It was discovered that the stimulation of M1 macrophages promoted fibroblast proliferation and fibrosis formation in vitro, accompanied with a disorder of the APN/CPT1A pathway, an overproduction of lipid peroxides, and a low level of autophagy in HFL-1 cells. Thereafter, APN treatment or CPT1A overexpression greatly suppressed above lipid peroxide accumulation, fibroblast proliferation, and fibrosis but activated autophagy in vitro. Furthermore, an in vivo IPF rat model was established by injection of bleomycin (BLM). Consistently, CPT1A overexpression exerted a protective role against pulmonary fibrosis in vivo; however, the antifibrosis property of CPT1A was partly abolished by 3-methyladenine (an autophagy inhibitor). In summary, APN/CPT1A-mediated fatty acid metabolism exerted its protective role in IPF partly through activating autophagy, shedding a new prospective for the treatment of IPF.Idiopathic pulmonary fibrosis (IPF) is a chronic progressive interstitial lung disease that leads rapidly to death. The present study is aimed at discovering the in-depth pathogenesis of IPF, exploring the role of adiponectin/carnitine palmityl transferase 1A- (APN/CPT1A-) mediated fatty acid metabolism during the development of IPF, and excavating its potential mechanism. Here, THP-1 cells were differentiated into M0 macrophages, followed by polarization to M1 macrophages upon hypoxia. Subsequently, lung fibroblast HFL-1 cells were stimulated by M1 macrophages to simulate hypoxia-related IPF condition  The rats were randomly assigned into 5 groups  solution. The pathological morphology changes were observed under a light microscope .After fixing with 4% paraformaldehyde for 24 h, the tissue samples were dehydrated in ethanol, embedded in paraffin, cut into 4 t-test, and the difference among more than two groups was evaluated by one-way ANOVA analysis, followed by Tukey's post hoc test. A p value less than 0.05 was considered to be statistically significant.Data were expressed as mean ± standard deviation (SD) from at least three independent experiments or groups of 6 rats each. The normal distribution of variables was assessed by the Shapiro-Wilk test. The differences between two groups were determined by Student's α and mammalian target of rapamycin (mTOR)  . mTOR isn (mTOR) . Furthern (mTOR) . All theβ, α-SMA, and Collagen I for the M1-treated group  was also introduced to HFL-1 cells. The subsequent western blot assay illustrated that addition of APN or overexpression of CPT1A could greatly elevate the protein expression of AdipoR1 and CPT1A in HFL-1 cells (μg/ml) and CPT1A overexpression remarkably lowered HFL-1 cell viability cocultured with M1 macrophages  (Figures Next, to further explore the regulatory role of fatty acid metabolism during the progression of pulmonary fibrosis, particularly to the induction of macrophages, APN/CPT1A signaling was activated to modulate fatty acid metabolism in HFL-1 cells with the coculture of M1 macrophages. As shown in -1 cells , suggestrophages . In addi Figures . Further Figures . These aβ, α-SMA, and Collagen I in pulmonary tissues, whereas C75 treatment lowered this rise, indicating that C75 protected rats against fibrosis; however, this antifibrosis effect of C75 was then partly weakened by the simultaneous treatment of 3-MA (Finally, to validate the critical regulation of APN/CPT1A during the progression of IPF, we successfully established an IPF rat model using BLM injection. In addition, the BLM-induced rats were injected with C75 and/or 3-MA for treatment. The histological analysis presented in  of 3-MA . In addi of 3-MA . What is of 3-MA . The fol of 3-MA . Further of 3-MA . Taken tin vitro, accompanied with a disorder of the APN/CPT1A pathway which is critical for lipid acid metabolism, an overproduction of lipid peroxides, and an activation of autophagy. Thereafter, APN treatment or CPT1A overexpression greatly suppressed lipid peroxide accumulation, fibroblast proliferation, and profibrotic protein expression but activated autophagy in vitro. The following in vivo experiments further validated the protection of CPT1A overexpression in pulmonary fibrosis; however, the antifibrosis property of CPT1A was partly abolished by 3-MA, indicating that APN/CPT1A-mediated fatty acid metabolism exerted its protective role in IPF partly through activating autophagy.IPF is a chronic progressive interstitial lung disease that leads rapidly to death. The incidence of pulmonary fibrosis keeps increasing and brings great burden to global healthcare. Thus, exploring the pathogenesis of IPF and discovering novel effective targets for IPF treatment are essential. Currently, accumulating evidence discloses that the alternations of fatty acid metabolism are commonly detected in fibrosis diseases. In the present study, we uncovered a critical function for APN/CPT1A-mediated lipid acid metabolism in the development of pulmonary fibrosis. Here, we disclosed that hypoxia induced macrophage polarization to M1, which was beneficial to fibroblast proliferation and fibrosis formation β [in vivo [β, α-SMA, and Collagen I, revealing that hypoxia induced the generation of M1 macrophages, which subsequently promoted the fibroblast proliferation and fibrosis. Nevertheless, in this study, in addition to verifying the influences of macrophages on fibroblast proliferation and fibrosis, more attention was focused on the influences on autophagy, oxidative stress, and fatty acid metabolism following hypoxia.Fibroblasts are effector cells driving the progress of fibrotic diseases including IPF featured as unrestrained deposition and stiffening of the extracellular matrix. Despite diverse and varied etiologies of fibrotic diseases, it is pointed out that macrophages play a critical role in the initiation and development of fibrosis . Macrophβ . In addi[in vivo . Thus, min vitro and in vivo, accompanied with the elevated expression of profibrotic proteins, including TGF-β, α-SMA, and Collagen I, but also demonstrated that strengthening of APN/CPT1A signaling was beneficial to restore the balance of lipid peroxides and profibrotic proteins, so as to suppress oxidative stress and alleviate pulmonary fibrosis.Emerging evidence uncovered that alterations in fatty acid metabolism are core pathways and common mechanisms which play a crucial role in multiple fibrotic disorders, including kidney fibrosis, liver fibrosis, and pulmonary fibrosis . Excessiβ-oxidation and accumulation of excessive lipids, suggesting that fatty acid oxidation may be mediated through an autophagy-dependent mechanism [in vivo and in vitro, and APN exerts as a novel therapeutic target of diabetic retinopathy through inhibiting autophagy [Last, but not least, fatty acid metabolism is closely associated with autophagy. It has been proposed that cells may be unable to deal with lipid droplets via acid lipases in the absence of autophagy, leading to the reduction of mitochondrial echanism , 46. Coiutophagy ; howeverutophagy , emphasiutophagy ; Qi et autophagy . ConsistIn summary, we demonstrated not only that hypoxia-induced macrophage polarization to M1 could induce pulmonary fibrosis but also that the activation of APN/CPT1A-mediated fatty acid metabolism suppressed lipid peroxide accumulation and fibroblast proliferation but activated autophagy in IPF. Mechanically speaking, the protective role of APN/CPT1A-mediated fatty acid metabolism was autophagy-dependent. Therefore, targeting fatty acid metabolism is an effective approach for the treatment of IPF.


