a temperature above the copper melting point (1100 °C) under a pressure of 2 kPa followed by a 5 min soak.d.Cooling the batch to ambient temperature under the same conditions.Graphene synthesis on a Cu-Ni bimetallic substrate in a vacuum generator SuperCarb, Seco/Warwick SA, Świebodzin, Poland, according to the procedure:HSMGe patent . The pro2 in size. The foils were previously washed in an ultrasonic cleaner in isopropanol and then in acetone.The CVD graphene synthesis process on copper solid-state foils was conducted by creating optimal conditions for carburizing in the SuperCarb vacuum generator. An acetylene, ethylene, and hydrogen (2:2:1) gas mixture was used with gas flow rates of 4.0 L/min for acetylene and ethylene and 2.0 L/min for hydrogen. Graphene was synthesized on copper foils 0.1 mm thick and 50 × 50 mm1.Heating in vacuum (10 Pa) to a temperature of 1000 °C,2.Carburizing in an acetylene–ethylene–hydrogen atmosphere for 1 min.3.Heating in a vacuum at 1000 °C for 5 min.4.Carburizing in an acetylene–ethylene–hydrogen atmosphere for 0.5 min.5.Cooling in vacuum to ambient temperature.The process of producing CVD graphene was as follows:2 of graphene. The polymer was dried at a temperature of c.a. 40 °C for 30 min. The double support layer consisted of an additional top layer of poly(dimethylsiloxane), produced by applying about 20 mg of liquid PDMS per cm2 of graphene. The polymer was prepared from a silicone elastomer base and hardener SYLGARD® 184 in a ratio of 10:1. The drying time of the PDMS layer at 60 °C was 60 min. After that, the growth substrate with graphene and polymer was placed in the grip in a 0.5 M NaOH solution so that only its edge was immersed in the electrolyte. The process of graphene delamination was started by applying a constant voltage in the range of 2.5–5 V, causing hydrogen generation between the graphene and growth substrate (cathode). The progress of graphene separation was carried out at a constant speed of 0.01–0.05 mm/s set in the program so that the graphene separation line was at the level of the liquid level. The graphene was then washed twice in distilled water and air dried. Graphene quality was tested by measuring its resistance using the UNI-T UT70B multimeter, UNI-TREND Technology, China, with needle probes.Graphene was separated from the growth substrate using the electrochemical delamination method, which was preceded by drop/blade coating of graphene by a one or two-layer carrier polymer. A 0.05 M solution of polymethyl methacrylate  dissolved in 1 mL of chlorobenzene Chempur Piekary Śląskie, Poland) was used in the amount of 40 μL of solution per 1 cm2O3) was quartz (SiO2). Transfer on quartz is less problematic thus methods presented in the literature were adapted for the needs of graphene transfer to a porous aluminium oxide substrate. However, porous Al2O3 is such a different material that the adoption of the 1:1 transfer methodology resulted in poor quality graphene, the main reason being insufficient adhesion of graphene to porous ceramics. The standard wet transfer procedure with the elimination of the polymer in boiling acetone vapour, which works well in the case of quartz, did not work in this case. Attempts were made to increase the adhesion of graphene to ceramics by applying 2 mL of a 75% acetone/water solution on the sample before placing in it the bath [The reference point in the development of the graphene transfer methodology to alumina ceramics . The system consists of two metal discharge electrodes covered with a dielectric, the so-called dielectric barriers, separated from each other by a 2-millimetre discharge gap. After the system is powered by a high-voltage generator with a frequency of approx. 80 kHz, plasma is generated in the space between the electrodes.In order to improve the adhesion of graphene to ceramics, an attempt was made to change the properties of its surface by creating a barrier discharge powered with a voltage of 15 kV and a frequency of 80 kHz. The time of exposure of the samples to the plasma thus formed was 1 min.2O3 surface with oxygen plasma. For this purpose, the RF PACVD  method was used. The main components of the RF PACVD plasma surface modification equipment are: a working chamber with an HF (high-frequency) electrode, a diffusion pump, a gas dosing system, a pressure measurement system, a radio frequency (13.56 MHz) generator, and a pyrometer for temperature control. The process parameters are shown in In order to increase the adhesion between the transferred graphene and the target ceramic substrate, an attempt was also made to modify the AlChemical etching of the ceramics with an aqueous solution of hydrofluoric acid  was also used to increase the adhesion of graphene to the surface. Solutions of two concentrations were used: 5 and 9.5% HF; pickling time 15 min.2O3 samples were performed.The assessment of the change in wettability of the ceramic surface with distilled water after the modifications was made using a Krüss DSA10 goniometer, Hamburg, Germany. The contact angles were determined based on photos of liquid drops with a volume of 0.8 μL placed on the surface of the samples, taken with a camera set in the axis perpendicular to the surface of the sample. Investigations on non-porous AlTo measure the change in ceramic surface roughness after etching with hydrofluoric acid (HF), a contact profilometer HOMMEL TESTER T1000was used, supported by the Turbo Datawin NT 1.34 software, both from JENOPTIK Industrial metrology, Villingen-Schwenningen, Germany, enabling the determination of surface roughness and waviness parameters based on the obtained etching profiles. Based on the Ra measurement results, the effect of HF acid concentration on the change in the surface topography of the tested samples was determined.−1. Spectra were registered with a resolution of 4 cm−1 with the use of a DTGS detector. The measurements were made in a reflection mode, with the use of Sequelle DRIFT mode, working at a reflection angle equal to 20 degrees. In the case of each material, data from 128 scans were collected in order to construct a single spectrum.The chemical structure was determined by using Fourier-transform infrared spectroscopy (FTIR) using a Thermo Scientific iS50 Spectrometer, Waltham, MA, USA, with a spectral range from 4000 to 400 cmIn order to detect large defects on the surface, the transferred graphene layers were observed at low magnification using a Nikon AZ, Tokyo, Japan stereomicroscope with NIS-Elements software for automatic image registration and processing.2 was analysed each time. In this way, the share of defects in the total area of the analysed sample was calculated.A Nikon Eclipse MA200, Tokyo, Japan light microscope, equipped with NIS-Elements image analysis software, was used to accurately assess the defects in the surface of the transferred graphene in micro-areas. This type of microscope was used because, due to the strong representation of the topography of the ceramic surface, the analysis of the continuity of the graphene layer using a scanning electron microscope, both in the secondary electron (SE) and AEE mode , turned out to be ambiguous. ImageJ software, LOCI, Madison, WI, USA, was used to perform statistical evaluation of graphene defects after transfer onto a porous ceramic substrate. An area of 1 cm−1. Two-dimensional Raman maps were collected from 50 × 50 μm areas with 5 μm spatial resolution. All the collected Raman spectra and maps were preprocessed using WiRE 5.5 software .The Raman spectra and maps were carried out with an InVia Raman microscope  system. The graphene surfaces were exanimated using a 532 nm laser with a 50× objective lens . The laser power at the sample was restricted to 4 mW. For Raman maps, map image acquisition mapping was applied in the wavenumber range of 900 to 3200 cm2O3 ceramics before and after chemical modification was carried out. The analysis of the data presented in 2O3 with a 5% HF acid solution resulted in the levelling of its surface unevenness, manifested by a decrease in the value of the Ra parameter by 0.15 µm compared to its initial state. On the other hand, the almost 2-fold increase in the acid concentration caused a drastic increase in the surface roughness of the Al2O3 ceramics by more than 1µm compared to its state before the modification.To increase the contact area between the surface layer of ceramics and graphene as a result of etching the ceramic surface with 5% and 9.5% hydrofluoric acid (HF) solutions, an analysis of the Ra value of the surface of Al2O3) has weak hydrophilic properties, consistent with the literature data [Investigations of the ceramics’ wettability with the use of distilled water showed that corundum oxide  and the hydroxyl donor ion, which is the so-called Lewis principle. On the other hand, hydroxyl groups behave as Bronsted acid sites. However, high-energy plasma surface cleaning can cause two adjacent OH ions on the alumina surface to form an oxygen bridge, which, however, is the Lewis acid active site, as shown in the diagram below . Such a −1 disappeared almost completely.Moreover, surface modification in plasma also cleans/etches the surface of organic impurities. The band in the range of 3000–2800 cm−1, there was also a slight increase in the intensity of the peaks at the wave numbers 600 and 657 cm−1, which originate from the Al-O bonds characteristic of the alumina structure, which means that the surfaces may have undergone additional oxidation  which is presented in In the range of 1030–400 cm2O3 ceramics. The picture  ceramic substrates using the wet transfer method supported by a PDMS/PMMA layer. Then, a qualitative assessment of graphene on an aluminium oxide substrate was carried out. Images obtained with a stereomicroscope, at 40× magnification, showed the surface of both types of graphene after etching the ceramics with DBD barrier discharge plasma  was carried out under a light microscope. The analysis of the images showed that cleaning the ceramic substrate with oxygen plasma , G (at about 1580 cm−1), and 2D (about 2690 cm−1) peaks. The presented maps show the change in the ratio of D to G peaks on the studied surfaces, which, according to the literature [2O3 ceramics. On the studied surfaces, the lowest, highest, and intermediate degree of defect sites with the lowest (marked “1”), highest (marked “2”), and intermediate (marked “3”) ID/IG ratio values are indicated. The least damaged layer of HSMG® graphene was obtained after transfer to ceramics modified by oxygen plasma (RF PACVD). The most defective HSMG® graphene was on ceramics etched with 5% HF. The same was true for CVD graphene. The least damaged graphene was found on ceramics modified with oxygen plasma and a similar result was obtained for the barrier plasma. The degree of damage to the graphene layer on the substrate etched with 5% HF was the highest.graphene ,33,48,49terature ,49,50,51D/IG ratio was also determined for all points from the tested surfaces. When studying the transferred HSMG® graphene, the average ID/IG ratio was shown to be the lowest for the initial modification of substrates with RF PACVD oxygen plasma (0.63 ± 0.18). When DBD plasma was used, it increased to a value of 0.72 ± 0.17, and in the case of ceramic surface treatment with acids, it was already 0.77 ± 0.28. On the other hand, in the study of CVD graphene, the lowest ID/IG values were obtained for modification with DBD plasma (0.17 ± 0.09), while for samples treated with RF PACVD oxygen plasma or treated with acids, the values were higher and were 0.21 ± 0.08 and 0.21 ± 0.19, respectively.On the basis of the analysis carried out for each sample, the average value of the I2O3 ceramics, it is possible to transfer monolayer HSMG® and CVD graphene to this substrate. The surface modification strongly affects the change of the free energy of the ceramic surface. This surface, especially after plasma modification, became strongly hydrophilic. This had a direct impact on the possibility of transferring graphene