Here, we demonstrate a physical interaction between 5-HT2A and TrkB in vitro and in vivo using co-immunoprecipitation and biophysical and biochemical approaches. Heterodimerization decreased TrkB autophosphorylation, preventing its activation with agonist 7,8-DHF, even with low 5-HT2A receptor expression. A blockade of 5-HT2A receptor with the preferential antagonist ketanserin prevented the receptor-mediated downregulation of TrkB phosphorylation without restoring the TrkB response to its agonist 7,8-DHF in vitro. In adult mice, intraperitoneal ketanserin injection increased basal TrkB phosphorylation in the frontal cortex and hippocampus, which is in accordance with our findings demonstrating the prevalence of 5-HT2A–TrkB heteroreceptor complexes in these brain regions. An expression analysis revealed strong developmental regulation of 5-HT2A and TrkB expressions in the cortex, hippocampus, and especially the striatum, demonstrating that the balance between TrkB and 5-HT2A may shift in certain brain regions during postnatal development. Our data reveal the functional role of 5-HT2A–TrkB receptor heterodimerization and suggest that the regulated expression of 5-HT2A and TrkB is a molecular mechanism for the brain-region-specific modulation of TrkB functions during development and under pathophysiological conditions.Serotonin receptor 5-HT Tropomyosin receptor kinase B (TrkB) is a major transducer of intracellular signals from mature brain-derived neurotrophic factor (BDNF). TrkB is predominantly expressed within the CNS, especially in the frontal cortex, hippocampus, cerebellar cortex, visual system, hypothalamus, striatum, substantia nigra, and dorsal raphe nucleus ,3,4,5,6.2A) belongs to the G-protein-coupled receptor (GPCR) family and is broadly distributed within the brain, showing high expression in the olfactory tubercle, cortical areas, and dentate gyrus [2A is involved in neurogenesis and can modulate synaptic plasticity via long-lasting increases in the excitability and firing rate of glutamatergic and GABAergic neurons [2A receptor is implicated in numerous CNS disorders, including bipolar disorder, depression, and schizophrenia [2A-blocking antipsychotic drugs elicit better clinical responses in bipolar or treatment-resistant depressive patients [Serotonin receptor 2A , leading to increased Ca2+ release from the endoplasmic reticulum. In neurons, Ca2+ influx can activate cyclic AMP (cAMP) response element-binding protein (CREB), which, in turn, boosts the transcription of BDNF [2A stimulation [2A and TrkB signaling pathways. Although the underlying mechanisms are not completely understood, a direct interaction between 5-HT2A and TrkB may be an intriguing explanation. Research groups have demonstrated that 5-HT2A can form heterodimers with other GPCRs, including the 5-HT1A receptor [4 and 5-HT7 subtypes, can form heterodimers with adhesion molecule L1 [2A interacts with non-GPCR proteins.The coupling of 5-HT of BDNF . Interesmulation ,27,28,29receptor , dopaminreceptor , and metreceptor . Moreoveecule L1 , CDK5 [3ecule L1 , or CD44ecule L1 , respect2A and TrkB form heteroreceptor complexes both in vitro and in vivo. We further demonstrated the functional effects of 5-HT2A and TrkB receptor heterodimerization.In the present study, we found that 5-HT2A antagonist ketanserin  or the selective 5-HT2A agonist 25CN-NBOH  was dissolved in saline, and each was administered i.p. at a dose of 1 mg/kg of body weight (eight mice in each group). The control group (eight mice) received an equivalent saline injection. At 10 min (for 25CN-NBOH) or 30 min (for ketanserin) after the injection, the animals were euthanized, the brains were removed on ice, and the frontal cortex, hippocampus, and striatum were dissected.Adult (P60) male C57BL/6J mice  were used for acute treatment with ketanserin or 25CN-NBOH. The mice were housed under standard laboratory conditions in the natural light–dark cycle (16 h of light and 8 h of dark) with free access to water and food. The preferential 5-HT2 in DMEM  that contained 10% of fetal bovine serum  and penicillin/streptomycin . Transient transfection was performed with Lipofectamine 2000 (Invitrogen) according to manufacturer’s protocol, and after transfection cells were incubated in the serum-free medium. For treatments, we applied 500 nM selective TrkB agonist 7,8-DHF , 1 μM 25CN-NBOH , and/or 1 μM ketanserin . The doses of all drugs were chosen based on literature data. In one work, it was shown that a 7,8-DHF concentration of 500 nM is maximally effective for TrkB activation [2 sites [2A receptor [Mouse N1E-115 neuroblastoma cells from the American Type Culture Collection  were grown at 37 °C and 5% COtivation . 25CN-NBreceptor .2A cDNA was cloned into the pcDNA3.1(+) donor vector  carrying an HA-tag. TrkB_mEGFP was kindly gifted by Ryohei Yasuda ; RRID: Addgene_83952).Murine 5-HT2A and GFP-tagged TrkB was performed as described previously [Co-immunoprecipitation in lysates of N1E-115 cells that co-expressed HA-tagged 5-HTeviously  with an 2A  followed by incubation with Protein A-Sepharose and Western blotting with an anti-TrkB antibody .Co-immunoprecipitation from hippocampal, cortical, and striatal homogenates was performed according to a protocol described elsewhere . BrieflyAdult (P90) male and female C57BL/6J mice were subjected to this assay. The mice were bred and housed at the animal facility of the University of Hohenheim at a controlled temperature (25 °C) and photoperiod (12/12 h light/dark cycle) and were allowed unrestricted access to standard food and tap water.2 asphyxiation, the brains were removed, postfixed overnight at 4 °C, processed routinely, and embedded in paraffin. Next, 5 µm-thick sections were prepared on a Leica RM2255 microtome  and transferred to SuperFrost®Plus glass slides . The sections were air-dried at least overnight at 37 °C and then subjected to the immunofluorescence assay.Mice were euthanized by CO2A receptor  and goat polyclonal anti-TrkB  antibodies diluted in PBS was carried out overnight at 4 °C. After a wash in PBS, appropriate secondary antibodies were applied for a 1 h incubation at room temperature: a donkey anti-rabbit IgG antibody conjugated with Alexa Fluor® 488  and a donkey anti-goat IgG antibody conjugated with Alexa Fluor® 594  both diluted 1:800 in PBS. After a subsequent wash in PBS, cell nuclei were visualized with a bis-benzimide solution . Finally, the sections were mounted in an antiquenching medium and examined under a fluorescence microscope Zeiss Axiovert 200M  with 20× air objectives.The brain sections were deparaffinized according to a standard protocol and washed with PBS. Antigen retrieval was performed by immersion in 0.01 M sodium citrate buffer (pH 9.0) heated at 80 °C in a water bath for 30 min. The blocking of nonspecific binding sites was performed for 1 h at room temperature in PBS containing 5% normal donkey serum , 0.1% Triton X-100 , and 0.02% sodium azide . Incubation with rabbit polyclonal anti-5-HT2A-mTurquoise2 and TrkB-YPet served as the donor and acceptor, respectively. As a negative control, cells were co-transfected with mTurquoise2-tagged CD86 and YPet-tagged CD86. After 16 h, the cells were imaged with a Zeiss LSM 780 microscope equipped with a C-Apochromat 40×/1.2 W Korr water immersion objective via the excitation of the fluorescent proteins at 440 and 514 nm according to the protocol. For the image analysis and evaluation, custom-written MATLAB scripts were employed. We calculated the predicted apparent FRET efficiency as EfDA = ½ EfD/(1 − xD) = ½ EfA/xD, assuming a standard dimerization model [These measurements were performed on live N1E-115 neuroblastoma cells as described elsewhere . 5-HT2A-on model ,41.2A-mTurquoise2, or both. We acquired time series data under the Zeiss LSM 780 microscope for 10 min per recording (5 s per frame). Ca2+ activity was assessed by means of GCaMP6f fluorescence signals (F) and changes calculated as F/Fmax. To raise Ca2+ levels to saturated, 10 µM ionomycin was applied after 6 min. This saturated Ca2+ signal was used as Fmax for the calculation of basal Ca2+ levels.This assay was performed on N1E-115 cells expressing TrkB-YPet, 5-HTA PLA assay was performed with the Duolink in situ PLA Probes and Duolink in situ Detection Reagents Red Kit (cat. # DUO92008). Brain sections of 20 μm thickness were utilized in the PLA. Antibodies, the same as those used for immunohistochemistry, were diluted 1:100 with the Antibody Diluent provided with the kit. For imaging, the slides were dried and mounted with a cover slip by means of ~7 μL of the Duolink in situ Mounting Medium with 4′,6-diamidino-2-phenylindole (DAPI). The imaging and analysis of the stained brain sections were carried out under the fluorescence confocal microscope (Zeiss LSM 780) using a 40× objective and excitation wavelengths of 561 nm for PLA and 405 nm for DAPI. In each brain section, 3–4 measurements in the frontal cortex, hippocampus, and striatum were performed. Each image was analyzed in ImageJ  to count individual fluorescent spots. The data are presented as the number of spots per cell, normalized to the number of cells.Polr2a) cDNA, which served as an internal standard [Total RNA was extracted from the brain tissue of C57BL/6J mice using ExtractRNA , treated with RNA-free DNase , and diluted to 0.125 µg/µL with diethyl pyrocarbonate-treated water. One microgram of total RNA was subjected to cDNA synthesis with a random hexanucleotide mixture ,43,44. Tstandard ,43,44. A3VO4, 2 mM phenylmethylsulfonyl fluoride, and a protease and phosphatase inhibitor cocktail. The protein concentration was estimated spectrophotometrically using the Pierce BCA Protein Assay Kit  and a NanoDrop 2000C spectrophotometer , followed by the adjustment of samples to equal concentrations with 2× Laemmli sample buffer. After denaturation by boiling for 10 min at 95 °C, the cell extracts  were resolved on 10% SDS-PAGE and blotted onto a nitrocellulose membrane . The membrane was incubated with a primary antibody , anti-pTrkB , or anti-5-HT2A antibody ) at 4 °C overnight, then the membrane was washed in Tris-buffered saline supplemented with 0.05% Tween 20 (TBS-T) and incubated with an HRP-conjugated secondary antibody. After protein detection, the blot was stripped and then re-probed with an anti-β-tubulin antibody  as a loading control. Immunoreactive bands were detected by means of the Clarity Western ECL Substrate . Protein bands were documented on a C-DiGit Blot Scanner  and quantified in the Image Studio software . Target protein levels were assessed in chemiluminescence relative units and normalized to β-tubulin chemiluminescence relative units.The extraction of total protein from N1E-115 cells was performed in RIPA buffer  with the addition of 1 mM Nat test after a Gaussian distribution evaluation by the D’Agostino–Pearson normality test. Groupwise comparisons were made by one-way ANOVA followed by Fisher’s post hoc test. Cell groups co-expressing different amounts of 5-HT2A receptors and treated with 7,8-DHF were compared by two-way ANOVA followed by Fisher’s post hoc test. In figures, significance is displayed as p < 0.05 (*), p < 0.01 (**), and/or p < 0.001 (***).The data are presented as means ± the standard error of the mean (SEM). Unless stated otherwise, the significance of pairwise differences was assessed by Student’s 2A and TrkB, we performed co-immunoprecipitation experiments with neuroblastoma N1E-115 cells co-expressing HA-tagged 5-HT2A and GFP-tagged TrkB. It is noteworthy that non-transfected N1E-115 cells express neither 5-HT2A nor TrkB (data not shown). After immunoprecipitation with an antibody against the HA tag, GFP-tagged TrkB was detectable only in cells co-expressing both HA- and GFP-tagged receptors (2A or GFP-TrkB) were mixed prior to lysis and analyzed in parallel. While individual receptors could be detected by the same antibody, co-immunoprecipitation did not occur -based approach. We measured the apparent FRET efficiency (EfDA) between mTurquoise2-labeled 5-HT2A  and YPet-labeled TrkB  in living N1E-115 cells using