sheets. The quality of the transferred graphene, in turn, is affected by the modification method used. The conducted tests showed that the best quality HSMG® graphene layers were the ones transferred to a substrate modified with low-temperature oxygen plasma (RF PACVD). Additionally, for CVD graphene, the best option to modify the surface of ceramics was barrier plasma (DBD). As indicated by the FTIR research, this results from the chemical cleaning of the surface, consisting of the removal of organic impurities, and most of the hydroxyl groups are related to the chemical activation of this surface. FTIR studies of ceramics modified with barrier plasma did not confirm such surface changes as was the case with oxygen plasma. This may be related to the shorter duration of the modification effect in the case of DBD, which did not allow the study of this effect on FTIR. Qualitatively, both HSMG® and CVD graphene were characterized by a low degree of defects after modification with oxygen or barrier plasma. Large damage to the graphene layers, both on a macroscopic and microscopic scale, was observed for samples subjected to chemical modification. This is probably due to the different nature of these changes, which in this case are related to the development of the ceramic surface. A slight effect of the graphene production method on its quality after transfer to ceramics was also found. Research indicates that graphene produced by CVD in this case was characterized by a slightly lower defect degree after transfer to Al2O3. It should be emphasised that the degree of defects in graphene layers is not only related to transfer. Damage to the graphene layers arises first during its synthesis, and again during the transfer. Based on both microscopic and Raman spectroscopy studies (the probed D band), the source of origin cannot be unequivocally indicated.The presented work proves that by modifying the surface properties of Al2O3 ceramics. The preparation of such novel composites could find practical applications as stable membrane materials for the filtration of liquids and gases at extreme temperatures and pH or under high pressure in many industries, such as the biotechnology, pharmaceutical, food, chemical, and petrochemical industries. However, for their practical use in filtration, further research is needed to assess their effectiveness. This work focused primarily on the development of an effective method of transferring large-area graphene to a porous ceramic substrate with Al2O3 and assessing its quality after transfer on a macro- and microscopic scale. Further research should include the assessment of graphene defects on the ceramic substrate at the nanoscale and the determination of the filtration characteristics of these membranes.In conclusion, the obtained research results allow for obtaining composite membranes based on graphene and Al


Given the potential large ethical and societal implications of human germline gene editing (HGGE) the urgent need for public and stakeholder engagement (PSE) has been repeatedly expressed. In this short communication, we aim to provide directions for broad and inclusive PSE by emphasizing the importance of futures literacy, which is a skill to imagine diverse and multiple futures and to use these as lenses to look at the present anew. By first addressing “what if” questions in PSE, different futures come into focus and limitations that arise when starting with the “whether” or “how” questions about HGGE can be avoided. Futures literacy can also aid in the goal of societal alignment, as “what if” questions can be answered in many different ways, thereby opening up the conversation to explore a multitude of values and needs of various publics. Broad and inclusive PSE on HGGE starts with asking the right questions. Decisions about whether or not to pursue human germline gene editing (HGGE; Table PSE has the potential to enable policy making that is informed by the values and needs of society at large (Scheufele et al. Inclusivity is described as one of the ideals for effective PSE in developing HGGE policies (Iltis et al. Bucchi and Trench  defined ‘Globally’, as Françoise Baylis writes, ‘the political consensus on heritable human genome editing—such as it is—inclines toward an outright ban, and if not a ban, at least a moratorium’ Baylis . AlthougThe “what if” questions require time for the public to reflect, slow down, use imagination and think beyond their current lived experiences. They allow the public to think about the ways in which HGGE could improve their lives and the lives of their children, as well as which futures would be undesirable and inconsistent with their values. This speculative “if yes, then how” question should not be confused with the question of “how” HGGE should be implemented. Therefore, it is important to be transparent about this distinction at the outset of PSE. The questions “whether” and “how” provide valuable insight in how practice and policy around HGGE should be organized, which will become relevant once the scenarios of what our society may become are deliberated on. Until then, there is a widely shared responsibility to enable this deliberation and opinion formation (Houtman et al. Broad and inclusive social conversation around HGGE starts with asking the right questions. By focusing on questions of “what if” before “whether” and “how” in PSE we can open up the conversation by creating various future narratives. Consequently, we reduce directivity and framing as initiators of PSE, and increase inclusiveness as this approach does not exclude in advance those who hold fundamental objections against HGGE or those who have yet to make up their mind for various reasons. Actual participation in PSE by the aforementioned groups is something that should be evaluated and monitored continuously. Asking “what if” will surface the values and needs that are at stake, which can then be taken into consideration when the questions of “whether” and possibly “how” are deliberated on.