the linear unmixing FRET (lux-FRET) method combined with confocal microscopy (DA = 14.6%) for 5-HT2A-mTurquoise2 and TrkB-YPet. In contrast, cells expressing a monomeric fluorophore-tagged CD86 protein [DA values  . In all and TrkB C. The qustriatum D.2A heterodimerization, we first assessed Ca2+ activity in N1E-115 cells expressing TrkB-YPet, 5-HT2A-mTurquoise2, or both. To this end, we used the Ca2+ indicator GCaMP6f [2+ dynamics with the multi-threshold event detection approach [2+ signal (Fmax) indicated that basal Ca2+ levels were significantly higher following heterodimerization compared to control cells  protein in N1E-115 cells, using a phospho-specific antibody that specifically recognizes the Y515 site [2A , either alone or together with an increasing concentration of HA-tagged 5-HT2A C. Basal f 5-HT2A B.2A–TrkB oligomerization impacted TrkB function in a manner depending on the receptors’ expression ratio. Therefore, we next determined the expression profiles of 5-HT2A and TrkB in mouse hippocampus, striatum, and cortex at different stages of postnatal development using real-time quantitative RT-PCR. An analysis of the Ntrk2 transcript encoding TrkB revealed that the Ntrk2 mRNA level was continuously elevated from P1 to P30 in the hippocampus and frontal cortex and that this increase was sustained in the striatum until P60  concentrations of HA-tagged 5-HT2A. These experiments revealed significantly reduced basal phosphorylation of TrkB in the cells expressing 5-HT2A at concentrations ≥0.05 μg  C57BL/6J mice with either ketanserin (1 mg/kg) or the selective 5-HT2A agonist 25CN-NBOH (1 mg/kg). TrkB phosphorylation was significantly increased in the hippocampus and frontal cortex of mice treated with ketanserin but not those treated with 25CN-NBOH  in the mouse hippocampus [2A-TrkB heteroreceptor complexes both in vitro and in vivo.Several research articles have reported the modulation of BDNF expression by the 5-HTcircuits ,28,29, bcultures . We recese brain , indicatecule L1 , CDK5 [3ecule L1 , or CD44ecule L1 . The exiecule L1 ,58. Morepocampus . In the 2A receptor functions. Importantly, our data demonstrated that the degree of heterodimerization played a pivotal role in this process. This suggests that changes in the relative amounts of both receptors and the corresponding changes in heterodimerization rates can provide an intriguing mechanism for the differential regulation of TrkB functions in health and disease. Several studies have described substantially decreased TrkB expression and lower TrkB phosphorylation in the hippocampus of depressive suicidal victims [2A levels in some brain structures, including the frontal cortex and hippocampus, in post-mortem samples from patients with major depressive disorder and suicide victims [2A expression is reportedly sensitive to basal 5-HT concentration, with the 5-HT2A receptor becoming more abundant in response to a diminished synaptic 5-HT concentration and vice versa [2A expression in the brain of suicide victims may be explained by an adaptive response to deficient 5-HT2A signaling due to a reduced 5-HT level in depression. Based on the above-mentioned data, it might be hypothesized that under pathological conditions , the balance between 5-HT2A and TrkB becomes shifted toward increased levels of 5-HT2A–TrkB heteroreceptor complexes, with a subsequent loss of TrkB function. The prevalence of 5-HT2A–TrkB heteroreceptor complexes in the hippocampus, even under the basal conditions obtained in this study, suggests that this brain structure is among the most vulnerable to the above scenario.From a functional perspective, this heterodimerization suppressed basal TrkB autophosphorylation and prevented agonist-mediated TrkB activation without affecting 5-HT victims ,61,62,63 victims ,65,66. Mce versa ,68,69. I2A expression was drastically increased (up to five-fold) in all analyzed brain regions, while TrkB expression exhibited only a moderate change (up to a two-fold increase). Considering that even a very low relative amount of 5-HT2A receptor elicited inhibitory effects on TrkB, such a shift in the receptor ratio could result in the decreased ability of BDNF to activate TrkB.Notably, our findings may also explain why the ability of BDNF to activate TrkB gradually declines during early postnatal development in mice . Startin2A–TrkB interaction could also play a role in the action of antidepressants. It is generally accepted that BDNF–TrkB signaling is a critical component of an antidepressant response [2A receptor inactivation may also facilitate antidepressant action [2A receptor antagonist ketanserin increased neurogenesis in the adult rat hippocampus [2A receptors because a combined treatment with fluoxetine and ketanserin applied at lower dosage  failed to produce a neurogenic effect while it boosted the expression of Bdnf mRNA in the rat hippocampus [2A antagonism can stimulate BDNF expression and/or secretion [2A receptor activation can suppress Bdnf transcription in the hippocampus [2A and BDNF could thus explain the functional crosstalk between these receptors at the molecular level. Indeed, we found that an acute blockade of 5-HT2A with ketanserin reversed basal TrkB phosphorylation in vitro. We also demonstrated that acute treatment with ketanserin led to increased TrkB phosphorylation in the mouse hippocampus and frontal cortex in vivo, which is in accordance with our findings regarding the prevalence of 5-HT2A–TrkB heteroreceptor complexes in these brain regions.The 5-HTresponse ,71,72,73t action ,75,76,77pocampus . Howeverpocampus . Moreoveecretion ,88,89,90pocampus . Our pre2A receptor, a member of the GPCR family, could form heterodimers with the non-GPCR receptor TrkB, both in vitro and in vivo. Heterodimerization considerably decreased TrkB autophosphorylation and prevented TrkB activation by its ligand, and these effects could be reversed by a pharmacological blockade of 5-HT2A with ketanserin. Importantly, our findings suggest that the regulated and balanced ratio of heterodimerization in different brain structures may be crucially involved in both the onset and treatment responsiveness of psychiatric diseases such as depression and anxiety.Overall, our present results demonstrated that the 5-HT2A receptor, a member of the GPCR family, and the non-GPCR receptor TrkB, both in vitro and in vivo.In the present study, we provide a multilevel analysis demonstrating, for the first time, the physical interaction between the 5-HT2A receptor functions. Importantly, these detrimental effects on TrkB functions were reversed by a pharmacological blockade of the 5-HT2A receptor with ketanserin, a drug widely used to treat hypertension. Moreover, our data demonstrated that the stoichiometry of heterodimerization played a pivotal role in this process. This suggests that changes in the relative expression of both receptors and the corresponding changes in heterodimerization rates can provide an intriguing mechanism for the brain-region-specific regulation of TrkB functions in health and disease.From a functional perspective, heterodimerization suppressed basal TrkB autophosphorylation and prevented agonist-mediated TrkB activation without affecting 5-HT


A subgroup of IPF patients can meet IPAF criteria (features suggesting an underlying autoimmune process without fulfilling established criteria for a CTD). This study was aimed to evaluate whether IPAF/IPF patients compared to IPF patients differ in clinical profile, prognosis and disease course.This is a retrospective, single center, case–control study. We evaluated 360 consecutive IPF patients  and compared characteristics and outcome of IPAF/IPF to IPF.more frequently females , suffered more frequently from gastroesophageal reflux , and showed a higher prevalence of arthralgias , myalgias  and fever . The serologic domain was detected in all cases (the most frequent were ANA in 17 and RF in nine cases) and morphologic domain (histology features) was positive in 6 out of 10 lung biopsies (lymphoid aggregates). Only patients with IPAF/IPF evolved to CTD at follow-up . The presence of IPAF was a positive prognostic determinant , whereas the isolated presence of circulating autoantibody did not impact prognosis .Twenty-two (6%) patients met IPAF criteria. IPAF/IPF patients compared to IPF were The presence of IPAF criteria in IPF has a major clinical impact correlating with the risk of evolution to full blown-CTD during follow-up and identifying a subgroup of patients with a better prognosis. In 2015, ATS/ERS international task force defined the criteria describing a new research entity named IPAF  , 4. DespIPF can be reclassified as IPAF when, in addition to the usual interstitial pneumonia (UIP) pattern, have a combination of one feature from at least two of three different domains; clinical, serologic or morphologic [either pathological  or related to a multi-compartment involvement ] , 5.The paucity of studies investigating the impact of IPAF features on IPF natural history provides a strong rationale for the present study that was conducted in a large and well-defined IPF cohort using rigorous inclusion criteria for IPAF and was aimed to evaluate whether IPAF/IPF patients compared to IPF patients have a different prognosis and disease course.In this single-center, retrospective, investigator initiated comparative study, we evaluated consecutive patients presenting to the pulmonary unit of the GB Morgagni Hospital  with suspected interstitial lung disease who received a multidisciplinary diagnosis of IPF . Patients with incomplete clinical data, less than 3 months of follow-up and those without a complete autoimmune clinical and serological evaluation performed at our center were excluded. Baseline and follow-up data were collected as detailed in the This study was approved by the Comitato Etico di area vasta ROMagna, Italy .The primary endpoint was the prognostic significance of the presence of IPAF among IPF patients. This was measured by comparing transplant-free survival for IPF with IPAF to that of IPF without IPAF.Secondary endpoints included:(1)The prognostic significance of the presence of positive autoimmune serology alone  in IPF patients, compared to IPAF/IPF and to IPF only . This was measured comparing transplant-free survival for IPF with positive autoimmune serology to that of IPF with IPAF and to that of IPF only.