BackgroundEpistaxis is a very common symptom. The occurrence of epistaxis may be affected by dry environments, but there are some differences among previous reports and this view is controversial.ObjectiveWe investigated the relationship between the number of epistaxes and daily average relative humidity.MethodsData on patients with epistaxis between March 2011 and February 2021 were collected from two hospitals. The daily average relative humidity was examined, and the change in the number of patients with epistaxis due to humidity was investigated using a generalized linear mixed model.ResultsA total of 4184 cases of epistaxis were identified. The number of epistaxis cases per day was significantly associated with the daily average relative humidity (p < 0.001). One percent increment in average relative humidity decreases the number of epistaxis cases per day by 1.1%.ConclusionA negative correlation was found to exist between daily average relative humidity and occurrences of epistaxis. Epistaxis is one of the most common complaints in otolaryngology. Although the detailed pathological causes of epistaxis are unknown, it is commonly believed that epistaxis occurs during the colder, drier months . SeveralPatientsThe subjects of this study were all patients at Tsukuba University Hospital Mito Medical Center, Mito Kyodo General Hospital, Mito, Japan  and Tsukuba Gakuen Hospital, Tsukuba, Japan , two important acute care hospitals in Ibaraki Prefecture, Japan. The patients were diagnosed with epistaxis from March 2011 to February 2021. The data items collected from each hospital included disease name, age, sex, and date of diagnosis. For statistical analysis, the date of diagnosis was considered as the date of onset of epistaxis.The study was carried out in accordance with the tenet of the Declaration of Helsinki and the work was compliant with the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement. The institutional review boards of the respective participating institutions approved this study . By opting out, the institutional review board exempted this retrospective study from the requirement of obtaining informed consent from the individual participants.Meteorological datahttps://www.jma.go.jp/jma/index.html). The average relative humidity data was adopted for each hospital area. The number of days with the same humidity (%) over a 10-year period was counted. In addition, the number of patients with epistaxis who visited on days with the same humidity (%) was also counted.The average relative humidity values analyzed in this study were obtained from the Japan Meteorological Agency website  for categorical variables, and as median (range) for continuous variables. A generalized linear mixed model with Poisson distribution and log link function was used to explore the association between the incidence of epistaxis and average relative humidity, after adjusting for the number of days with the same humidity. In the model, the response variable was the number of patients per day and the fixed effect was the average relative humidity. The hospital was used as a random effect. All statistical analyses were performed with SAS version 9.4 .Patient characteristicsOverall, 4184 cases of epistaxis at the participating centers were enrolled in this study. The median age of the patients was 63 years (range: 0 to 97 years). Of the patients, 2520 (60.2%) were male and 1664 (39.8%) were female. The sex ratio and median age were comparable between the two facilities . One percent increment in average relative humidity decreased the number of epistaxis cases per day by 1.1% Figure .In this study, we observed the negative correlation between daily average relative humidity and the number of patients with epistaxis by examining a large amount of data from two hospitals over 10 years. One percent increment in average relative humidity decreased the number of epistaxis cases per day by 1.1%. That is, a 10% increase in average relative humidity resulted in a 10.5% decrease in the number of patients with epistaxis, and a 20% increase resulted in a 19.8% decrease.Several studies have investigated the relationship between humidity and epistaxis -7. The mEpistaxis has a bimodal age distribution, being more common in children under the age of 10 and adults in their 70s, and occurs more frequently in males . One of It is difficult to identify a pathophysiological explanation as to why a decrease in humidity increases the risk of epistaxis. The nasal gland system, stimulated by dry air, protects the lower airways by humidifying the air by producing a variety of secretions . The venIn this study, due to the large sample size, we could not review individual charts; therefore, cases of varying severity were included. Information on treatment was not available in the database used for the study. The date of diagnosis of epistaxis was investigated; however, the date of occurrence and the date of diagnosis may not necessarily be the same. Moreover, although our study included the most important acute care hospitals in the region, the extent to which the results of this study can be generalized to other countries with different environments is limited. Despite these limitations, this study provides strong support for the hypothesis that dryness is a risk factor for epistaxis. Our result may be useful for patients who are prone to epistaxis and additional research is needed.In this study of a large population in a specific area belonging to the humid subtropical climate, there was a strong negative correlation between daily average relative humidity and the number of patients diagnosed with epistaxis in selected hospitals. This result may be related to increased intranasal resistance and decreased mucociliary movement due to dry air. This result supports the hypothesis that dryness is a risk for epistaxis.


Emergency departments are key settings for suicide prevention. Most people are deemed to be at no or low risk in final contacts before death.To micro-analyse how clinicians ask about suicidal ideation and/or self-harm in emergency department psychosocial assessments and how patients respond.Forty-six psychosocial assessments between mental health clinicians and people with suicidal ideation and/or self-harm were video-recorded. Verbal and non-verbal features of 55 question–answer sequences about self-harm thoughts and/or actions were micro-analysed using conversation analysis. Fisher's exact test was used to test the hypothesis that question type was associated with patient disclosure.N = 46/55) were closed yes/no questions about self-harm thoughts and/or feelings, plans to self-harm, potential for future self-harm, predicting risk of future self-harm and being okay or keeping safe. Patients disclosed minimal information in response to closed questions, whereas open questions elicited ambivalent and information rich responses. (b) All closed questions were leading, with 54% inviting no and 46% inviting yes. When patients were asked no-inviting questions, the disclosure rate was 8%, compared to 65% when asked yes-inviting questions (P < 0.05 Fisher's exact test). (c) Patients struggled to respond when asked to predict future self-harm or guarantee safety. (d) Half of closed questions had a narrow timeframe  or were tied to possible discharge.(a) Eighty-four per cent of initial questions (Across assessments, there is a bias towards not uncovering thoughts and plans of self-harm through the cumulative effect of leading questions that invite a no response, their narrow timeframe and tying questions to possible discharge. Open questions, yes-inviting questions and asking how people feel about the future facilitate disclosure. Meta-analysis indicates that only 28.7% of people disclose suicidal intent to healthcare professionals before suicide.2 Many patients are reported to ‘deny suicidal ideation’ in the days or weeks before a suicide attempt.4 Importantly, UK professionals judge immediate risk of suicide to be low or not present in 85% of assessments in the final appointment before death.5 Self-harm, self-injury or self-poisoning irrespective of intent6 is the strongest risk factor for suicide.7 Most studies have focused on professional reports or patients’ medical records rather than direct research on communication about self-harm.Suicide prevention is a global public health priority, with over 700 000 deaths by suicide annually.8 Self-harm remains highly stigmatised, and patients report shame, guilt, fear about loss of autonomy9 and onward consequences such as admission or removal of children from their care.10Communicating about self-harm is challenging. Sociological studies have shown a strong preference for ‘saving face’ and not disclosing face-threatening and stigmatised information in social interactions.11 e.g. ‘Are you feeling suicidal?’ is framed positively, inviting agreement to ‘feeling suicidal’.12 Conversely, ‘Not feeling suicidal?’ is negatively framed, inviting agreement to ‘not feeling suicidal’. Word choice further reinforces the direction of the question. Words such as ‘any’, ‘ever’, ‘at all’ are negative polarity items  that invite a ‘no self-harm’ response and affect patients’ likelihood of responding ‘no’.13Recent studies have highlighted that clinicians’ questions about self-harm can have a considerable impact on disclosure of thoughts and plans. Yes/no questions are prevalent in medical interaction. Although medical communication training encourages professionals to ask non-leading questions, there is no such thing as a non-leading yes/no question. Yes/no questions communicate an expectation in favour of either ‘yes’ or ‘no’ responses through wording and grammatical structure,14 In secondary out-patient mental healthcare, more than 75% of questions invited patients to confirm the absence of self-harm thoughts and/or actions.15 Patients were significantly more likely to say no in response to no-inviting questions (compared with yes-inviting questions), even though some disclosed suicidal thoughts in self-report measures.In primary care, most questions invite ‘no self-harm’ answers, with patients showing difficulty reporting self-harm thoughts and/or actions when clinicians ask these leading questions .6 around 220 000 episodes of self-harm by 150 000 people are managed by emergency departments in England annually.16 Most emergency departments have a psychiatric liaison team staffed by specialist mental health clinicians. They conduct psychosocial assessments after self-harm and make onward referrals.6 Emergency departments are high-pressure environments with 4 h targets for patients to be seen, treated and admitted or discharged. Although the number of people seeking help for mental health problems has risen year on year, numbers of hospital beds have fallen,17 increasing the pressure to discharge patients. To date, no studies have observed psychosocial assessments for self-harm in emergency departments. This study addressed the following questions.How do clinicians ask patients about suicidal ideation and/or self-harm in emergency department psychosocial assessments?How do patients respond verbally and non-verbally?A key setting for suicide prevention is the emergency department:This was a cross-sectional non-participant observational study of clinical practice, involving video-recording psychosocial assessments between people presenting to the emergency department for suicidal ideation and/or self-harm and psychiatric liaison clinicians.6, providing the basis for a decision on whether to discharge and a management plan for further support.The study took place in an emergency department in England. As per usual practice, people seeking care for suicidal ideation and/or self-harm met with emergency department triage staff to assess clinical urgency and undergo any physical interventions before referral to the emergency department liaison psychiatry service for a psychosocial assessment, including risk of harm to self18All procedures contributing to this work comply with the ethical standards of the relevant national and institutional committees on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008. All procedures were approved by London Central Research Ethics Committee (17/LO/1234). Study design, materials and recruitment processes were developed in collaboration with a lived experience group including one carer, one mental health nurse and