(2)Evaluation of natural history: development of full-blown CTD at follow-up. We described the baseline characteristics and compared the prevalence of CTD at follow-up between three patients subgroups: IPF with IPAF, IPF with positive autoimmune serology alone  and IPF only .t-test or Chi2 test, as appropriate. We used exact probability values  and an alpha level of 0.05.For baseline data, the summary descriptive statistics were be generated with categorical data displayed as absolute numbers and relative frequencies. Continuous data were shown as mean (SD) for normally distributed data or as median (interquartile range) for skewed distribution. Comparisons between groups was performed using a p-values ≥0.2 and excluding covariates with significant collinearity (r > 0.8) at univariate analysis. The proportional hazard assumption for each predictor was tested using approximate score statistic of linear correlation between the rank order of failure times in the sample and Schoenfeld partial residuals. We calculated hazard ratios (HRs) for overall mortality analyses. Patients were censored at death, lung transplant, or date of last known follow-up. Data cut-off was December 28, 2016. Results are reported as HRs, 95% CIs and p values, and are shown graphically as Kaplan–Meier survival curves. All statistical analyses were performed using STATA 15.For regression analysis, sample size calculation met the rule of thumb of at least 10 observation per variable, with 170 observed events we could evaluate 17 variables without overfitting. The small size of missing data allowed an analysis restricted to individuals with complete information on all variables of the main analysis . The fundamental method of univariable/multivariable analysis was Cox regression. Causal model based on previous literature was used to identify confounders: age, gender, smoking status, comorbidities and disease severity. The models were formulated by systematically removing predictors that were not significant using a backward selection procedure removing variables with N = 274) was excluded due to the lack of rheumatologic and/or serologic evaluation performed at our center. The flow chart of cases inclusion is presented in the p = 0.003), as detailed in the We extracted from the Forlì database 703 consecutive IPF patients, between January 1, 2002 and December 28, 2016. Among those 360 met protocol requirements and were included in the study. A vast minority , p = 0.02. No significant differences were noted in age, smoking history, family history and comorbidities profile with the notable exception of GERD that was significantly more prevalent in IPAF/IPF compared to non-IPAF/IPF . The pulmonary function profile showed a slight but significantly higher DLco in the IPAF/IPF patients  and a marginally higher FVC not statistically significant  without significant differences in the GAP stage distribution. Similarly to non-IPAF/IPF patients, IPAF/IPF patients presented in the vast majority with a chronic onset (75%) of dyspnea (86.4%) and/or cough (59.1%). However, in contrast to what observed in non-IPAF/IPF patients, all IPAF/IPF patients presented with at least one systemic symptom, with a strikingly higher prevalence of arthralgias , myalgias  and fever  compared to non-IPAF/IPF. Further rheumatologic evaluation of arthralgias revealed inflammatory arthritis only in IPAF/IPF cases, as shown in Comparison between IPAF/IPF patients and non-IPAF/IPF patients is shown in Among the 338 IPF patients that did not meet IPAF criteria, 43 (12.7%) showed a positive autoimmune serology.All cases showed isolated positivity for a single class of autoantibodies. Four patients  showed ANCA positivity and one of them developed a full-blown vasculitis after 10 years of follow-up . Seventeen (39%) patients had an isolated anti-thyroid positive autoimmunity, all were clinically identified as autoimmune thyroiditis without evidence of systemic autoimmune disease. 11  patients showed isolated ANA positivity, two ENA, two anti-CCP, and 7 RF positivity. None of them developed CTD at follow-up.Comparison of clinical characteristics of non-IPAF/IPF cases with and without positive autoimmune serology are reported in p values >0.05, not shown) with two notable exceptions: (1) higher prevalence of arthralgias in the non-IPAF subgroup with positive autoantibodies  compared to non-IPAF with negative autoantibodies , p < 0.0001. Those patients were not classified as IPAF because arthralgias was interpreted by the rheumatologist as non-specific. (2) higher prevalence of digital clubbing in the non-IPAF subgroup with positive autoantibodies  compared to non-IPAF with negative autoantibodies , p = 0.003.N = 22), the difference was robust both by univariate analysis HR 0.17  and after adjusting for age, sex, lung cancer, pulmonary function variables (%pred FVC and %pred DLco) and diagnosis before or after 2011, HR 0.22 . Univariate and multivariate analyses are reported in The presence of IPAF criteria in patients who received a multidisciplinary diagnosis of IPF was associated with significantly lower overall mortality compared to IPF patients lacking IPAF criteria. Despite the small number of cases (N = 338) the presence of positive autoimmune serology (N = 43) did not impact patients’ prognosis, unadjusted HR 1.01 (95% CI 0.69–1.48), p = 0.96 and HR 1.00 (95% CI 0.67–1.49), p = 0.99 after adjusting for age, sex, lung cancer and pulmonary function variables (%pred FVC and %pred DLco). Overall mortality rate per 100 person-year and survival at 1, 3, and 5 years were all similar between the two groups, data are reported in When analysis was confined to IPF patients not meeting IPAF criteria  developed CTD: rheumatoid arthritis , scleroderma  and one Sjogren. Mean latency time from IPAF diagnosis to CTD diagnosis was 21.5 months (range 6–60 months).The characteristics of the 22 IPAF cases are reported in To the best of our knowledge this is the first study evaluating IPAF features and clinical meaning in a well-defined cohort of IPF patients, showing that IPF patients can present IPAF features in a minority of cases (6%) with a clinical profile and natural history strikingly divergent from that of IPF. IPAF/IPF compared to IPF is characterized by:(1)a significantly better prognosis;(2)a high risk of evolution to full blown CTD (45.5%), that among IPF patients seems exclusive of those presenting with IPAF features;(3)a specific clinical profile with a higher prevalence of females and gastroesophageal reflux. Most notably, systemic signs and symptoms, rare in IPF, are universally present in IPAF/IPF  and are associated with at least one positive autoimmune finding on serology (most commonly elevated levels of ANA and/or RF).To define IPAF in this cohort of IPF patients we have meticulously applied the current IPAF ERS/ATS criteria to the clinical and serological features. The morphologic domain was defined by histopathology patterns  of UIP with interstitial lymphoid aggregates with germinal centers and/or diffuse lymphoplasmacytic infiltration (with or without lymphoid follicles), and these features were present in one third of IPF/IPAF cases (6/22). The multi-compartment involvement was defined using more stringent criteria: unexplained pleural or pericardial effusion or thickening, or by the presence of unexplained airway disease as seen by histopathology (follicular bronchiolitis or constrictive bronchiolitis). Interestingly none of our IPF/IPAF patients met the multi-compartment features following these restricted criteria. This punctilious approach led to a prevalence of IPAF among IPF cases lower than previously described, but with a strikingly prognostic divergence that previous studies could not detect .p = 0.003). Age, lung cancer, pulmonary function (% of pred FVC and % of pred DLco), diagnostic time period (before/after 2011) and IPAF were all significant prognostic factors at both univariate and multivariate survival analysis. IPAF/IPF patients compared to IPF had a significantly lower overall mortality rate per 100 persons year (3.13 vs. 16.26) and a significantly better prognosis . Previous studies have compared the prognosis of IPAF to that of historical IPF patients cohorts, showing that IPAF carries a better prognosis compared to IPF  (The Oldham study  is the m= 0.009) .In the absence of IPAF criteria the sole presence of autoantibodies in IPF did not influence survival. It is difficult to compare our results with the existing literature on this topic because our study was not powered to detect the prognostic significance of specific antibodies. In the literature the effect of ANA and RF on all causes mortality does not seem significant.  The subsN = 22) imbalanced compared to the controls groups . Biases related to the observational retrospective design of this study spanning over a wide time period (2002–2016) are alleviated by the consecutive enrollment of patients, the reassessment of all IPF diagnosis based on ERS/ATS 2011 guidelines and the introduction in the multivariate survival analysis of the diagnostic time period (before/after the year 2011) as a dummy variable. However, concerns about the high dropout rate, a price we had to pay to achieve a highly accurately selected IPAF population with serology and rheumatology evaluation completed at our center, are only partially mitigated by the observation that the excluded cases compared to included cases had homogeneous clinical profile, although a slightly divergent prognosis.This study has several limitations: the retrospective and monocentric study design, the high rate of cases exclusion , the very small sample size of IPAF cases  and identifying a subgroup of patients with a clearly better prognosis .Future prospective and larger studies will help to better define IPAF diagnostic criteria and their utility to identify in IPF specific subgroups with different prognosis and treatment response.The original contributions presented in the study are included in the article/This study was approved by the Comitato Etico di area vasta ROMagna, Italy . Written informed consent [from the patients/participants OR patients/participants legal guardian/next of kin] was not required to participate in this study in accordance with the national legislation and the institutional requirements.ST, CR, SPu, SPi, AW, JR, MB, and VP: conception and design. ST, SPu, CR, MB, AW, JR, AD, SPi, FG, FL, ER, VL, MC, and VP: acquisition, analysis or interpretation, and drafting the manuscript for important intellectual content. VP had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. All authors contributed to the article and approved the submitted version.ST declares speaker’s fee from Boehringer-Ingelheim, Roche, Erbe, PulmoniX; and VP declares speaker’s fees from Boehringer-inghelhem, Erbe, Ambu, and Roche.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The three-dimensional full-field morphologies at different compressive loads were obtained. The evolution of flaws, including pores, transverse cracks, and vertical cracks, during compressive loading was observed. The number and volume of pores, transverse cracks, and vertical cracks were extracted. It was found that most pores and transverse cracks tend to be compacted. However, high compressive loads cause vertical cracks near the upper surface to expand, leading to the failure of the specimen. Real flaws with morphological and positional information were introduced into the finite element models. The influence of different types of flaws on the mechanical behavior is discussed. It was found that vertical cracks have a greater influence on mechanical behavior than do transverse cracks under compression. The presence of transverse cracks contributes to the evolution of vertical cracks. This study may be helpful for process optimization and performance enhancement of additively manufactured ceramic materials.The additive manufacturing process may create flaws inside ceramic materials. The flaws have a significant influence on the macroscopic mechanical behavior of ceramic materials. In order to reveal the influence of flaws on the mechanical behavior of additively manufactured ceramic, flaw evolution under mechanical loads was studied by in situ X-ray computed tomography (XCT) in this work. In situ compression XCT tests were conducted on stereolithographic additively manufactured Al The higiC, etc. ,8,9.2O3 depends on the relation between the printing and loading directions. The microstructural characteristics and fracture strength of yttria-stabilized tetragonal zirconia polycrystal fabricated by stereolithographic additive manufacturing and traditional subtractive manufacturing were studied. Although both materials showed similar microstructures considering grain size and phase composition, significant differences in critical defects were observed [Stereolithography additive manufacturing technology for ceramics generally comprises shaping, debinding (binder burnout), and sintering processes ,8,9. Durobserved . Anotherobserved . The infSEM, as a commonly used method for microstructural characterization, provides only two-dimensional information of outer surfaces. To obtain three-dimensional information of the whole body, successive sectioning and subsequent microscopy are adopted. However, the sectioning process may cause damage to materials. X-ray computed tomography (XCT) technology can obtain internal information of materials and has become an established technique for nondestructive analysis ,17,18. I2O3 ceramics. The flaw evolution behaviors under compressive loads were investigated. The influence of different types of flaws on the mechanical behavior is discussed. It is believed that this study may provide guidance for the process optimization and performance enhancement of additively manufactured ceramic materials.In this paper, compression tests with in situ XCT were conducted on additively manufactured Al2O3 ceramics is presented in 2O3 green bodies was reported in a previous work [2O3 coarse grain particles of 50 vol.