six people who had presented to the emergency department for suicidal ideation and/or self-harm. Written informed consent was obtained from all participants.N = 11), a student nurse (N = 1), doctors (N = 2), junior doctors (N = 7), social workers (N = 2), occupational therapists (N = 2), healthcare assistants (N = 2), consultant psychiatrists (N = 2), psychiatry trainees (N = 2) and unspecified (N = 2). Their mean age was 38.3 years , and they were mostly female (N = 20/33) and White British (N = 28/33).All 43 psychiatric liaison clinicians in the psychiatric liaison team who conducted psychosocial assessments were invited to participate, and 33 consented (76.7%). Clinician participants comprised mental health nurses , self-harm by overdose (N = 23/46) or self-harm by ligature or attempted drowning (N = 3/46). Patient mean age was 35.5 years . They were mostly female (N = 31/45) and White British (N = 43/45). Caregivers, typically parents, were involved in eight assessments.Recruitment took place from September 2018 to April 2019. A total of 260 referrals were screened, 82 individuals were approached regarding participation and 48 consented. Three patients were excluded after consenting, as they did not present with suicidal ideation or self-harm. One patient re-presented, resulting in a total of 46 assessments. Referrals were for suicidal ideation  as the focus was on initial disclosure of thoughts, actions or plans. Questions about acting on thoughts or repeating self-harm were typically asked at the end of the assessment, alongside questions about next steps and discharge.19 to micro-analyse clinicians’ questions and patients’ responses. Standardised methods were used to transcribe and analyse the content of speech and characteristics such as pauses, overlap, stress, intonation, pace and non-verbal communication.20 This paper contains simplified transcripts with names and locations changed. Numbers in brackets denote silence in seconds. We coded the following.(i) open or closed20(ii) inviting a yes or no responseClinicians’ questions as:no self-harm responseself-harm response21‘non-answer’ that did not provide an answer to the question, e.g. ‘I don't know’23resisting the question or an assumption in the question, e.g. ‘I'd like to say no but that's one of those things that – I – I don't think I could guarantee it’.Patients’ responses as:How the question affected the patient's response. As previous work has shown that questions bias patients’ responses, we used Fisher's exact test to test the hypothesis that no-inviting questions were more likely to elicit ‘no self-harm’ responses.We used a qualitative method, conversation analysis,24 Disengagement was classified as ≥3 markers of disengagement.In the videos, patients did not always show conviction or certainty in responses that claimed ‘no suicidal ideation’ or ‘no self-harm’. Hence, we identified (a) statements indicating lack of knowledge or certainty (e.g. ‘I don't know’), and (b) signs of patient disengagement, which could indicate difficulty or reluctance to disclose more sensitive information. This included flat prosody, quiet or slow speech relative to the patient's typical speech, minimal lexical responses (e.g. ‘No. [2 s]’), averting gaze, flat or unchanging facial expression, and physically turning away from the clinician.N = 19), plans to self-harm (N = 13), the potential for future self-harm (N = 13), predicting risk of future self-harm (N = 5) and being okay and/or keeping safe (N = 5).There were 55 initial questions about current thoughts or feelings of self-harm (N = 46/55). However, in 16% cases (N = 9/55), initial questions were open questions, starting with ‘what/when/how’, inviting the patient to provide a longer response . For example:Video extract 1Clinician:  So, do you have any thoughts of ending yourlife as we speak now?Patient:    (2 s)No. (1 s)Video extract 2Clinician:  And are you feeling suicidal?Patient:   (2 s)Mhm. Yeah. (4 s)Video extract 3Clinician:  You'd take it [overdose] again if you could?Patient:   nods (1 s)Closed questions ask patients to choose yes or no, and are designed to constrain responses. Patients typically provided minimal answers followed by silence and did not elaborate on their experiences (Video extract 4Clinician: What's your thoughts about wanting to die?Patient:  (1 s)They're, I still kind of want to die. Butthen Jack thinks that I can't die ‘cause I gotmy boyfriend and there are people who care aboutme. (1 s) It's hard. I'm sort of in the middle.Video extract 5Clinician: How do you feel about ending your life as wespeak now?Patient:  It's still on the forefront of my mind. This seemslike a sticking plaster coming up here. And (1 s)I'm afraid to go home and be on my own again.Because I think there'll just be another day whereI'm not strong enough to override it and call forhelp and they'll – they'll just find me.Video extract 6Clinician: What plans do you have now?Patient:   Like I'm not prepared for the pain that I knowthat ligaturing is gonna cause. Like that'sgonna be painful, I know it is.By contrast, open questions elicited longer information-rich responses, with patients frequently describing conflicting or ambiguous thoughts.12 Of 46 closed questions, more than half  invited a no-self-harm response, with fewer than half (46%) inviting a yes-self-harm response. No-inviting questions used the negative polarity markers any/ever/at all  compared with the 65% disclosure rate in response to yes-inviting questions   .Table 325 For example, in extract 7, there are long delays , the patient breaks eye contact (line 3) and eventually confirms (‘I do’) but downplays the extent (‘a few’) and does not describe his ideas or plans in any detail.Video extract 7Clinician: Can I ask (1 s) Do you have any        plans todo anything to harm yourself?(2 s) patient looks downClinician: Or to take your life?(4 s)Patient:(no eye contact) I do have a few ideas.(1 s)There is further evidence on how patients respond to no-inviting questions demonstrating that such questions are highly problematic for disclosing suicidal ideation and/or self-harm: patients often delayed their response, qualified or downplayed their response, looked away and provided minimal information. These features mark difficulty responding.24 Patients gave a disengaged response 35% of the time (N = 7/20) when giving a no-problem answer to a no-inviting question versus 11% of the time (N = 1/9) when giving a no-problem answer to a yes-inviting question.Video extract 8Clinician: You're seeing [your therapist] tomorrowaren't you?Patient:   Yeah.