%  were used as the raw material to prepare Al2O3 ceramic slurry. Al2O3 fine grain particles  were added as fine grains. Al2O3 slurry was dispersed in a pre-photosensitive resin suspension that consisted of photosensitive resin monomers (HDDA and TMPTA), photoinitiator (1 wt% TPO), dispersant (2 wt% KOS110), and sintering additives (3 wt% TiO2 and 1 wt% MgO). The photosensitive slurry was mixed via ball milling at 400 rpm for 24 h. Al2O3 green bodies were then fabricated using stereolithographic additive manufacturing equipment with a 405 nm ultraviolet light projector . The light intensity, exposure time, and slicing thickness were set to 14,000 μw/cm2, 4 s, and 100 μm, respectively. After that, the Al2O3 green bodies were debound at 550 °C for 2 h (a heating rate of 0.5 °C/min) and pre-sintered at 1000 °C for 2 h in a furnace . Finally, the debound bodies were sintered at 1650 °C for 3 h in air in a furnace . Based on the above process, Φ 5 mm × 12.5 mm (diameter × height) sintered specimens that were built with layers in the height direction were prepared for in situ XCT tests. The microstructures of the specimens were observed using a scanning electron microscope .A schematic for the preparation of additively manufactured Alous work . Al2O3 c2O3 ceramics using an in situ X-ray computed tomography apparatus assembled in our laboratory, as shown in In situ compression tests were conducted on additively manufactured Al2O3. The relative density of the sintered specimen was 96.63 ± 0.52%. The layer-by-layer characteristic was observed on the surface of the material in layer-by-layer processing of stereolithography additive manufacturing. It can be seen that the material had a relatively fine-grained microstructure. The grain size in the specimen ranged from 1 to 6 μm. The crystallization of Al2O3 particles was also observed.2O3 specimens.Load and displacement data were recorded during the compression tests. A typical load–displacement curve is shown in 2O3 ceramics, and the flaws evolve with increasing compressive load. At 10,000 N, flaws near the upper surface evolve into large flaws, resulting in the failure of the specimen. To quantitatively analyze the evolution behavior of the flaws during compressive loading, the number and volume of the total flaws at different compressive loads were also obtained, as shown in Three-dimensional volume rendering of total flaws at different compressive loads was obtained by stacking two-dimensional images, as shown in To gain insight into the evolution behavior of flaws with the load, the flaws were classified and analyzed. As mentioned before, flaws mainly include pores and cracks. The sphericities of pores were calculated to distinguish between pores and cracks. Flaws with a sphericity value less than 2.9 were classified as pores, and those with a sphericity value larger than 2.9 were classified as cracks. Three-dimensional volume renderings of the pores at different compressive loads were individually extracted and are shown in z-axis was located along the height direction of the specimen and parallel to the loading direction. Cracks with a maximum size greater than 200 μm in the z-direction were defined as vertical cracks, and those with a maximum size less than 200 μm in the z-direction were defined as transverse cracks. As is widely known, the relationship between crack orientation and loading direction may have an important influence on the crack evolution. Thus, cracks were further subdivided according to crack orientation. A coordinate system was created and is shown in Three-dimensional volume renderings and the number and volume of transverse cracks at different compressive loads were also obtained, as shown in To explain the change in volume with the load, some transverse cracks with a large volume were chosen and extracted separately for analysis, as shown in 2O3 ceramic specimens. Tensile stresses on the tip of vertical cracks induced by compressive loads can lead to specimen failure.Under compressive loads, most pores and transverse cracks tend to be compacted. However, the vertical cracks have an important influence on the failure of additively manufactured Al2O3 ceramic specimens. Four three-dimensional (3D) finite element models, as shown in Finite element analysis was adopted to further investigate the influence of different types of flaws on the mechanical behavior of the additively manufactured AlAlthough the displacement data could be obtained from the sensor of the material testing machine, the measured displacement was the displacement of the crossbeam rather than that of the specimen. Two-dimensional XCT images were used to calculate the true displacement of the specimens, as shown in Load–displacement curves corresponding to the four models were obtained from the finite element analysis, as shown in 2O3 ceramic. We conclude the following:(1)2O3 ceramic specimens. In situ XCT compression tests were conducted on additively manufactured Al2O3 ceramic specimens. Details on the three-dimensional full-field morphology of the specimens at 0, 3000, 6000, and 10,000 N were obtained by stacking two-dimensional XCT images.Stereolithography additive manufacturing technology was used to prepare Al(2)Different types of flaws, including pores, transverse cracks, and vertical cracks, were studied. The number and volume of the pores, transverse cracks, and vertical cracks were analyzed. It was found that the number of flaws tended to decrease with increasing compressive load, resulting from the compaction effect. At 10,000 N, vertical cracks near the upper surface expanded and formed large vertical cracks, leading to the failure of the specimen.(3)Real flaws with morphological and positional information were introduced into the finite element models to study the influence of different types of flaws on the mechanical behavior. The true displacements were obtained by analyzing XCT images. The failure mode obtained from the model including two vertical cracks and one transverse crack was similar to the experimental result. It was found that the influence of vertical cracks on the mechanical behavior was greater than that of transverse cracks. The presence of transverse cracks contributes to the evolution of vertical cracks.In this paper, we investigated the influence of manufacturing flaws on the macroscopic mechanical behavior of additively manufactured Al


Public resources to answer pertinent research questions about the impact of illness and treatment on people with mental health problems are limited. To target funds effectively and efficiently and maximize the health benefits to populations, prioritizing research areas is needed. Research agendas are generally driven by researcher and funder priorities, however, there is growing recognition of the need to include user‐defined research priorities to make research more relevant, needs‐based and efficient.To gain consensus on top priorities for research into early intervention in psychosis through a robust, democratic process for prioritization enlisting the views of key stakeholders including users, carers and healthcare professionals. We also sought to determine which user‐prioritized questions were supported by scientific evidence.We used a modified nominal group technique to gain consensus on unanswered questions that were obtained by survey and ranked at successive stages by a steering group comprising users, carer representatives and clinicians from relevant disciplines and stakeholder bodies. We checked each question posed in the survey was unanswered in research by reviewing evidence in five databases .Two hundred and eighty‐three questions were submitted by 207 people. After checking for relevance, reframing and examining for duplicates, 258 questions remained. We gained consensus on 10 priority questions; these largely represented themes around access and engagement, information needs before and after treatment acceptance, and the influence of service‐user (SU) priorities and beliefs on treatment choices and effectiveness. A recovery SUtheme identified specific self‐management questions and more globally, a need to fully identify factors that impact recovery.Published research findings indicated that the priorities of service users, carers and healthcare professionals were aligned with researchers' and funders' priorities in some areas and misaligned in others providing vital opportunities to develop research agendas that more closely reflect users' needs.Initial results were presented at stakeholder workshops which included service‐users, carers, health professionals and researchers during a consensus workshop to prioritize research questions and allow the opportunity for feedback. Patient and public representatives formed part of the steering group and were consulted regularly during the research process. Research agendas are typically agreed upon at strategic levels by government officials and experts. Few strategic research plans exemplify community participation and the criteria and mechanisms that guide how decisions are made lack transparency.Uncertainty can influence patient outcomes across all areas of decision‐making in mental health including assessing intake acceptance, assigning diagnoses, attributing causal explanations and treatment and care planning.Researchers are beginning to acknowledge that scientifically important questions may not be directly relevant to patientsThe aim of this project was to generate a set of user‐identified research priorities for individuals in the early phase of psychosis. This is a fertile area of research. The development of specialist research units, regional, pan‐continental, and international networks, field‐specific conferences, journals and citation indices evidence this. Some researchers argue that bringing research interest and priorities closer to the need and demand for services is essential to identify and prioritize gaps in knowledge,2Using mixed methods, we systematically identified and prioritized unanswered questions in early psychosis research. We implemented a staged process of surveying to gather priorities, systematic literature searches to determine unanswered questions and a modified nominal group workshop to determine priorities. Our methods were informed by the recommendations of Chalmers et al.2.1http://www.vip.bmh.manchester.ac.uk/).The steering group (VIP‐SG) was established to shape the survey question and design the survey, determine the scope of the project and comment on the processes and procedures for identifying, ranking, and prioritizing uncertainties, agreeing on methods and providing expertise in gaining final consensus. The VIP‐SG comprised service user representatives, clinical representatives, information specialists, leadership and administrative support. Representatives were selected for their involvement with relevant professional groups to represent multiple professional disciplines, user–researcher involvement, and user and carer representation.We identified key themes among relevant policies, guidelines and performance targets as a starting point for discussions around the scope of the project, initially asking the group to consider the appropriateness of the themes and generate further discussion. These included the Mental Health Programme Implementation Guide 2001,2.2Responses to questions were gathered during two phases of the survey and these were delivered online on both occasions. We piloted the survey in the first phase to identify any implementation issues and ensure high‐quality data. The Phase 1 survey was developed by the VIP‐SG adopting a comprehensive approach to generating uncertainties reflecting the wide‐ranging research areas within EI. These included all aspects of illness, recovery, and treatment given that psychotic illnesses are complex, and outcomes of psychosis and subthreshold illnesses framed in a transdiagnostic framework are heterogeneous. Questions were developed in six themes covering community engagement, recovery, treatment refractoriness, effective interventions, EI and physical health improvement . The first author reviewed submissions twice, initially developing indicative questions and on the second review developing and assigning each question to a theme and subtheme which were used to organize the data and identify duplicates. Indicative questions were developed where there were multiple questions with similar meanings and these were assigned red, amber and green classifications based on the degree of confidence held by the researcher regarding how accurately the question retained its intended meaning based on the participants' submission. A consensus meeting between L. R. and R. L. M. was held to determine iterative questions and questions, not in remit which was removed, for example, questions out of scope, philosophical questions not answerable by research and basic research. Iterative questions are determined following an iterative process of refining the questions and the purpose was twofold; we aimed to refine questions to ensure they were researchable and answerable and to ensure that we retained the meaning of each question posed, whether framed in commentary or question, to the greatest extent possible. When respondents asked about NHS service access we considered an effectiveness question to precede this, that is, if a question asked about cognitive behavioural therapy (CBT) access we included a question about whether CBT was effective. Other examples include home treatment, routine physical health screening, public and targeted education, self‐management and supported employment. The iterative process of refining and reformulating questions was based on the agreement between two researchers (L. R. and R. L. M.). See Table 2.4The search strategy was developed by an information specialist (O. S.) consulting with the wider specialist library team at Central Manchester Foundation NHS Trust and the first author (L. R.). For each question a systematic search of the literature was conducted in Medline, EMBASE, Cinahl, PsycINFO, Cochrane Database of Systematic Reviews and the Cochrane Controlled Register of Trials (CENTRAL) where applicable. Specific search strategies were developed for each database and for each question, using a combination of keywords and MeSH/Map terms to include the following: ‘psychosis’, ‘first‐episode psychosis’, ‘early intervention’ and question‐specific keywords. No limits were set on the publication date and level 1 evidence was retrieved comprising systematic reviews, meta‐analyses or qualitative metasyntheses . We included reviews that focused on psychosis and schizophrenia spectrum diagnoses to extract information about first‐episode illnesses if available. Searches were conducted by a team of five information specialists at Manchester Royal Infirmary Library at Central Manchester Foundation Trust and Wythenshawe Academy Library at University Hospital of South Manchester NHS Foundation Trust between February and May 2018. In addition, representatives from VIP‐SG were asked to contribute papers they believed were relevant to answering the questions we obtained. L. R. checked the results and supplemented them with known systematic reviews that were not obtained during searches where available. The search results were analysed and evidence summaries were developed for each question where available and a question was considered to have met the certainty criterion if a systematic review concluded that there was sufficient evidence to answer the question.2.5All questions taken forward to prioritization were ranked in order of the 30 most important questions via an online survey by each VIP‐SG member and these were collated to develop a shortlist of priorities among treatment and illness uncertainties. VIP‐SG members, patients and carers were then recruited to participate in the final prioritization workshop using a modified nominal group technique. Attendees ordered the organization of questions where necessary, voted on their priorities among similarly themed questions and gained consensus on final priorities through small‐group discussions. The final 10 were agreed upon by the entire group during the consensus workshop which comprised service‐users, carers, healthcare professionals, advocates and researchers. Each member was allocated equal voting weight.2.6We adhered to ethical principles contained in organizational policies, but no identifying personal data were obtained and approval by University Research Ethics Committee was not required.33.1Two hundred and seven people answered all or part of the free‐text surveys; there were 80 responses to the Phase 1 survey and 137 responses to the Phase 2 survey. See Table 3.2It took 248 h of librarian time to conduct searches of the databases against the iterative questions comprising over 1000 searches. Relevant systematic reviews were returned in searches for 52 (18.4%) of the iterative questions and the number of available reviews for each question that had a review ranged from 1 to 10 reviews with varying degrees of evidence provided in each review. There were several areas where the evidence was extensive and robust which were also equivocal given the nature of the question posed, for example, no outcome identified, too broad to consider the findings to answer the question wholly or not first‐episode specific. These areas were the effect of lifestyle and diet interventions, the effect of public education campaigns on enhancing awareness, increasing help‐seeking and reducing the duration of untreated psychosis, the effect of family interventions, the effect of interventions to promote occupation, labour force participation, recreation and social network enhancement, prevention of psychosis, tailored interventions to manage weight gain, the effect of psychological interventions and cognitive remediation, influence of cannabis use and causal mechanisms in psychosis. There were reviews that gathered data on schizophrenia, but conclusions could not be drawn about the first episode or the early phase of illness, for example, managing sexual risk and safety, the effect of home‐based treatment and the long‐term effect of novel pharmacological treatments at this stage of illness. There were several qualitative metasyntheses exploring views about medication effectiveness, choice and lifestyle interventions, positive aspects of psychotic experiences, and the role of social support and networks during early psychosis. None of the systematic reviews reported a conclusive answer and we brought forward all questions for prioritization.n = 4), caregivers/family members (n = 4), clinicians/clinical leads (n = 8), policy administrators (n = 1) and educators (n = 2). See Table From 258 priority questions, there were 237 that were endorsed at least once by members of the steering group. Members of the group ranked their top 30 priority questions and once collated, we took forward 42 questions for final prioritization; included questions were endorsed four or more times. The final consensus workshop was attended by service‐users (tors n =  and educ4A final list of 10 priorities was obtained by consensus agreement by stakeholders comprising users of EI services and clinicians and can be considered an important record of user‐defined uncertainties providing intelligence concerning unanswered questions fundamental to the EI paradigm. Knowledge of uncertainties and unanswered questions is a necessary foundation for identifying future directions in research and can support international research efforts towards advancing early psychosis care and treatment. A key uncertainty involved preventing delays and promoting access to treatment and few reviews focused on this despite its centrality to the EI paradigm. Remarkably few studies examined the needs of users for information before gaining access to treatment or school‐based programmes to detect emerging psychosis. Meta‐analytic and narrative reviews emerged evaluating complex, multifocal interventions, discrete professional and patient levels initiatives and different service configurations for shortening delays in accessing care.Prioritizing reducing delays aligns strongly with national research strategy and frameworks recommending a focus on prevention, earlier detection and treatment of mental illnessThe top 10 emphasized an absence of evidence for interventions to enhance community participation and increase users' ability to self‐manage. Reviews obtained in searches largely dealt with recovery using researcher‐defined outcomes.Indeed, a prominent question considered by the consensus group asked specifically about how research funding decisions were made, this question was not retained in the final 10 but captures the essence of this project in that ultimately these priorities will be used by funders and researchers to identify future research priorities and carve out strategic pathways to conduct research of relevance to service users, carers and healthcare professionals. Prioritization is only of value if research follows5This project was a substantial group effort from the VIP‐SG to engage relevant stakeholders in identifying priorities. Targeted activities such as stakeholder review with collaborative networks for applied research, engagement and presentation to user involvement groups, service‐user research groups and charities, and a strong online and social media presence have been a successful strategy eliciting pertinent questions from interested groups. Many questions posed were specific to the field and while relevant, iterative questions tended towards questioning the effectiveness, value and availability of general mental healthcare and treatment.To promote nontokenistic involvement, efforts need to be taken to curb power imbalance and ensure active involvement from service users in mixed stakeholder groups.Lastly, methodologies used to obtain evidence to answer user‐defined questions were predominantly articulated through an experience or values‐based lens before identifying iterative questions. A qualitative inquiry may be best suited to answering these questions but is misaligned with the level 1 evidence that is sought to determine whether there is sufficient evidence in support of particular questions. Among the available level 1 evidence, methods for determining whether a question is answered or not can be subjective and available methodologies require greater scientific consideration as well as an understanding of the necessity for this in user‐defined priorities. In terms of including qualitative evidence, including metasyntheses may mitigate against the mismatch between the available evidence and user‐identified priorities somewhat as there is an increasing acceptance of these synthesis methods in healthcare decision‐making.6Service‐user, carer and healthcare professional defined priorities focused on enhancing evidence for increasing community participation, ability to self‐manage and promoting recovery in daily life. Published research findings indicated that the priorities of service users, carers and healthcare professionals were aligned with researchers and funders in some areas and misaligned in others providing vital opportunities to develop research agendas that more closely reflect users' needs.Laoise Renwick drafted the manuscript. Laoise Renwick was responsible for the organization and preparation of the manuscript. Laoise Renwick was responsible for the concept of the priority‐setting exercise in collaboration with Rebecca Lauren Morris. Olivia Schaff devised the search strategy, and Olivia Schaff and Laoise Renwick at the Trust conducted the searches. Caitlin McWilliams and Susan Ramsdale assisted with project management. Laoise Renwick and Rebecca Lauren Morris were responsible to question development. Laoise Renwick had oversight of the steering committee, ranking and consensus workshop.The authors have no conflicts of interest to declare.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.