(3 s)Clinician: You- Are you gonna be okay overnight?Patient:   patient looks down(1 s)nods(2 s)Second, when patients did not disclose suicidal ideation or self-harm, they more frequently showed signs of disengagement and lack of conviction after no-inviting questions. For example, after being asked if she will be okay overnight, the patient in extract 8 confirms but immediately shows signs of disengagement: breaking off eye contact, looking at the floor and nodding silently.N = 19/55, 35%)  .Table 4N = 12) of these questions inviting the patient to confirm they would not be at risk or could keep themselves safe. Asking for a prediction is different to asking patients whether they currently have plans or thoughts about ending their life (e.g. ‘Do you have any plans to do this again?’).Again, there was a bias with 63% (Video extract 9Clinician: What's the rating that you would give – probability thatyou'll take another overdose?Patient:     (5 s)I don't knowExplicitly claiming lack of knowledge:Video extract 10Clinician: What do you think the chances are of you actually goingthrough with some of those suicidal thoughts that you havePatient:    (4 s)I think (3 s)My – You know – (2 s)couldn't do it to my momI thinkHedging their response using ‘I think’, false starts and cut-off words (‘My-’) and downgrading their certainty:Patients found these questions particularly difficult to answer, as evidenced by substantial delay and resisting the implication that they could predict future self-harm in the following ways.Video extract 11Clinician: You think you'll do it again?Patient:  (2 s)I'd like to say no but that's one of those things that – I –I don't think I could guarantee it.Patients also emphasised the difficulty of assessing the likelihood of future suicidal acts when this was something they did not want, e.g. in extract 11 the clinician asks if the patient thinks he will attempt suicide again.Instead of giving a straightforward yes or no as invited by the question, the patient states ‘I'd like to say no’. He then gives a hedged answer, citing his uncertainty ‘I don't think I could guarantee’ and difficulty predicting whether he will attempt suicide.Video extract 12Clinician: So is there part of you that's scared you will hurtyourself?Patient:     Yeah.By contrast, there are three instances where a clinician asks about the future without asking the patient to predict their future actions. In extract 12, the clinician asks whether the patient is scared she may hurt herself. She immediately confirms, without hesitation, backpedalling or uncertainty, showing no difficulty in making this disclosure.current thoughts/feelings of self-harm, e.g. ‘So are you thinking about ending your life at the moment?’ ‘Do you have any plans of ending your life once you leave here?’nine of 19 questions about plans to self-harm, e.g. ‘Have you got any plans at the moment to take another overdose or harm yourself?’four of 13 questions about potential for future self-harm, e.g. ‘Where are you at over the next twenty-four hours about hurting yourself?’one of 13 questions about the predicting risk of future self-harm, e.g. ‘And so do you think there's any chance of you doing something else to harm yourself today tomorrow?’two of five questions being okay, e.g. ‘Are you gonna be okay overnight?’two of five questions about Some questions were also temporally constrained with a narrow timeframe (18 of 46 closed questions), as follows:plans to self-harm, e.g. ‘If you were to go home, to do something that would result in your death? Or harm to yourself?’two of 13 questions about potential for future self-harm, e.g. ‘On a basic level if you went home now, would you do anything to harm yourself?’two of 13 questions about keeping safe, e.g. ‘If you were to go home do you think that you could keep yourself safe?’two of five questions about In addition, towards the end of the assessment, some questions about future self-harm were explicitly tied to possible discharge (6/46 closed questions):This placement indicates to the patient that their answer is tied to discharge, which is likely to affect how patients respond if they wish to be discharged.Across the psychosocial assessment, there is a bias towards not uncovering thoughts and plans of self-harm through the cumulative effects of leading questions that invite a no response, their narrow timeframe and tying questions to possible discharge. First, 84% of questions were closed yes/no questions inviting minimal yes/no responses. Second, more than half were leading questions inviting a ‘no’ self-harm answer. When asked no-inviting questions, patients disclosed self-harm in only 8% of cases compared with 65% when asked a yes-inviting question. When answering ‘no’ (e.g. no thoughts or plans of suicide) to no-inviting questions, patients often showed signs of disengagement and lack of conviction (35%). Third, approximately one-third of questions asked patients to predict whether they would harm themselves in the future. Patients showed difficulty responding to these questions, resisting the assumption that they could predict future thoughts or actions, which is underpinned by patients’ criticisms of these questions, including ‘guaranteeing safety’ questions. Finally, half of closed questions had a narrow timeframe  or were explicitly tied to possible discharge.26 Nonetheless, these findings may help to shed light on the ‘low-risk paradox’, i.e. that patients are usually judged as low or no risk prior to death by suicide27 and ‘deny suicidal ideation’ even in the days and weeks before death.4 Our findings highlight that communication is structured in a way that makes it difficult for patients to disclose self-harm. Clinicians are unaware that a subtle difference in the wording of questions biases the patient's response with the items ‘any, ever, at all’ making questions no-inviting. The narrow timeframes demonstrate that clinicians are focusing on short time periods, typically up to 24 h. At the same time, they are tying patient responses to discharge, i.e. if the patient states they have no immediate thoughts or plans they are safe to discharge. Given that most patients report wanting to leave the emergency department after long waits to be seen in the main emergency department before being referred and then undergoing a psychosocial assessment, there is a risk of patients saying what they need to say so they can leave, i.e. they are not thinking about or planning self-harm. As a whole, these findings shed light on the National