Comorbidities in sarcoidosis are common, especially cardiovascular and pulmonary diseases.In the diagnostic workup, a distinction must be made between sarcoidosis-related complaints and complaints caused by other separate disorders. It can be very difficult to distinguish between complications of sarcoidosis and other concomitant conditions.The coexistence of multiple conditions may complicate the diagnosis of sarcoidosis, affect its natural course and response to treatment.Sarcoidosis is a heterogeneous disease, which can affect virtually every body organ, even though lungs and intra thoracic lymph nodes are almost universally affected. The presence of noncaseating granulomas is the histopathological hallmark of the disease, and clinical picture depends on the organs affected. Data about interaction between sarcoidosis and comorbidities, such as cardiovascular and pulmonary diseases, autoimmune disorders, malignancy and drug-related adverse events are limited. Several lung conditions can be associated with sarcoidosis, such as pulmonary hypertension and fibrosis, making it difficult sometimes the differentiation between complications and distinctive pathologies. Their coexistence may complicate the diagnosis of sarcoidosis and contribute to the highly variable and unpredictable natural history, particularly if several diseases are recognised. A thorough assessment of specific disorders that can be associated with sarcoidosis should always be carried out, and future studies will need to evaluate sarcoidosis not only as a single disorder, but also in the light of possible concomitant conditions. Sarcoidosis is a heterogeneous disorder affecting several organs and tissues, although the lungs and intrathoracic lymph nodes are the most common localizations of the disease. The histopathological hallmarks are non-necrotising granulomas that may have some distinctive features from other granulomatous disorders, though in some cases they can be indistinguishable . It is gTo this end, a literature search was conducted by searching public databases .Sarcoidosis AND Lungs OR respiratory system OR pulmonary disease;Sarcoidosis AND Heart OR Cardiovascular system OR Cardiovascular risk OR Cardiovascular disease;Sarcoidosis AND autoimmune system OR autoimmunity;Sarcoidosis AND tumours OR neoplastic disease OR lymphoma OR haematologic disorder;Sarcoidosis AND endocrine system OR thyroid disease OR diabetes mellitus OR diabetes OR obesity OR overweight;Sarcoidosis OR Sarcoidosis-like reaction AND drugs OR therapy OR drug treatment OR cortisone therapy OR glucocorticoids OR methotrexate OR azathioprine OR leflunomide OR biological agents OR tumour necrosis factor-alpha inhibitors.Search queries were:Duplicated papers or papers that were not adherent to the topic of the review were excluded.The exact prevalence of sarcoidosis associated pulmonary hypertension (SAPH) remains uncertain due to significant variability in diagnostic methods and criteria used as well as the populations studied. Overall, PH affects 1–6% of sarcoidosis patients , but it th World Symposium, SAPH is placed in Group 5.2 along with other forms of PH deemed to multifactorial and/or unclear pathophysiologic mechanisms  study) had originally reported a prevalence of 5% of cardiac sarcoidosis (CS) . In the The clinical presentation of CS is highly variable as with all forms of sarcoidosis. It can range from an incidentally ECG or echocardiographic abnormality without any symptoms to heart failure and sudden cardiac death depending on the location of granulomatous inflammation. Advanced atrioventricular block  and ventricular tachycardia are the most common ECG abnormalities, while supraventricular arrhythmias may or may not be related with CS. Sudden cardiac death is well reported in the sarcoidosis population even in patients with preserved left ventricular function. In advanced forms of the disease, heart failure develops from extensive myocardial fibrosis and/or inflammation.The diagnosis of CS is often challenging. The lack of a diagnostic gold standard has been highlighted and several diagnostic guidelines have been published to address this issue . EndomyoThe role of CMR is invaluable for the diagnosis and management of CS. CMR is the gold standard tool in providing an accurate assessment of the myocardial structure and morphology as well as tissue characterisation. The detection of late gadolinium enhancement (LGE) on CMR in specific patterns compatible with CS is considered a hallmark of CS. CMR can also detect edoema. T2- weighted sequencing, which is associated with inflammatory activity, has prognostic implications and can be reflective of disease regression during treatment . CMR is FDG-PET scan is useful to detect the presence of active myocardial inflammation, and is usually performed if CMR shows evidence of cardiac involvement or there is a high degree of clinical suspicion of CS . DespiteUnfortunately, there is no optimal strategy developed to screen the sarcoidosis population for cardiac involvement. The American Thoracic Society clinical practice guidelines recommend that all sarcoidosis patients should be interrogated for cardiac symptoms including palpitations, episodes of syncope and pre-syncope as well as  chest pain at every outpatient clinic visit (if possible) and have a 12-lead ECG at baseline . NonetheManagement of CS is focussing on a) eliminating the existing myocardial inflammation, b) supporting the myocardial function when remodelling has occurred and c) preventing future life-threatening arrhythmias. The 10-year survival in CS was recently reported at 90% . Core meThe association between sarcoidosis and autoimmune disorders has been described in several case reports and in large cohorts studies . ConsideA more recent study on a European cohort of 1737 patients found 16% of patients presenting at least one associated immune-mediated disease, with a frequency almost 2-fold higher than in the general population . This suInterestingly, the above mentioned two large cohorts, of similar size but of Eastern and Western origin, strongly agree in indicating that around one out of six sarcoidosis patient may present an associated immune-mediated complication ,95. WhenIt is interesting to note that the authors of the above cited Spanish study used the term “immune-mediated” rather than autoimmune diseases, including in the list the two most common primary antibody deficiencies of the adulthood, namely Common Variable Immunodeficiency (CVID) and selective IgA deficiency. CVID, in particular, was found as the immune-mediated disease with the highest degree of association with sarcoidosis . The defIn conclusion, the occurrence of systemic granulomatous disease in the context of immunodeficiency, autoimmunity and immune dysregulation, chronic infection-driven immune stimulation, as well as after administration of monoclonal antibodies and small molecule inhibitors during the treatment of other autoimmune diseases deserves further mechanistic investigations, highlighting once more the complexity of the pathogenetic pathways underlying sarcoidosis granulomatous inflammation.An association between sarcoidosis and malignancy has been suspected for a long time. Brincker initially hypothesised the importance of this comorbidity in 1972 by observing an association between sarcoidosis cases and lymphomas . The asset al, who followed more than 500 sarcoidosis patients for a long period and similarly did not find a significantly increased incidence of malignant lymphoma  . M. M154]. Second-line agents, such as MTX, an agent originally intended for antineoplastic use, be used as an alternative to GCs ,164,165.To date, in the treatment of sarcoidosis GCs and MTX are the two most commonly used drugs. Currently, the ongoing PREDMETH study - a randomised controlled trial - aims to investigate the effectiveness and tolerability of MTX compared with prednisone as first-line therapy in patients with pulmonary sarcoidosis . The demAzathioprine (AZA) is a purine antagonist that is often used in the management of sarcoidosis when MTX is contra-indicated, such as in pregnancy, or has failed to induce significant improvement. AZA exerts an anti-inflammatory effect by reducing the proliferation of B- and T-cells. Most common ADRs include infections, gastro-intestinal complaints, hepatic function decline and bone marrow depression, but fever and fatigue may also occur. These latter symptoms can be hard to distinguish from sarcoidosis-associated symptoms, but their rapid resolution following drug discontinuation support an AZA-associated phenomenon. AZA is metabolised by thiopurine S-methyltransferase (TPMT), and patients with low TPMT levels can develop severe neutropenia. TPMT genotyping is therefore advised before starting treatment with AZA to reveal patients susceptible to toxicity .Monoclonal antibodies targeting TNF-α, particularly infliximab and adalimumab, are recommended as third-line treatment. Most commonly reported ADRs include infections. Concomitant use of immunosuppressants such as MTX reduces the risk of neutralising antibodies against TNF-α inhibitors and increases drug efficacy . A studyBeside the treatment of sarcoidosis polypharmacy by itself may have a substantial influence. Thus, drug-induced ADRs may cause additional comorbidities in patients with sarcoidosis . Drug-inBesides rare gastrointestinal (GI) manifestations of sarcoidosis (e.g. from the