Confidential Inquiry findings that of the 17 people who die by suicide each day in the UK, four out of five are judged to be at low or no risk of suicide.Suicide risk assessment is a multifactorial process, and assessment of self-harm is just one component.15 In suicide risk assessment, no-inviting questions are compounded by patient shame, stigma and the consequences of disclosing self-harm . Concurrent feelings of wanting to live and die and fluctuating feelings about the ability to manage suicidal thoughts can also make these questions difficult to answer. On the other hand, when clinicians asked open questions, patients disclosed more information, particularly around inconsistent or ambiguous feelings of self-harm. Clinician judgement about the likelihood of self-harm could inform how practitioners design their questions. In conversation analysis, this process of tailoring our talk for the person we are speaking to is known as ‘recipient design’. Although this may contribute to how questions are designed, in a previous study we found that practitioners were not more likely to ask a yes-inviting question of patients who self-reported more suicidal thoughts and/or behaviour14.These findings are consistent with studies in out-patient community mental healthcare and general practice, where professionals also mostly asked no-inviting questions.12 In a randomised controlled trial, doctors who asked ‘Do you have some other concerns you would like to discuss?’, inviting a yes, versus ‘Do you have any other concerns you would like to discuss?’, inviting a no, were significantly more likely to elicit and reduce unmet concerns when using yes-inviting questions.13No-inviting questions are not just a feature of suicide risk assessment but a feature of healthcare questioning more widely. Doctors systematically ask questions inviting the ‘best case’ answer (e.g. ‘No pain?’); this is known as the principle of optimisation.28 This includes making recommendations (e.g. discharge) because of limited resources as opposed to their clinical judgement. This can manifest in burnout, clinician turnover, or developing a more detached or even callous approach to care as a defence mechanism against the system's impact on their practice.Emergency department psychiatric liaison clinicians are faced with increasingly limited options for treatment . If a person ends their life, clinicians may be called to a coroner's court if a person deemed at risk does not receive adequate treatment. Anecdotally, clinicians report defensive practice and can experience ‘moral injury’ when they are expected to work in ways that contradict their moral compass.28 along with adequate supervision and training.29Nonetheless, our findings have clear clinical implications. Patients are more likely to disclose self-harm when asked yes inviting  questions. No inviting questions typically use the words ‘any’, ‘ever’ or ‘at all’  or a negative declarative format (e.g. ‘You're not planning on doing it again?’). Open questions e.g. ‘How do you feel about ending your life as we speak now?’, ‘What are your thoughts about wanting to die?’ yield rich responses on the type and strength of thoughts and reasons the patient would not (re)attempt self-harm. Categorising self-harm thoughts and/or plans as either yes or no is problematic, as most people have ambivalent feelings. Guidelines and training should highlight how subtle differences in the wording of questions significantly influence patient disclosure of suicidal ideation and self-harm. For real change, there must also be an understanding of how clinicians protect themselves and cope with the challenges of underresourcing,N = 21/45) and 60% were female. Further research with minority ethnic groups, different socioeconomic groups and other marginalised groups to look at assessments in these groups in more detail would be important to understand whether communication differs and, if so, how it differs. We focused on initial questions as they influence the trajectory of further enquiry. We did not focus on caregiver communication, which was beyond the scope of this analysis. This is the first detailed analysis of video-recorded communication about self-harm in the emergency department. Conversation analysis allowed us to analyse non-verbal communication and to identify how subtle differences in question design affect patient disclosure.Data were collected in one emergency department, which may limit the generalisability of the findings. However, the findings are consistent with communications about self-harm in secondary and primary care. Around three-quarters of clinicians and just under 60% of patients consented: those consenting may not be representative of clinicians and patients. As is typical of many studies, patient participants were mostly White British (In conclusion, there is a bias towards not uncovering self-harm thoughts and plans in the emergency department. Closed no-inviting questions and asking patients to predict the future are common and deter disclosure. Other problematic aspects are narrow timeframes and tying questions to possible discharge. Open questions and yes-inviting questions facilitate disclosure. Asking patients how they feel about the future rather than asking them to predict whether they will harm themselves also facilitates disclosure.


We report the genome sequences of two genetic variants of grapevine rupestris stem pitting-associated virus (GRSPaV) from Idaho, USA. The coding-complete, positive-strand RNA genome of 8,700 nucleotides contains six open reading frames characteristic of foveaviruses. The two Idaho genetic variants belong to GRSPaV phylogroup 1. Foveavirus (family Betaflexiviridae) (Vitis vinifera L. and its hybrids (––Grapevine rupestris stem pitting-associated virus (GRSPaV) has flexuous filamentous particles and belongs to the genus viridae) . GRSPaV viridae) . The vir hybrids –4. The vhybrids –, 5, 6; ihybrids ––9.V. vinifera reference genome using bowtie2 v2.4.4 in local mode  from British Columbia, Canada. The same Riesling sample also yielded three additional contigs of 2,514, 2,893, and 506 nt, which exhibited 98.3%, 98.6%, and 98.8% identity, respectively, to the GRSPaV isolate AMCF clone 3 (GenBank accession number MG938311) from France  contig (GRSPaV-ID1) that spanned six ORFs and, based on the BLASTn program , exhibited 98.5% identity to the GRSPaV isolate 12G412  under BioProject accession number PRJNA931750.The coding-complete genome sequences of GSPaV-ID1 and GSPaV-ID2 are available in GenBank under accession numbers