direct involvement of GI system from sarcoidosis lesions), some studies have indeed demonstrated that sarcoidosis is associated with an increased risk events such as upper GI ulcer and/or haemorrhage and diverticulitis. These manifestations, however, are more likely to be side effects of drugs such as NSAIDs and biologic agents. In particular, long-term use of NSAIDs to control chronic symptoms such as musculoskeletal pain, is associated with cyclooxygenase-1 (COX-1) inhibition, reduction of prostaglandins production and increased hazard of gastroduodenal ulcers. The observation that patients using biologic agents have the highest risk of upper GI events supports the hypothesis that dysregulation of the immune system represents a concomitant disease mechanism .In summary, it is important to consider the option of drug-induced damage, especially as the clinical situation deteriorated after the introduction of a certain drug. Patients should be closely monitored for the development of potential ADRs or drug-induced comorbidities. The variability in drug response among patients including sarcoidosis is multifactorial. Therefore, increased recognition and a high index of suspicion are key to proper diagnosis and prompt withdrawal of the offending drug where the mainstay of treatment is drug cessation. Though, early recognising and managing drug-induced morbidity is a real clinical challenge. A tailored approach that can be adopted in clinical practice, based on the disease severity and risk profiles, is warranted. There is a need to develop trials investigating the role of genomic variations in not only disease susceptibility and predicting prognosis, but also treatment response, and in tailoring drug treatment for individual patients. Comorbidities or ADRs of drugs used in the treatment of sarcoidosis are sometimes very hard to differentiate from symptoms of sarcoidosis itself, which may lead to diagnostic issues.The study of the possible interactions between sarcoidosis and other comorbidities is a field of great interest. The coexistence of multiple conditions complicates the diagnosis of the disease and might be responsible, at least in part, for the highly variable natural history of sarcoidosis, particularly if more than one diseases is present. Therefore, a thorough evaluation of all disorders that may coexist with sarcoidosis should be carried out.In particular, assessment organs at high risk of comorbidities, such as heart and lungs, is highly recommended. In turn, functional assessment of these major organs, if involved, should be performed in order to timely initiate appropriate treatment to mitigate disease manifestations and prevent long-term complications. Attention should also be paid to non-specific organ-related symptoms, such as fatigue and pain, which may be caused by adverse drug reactions rather than sarcoidosis. Future studies on sarcoidosis should specifically focus on the presence of comorbidities. In particular, population-based cohort studies should investigate the risk of comorbidities in patients with sarcoidosis, with the aim to predict the long-term risk of comorbidities and related outcomes.


A humidity sensor is a crucial device in daily life; therefore, in the present study, a novel humidity sensor was designed to increase its specific surface area to improve its humid sensing capacity and conductivity. Titanium dioxide nanoparticles (TiNP) consisting of zero-dimensional nanospheres and one-dimensional nanotubes were prepared by anodic oxidation. Rod-shaped cellulose nanocrystals (CNCs) with average length and diameter of 60 nm and 800 nm, respectively, were obtained by enzymatic hydrolysis and high pressure homogenization. TiNP/CNC composite films exhibited superior hydrophilicity and large specific surface areas based on Fourier transform infrared spectroscopy and nitrogen adsorption–desorption results. The humidity sensing characteristics of sensors based on TiNP/CNC flexible composite films with varying contents of TiNP were investigated under a relative humidity range of 11–97%. The 6% TiNP/CNC-based humidity sensor exhibited high humidity response, rapid response/recovery speed, and high stability. Furthermore, the humidity sensing mechanism of TiNP/CNC composite films was analyzed based on the density functional theory. TiNP/CNC-based humidity sensors could be applied in flexible and wearable electronics. Humidity (water vapor) is one of the key meteorological elements that indicate the amount of water vapor in the air and the degree of wetness. Humidity regulation influences many aspects of daily life, such as human health, living environment, and product storage ,2,3. TheIn general, in addition to humidity sensors possessing excellent humidity sensing properties, flexible humidity sensors fabricated with humidity-sensitive materials, interdigital electrode parameters, and flexible substrates, have a unique role in promoting the development of wearable devices . To achiSome researchers have observed that cellulose generally occurs in the form of cellulose fiber, cellulosic paper, or cellulosic film, which when combined with other materials creates a conductive network for humidity sensors . However2) is one of the well-known semiconductor oxides, and has attracted the attention of researchers due to its remarkable properties, including low cost, non-toxicity, and high stability [2 can be a suitable candidate for humidity sensing due to its high responsive conductivity and hydrophilic properties. Notably, the sensing properties of TiO2 materials largely depend on their morphologies, which in turn, are determined by the preparation methods [2 nanomaterials can vary from quantum dots, nanofibers, nanotubes, nanosheets to nanoflowers, and other nanostructures depending on their various synthesis methods, such as hydrothermal synthesis, template-assisted synthesis, and chemical decomposition, among others [2 nanomaterials with relatively large surface areas have numerous porous sites and exhibit better electron transitions, which implies that they may have superior humidity sensing properties.Although CNCs can act as effective scaffold materials for humidity sensing, the choice of humidity-sensitive materials also directly affects sensor performance. To date, a variety of materials, including porous ceramics, semiconducting metal oxides, polymers, metallic nanowires, and carbon-based nanomaterials, have been explored as humidity sensing materials ,15. Titatability . Further methods . The geog others . TiO2 na2 nanoparticles (TiNP) consisting of zero-dimensional (0-D) nanospheres and one-dimensional (1-D) nanotubes were synthesized by anodic oxidation. They can provide several adsorption channels for H2O molecules and facilitate the rapid adsorption and desorption of H2O molecules. The CNCs were obtained by enzymatic hydrolysis combined with high pressure homogenization. The biodegradable CNC materials were used for the preparation of an integrated humidity sensor to enhance the mechanical properties of the composite material and to further improve the sensing properties of the sensor because of the water molecules adsorption capacity of CNCs. Afterwards, material characterizations of TiNP/CNC composite films were performed using a field emission scanning electron microscope (FESEM), X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, and nitrogen (N2) adsorption–desorption analyses. Furthermore, humidity sensing properties of flexible humidity sensors fabricated using TiNP/CNC composite films at room temperature and RH of 11–97% were investigated. Finally, the humidity sensing mechanism of TiNP/CNC composite films was analyzed based on the density functional theory (DFT).In the present study, multidimensional TiO2 nanotube arrays were initially grown via electrochemical anodization of Ti metal foil (0.1 mm) grown in a glycol solution with 0.55 wt% ammonium fluoride and 20 wt% deionized water at 30 V for 6 h. Subsequently, the anodized Ti foil was annealed at 450 °C for 2 h. The calcined nanotube arrays were then broken down and ground manually to obtain TiNP with residual nanotube fragments.Highly ordered and uniform TiOAfter swelling in 50% aqueous propanetriol solution for 2 h, the cotton pulp was pretreated by enzymatic hydrolysis. Based on a total enzyme concentration of 10 U/mL, with cellulase and xylanase combined at a concentration ratio of 9:1, the pretreated pulp was obtained following hydrolysis at 50 °C for 12 h, followed by successive washing and centrifugation cycles. Afterward, final CNC suspensions were sonicated in a high pressure micro jet homogenizer  at 25,000 psi for 15 min.The aqueous CNC obtained was then diluted with ultrapure water to a concentration of 0.1 wt%. Thereafter, CNC dispersions  were blended with TiNP and dispersed using an ultrasonic disruptor  at 50% amplitude for 5 min.Subsequently, the TiNP/CNC composite films with various ratios were prepared by vacuum-assisted filtration using polyvinylidene fluoride membrane filters with pore sizes of 0.22 μm. After filtration, the formed TiNP/CNC gels were pressed to dry in an ambient laboratory atmosphere and then peeled off from the filter membranes. The TiNP/CNC sensors were developed by processing gold interdigitated electrodes on the composite films based on ion sputtering. The synthesis and fabrication processes of TiNP/CNC-based humidity sensors are illustrated in The surface morphology of the TiNP/CNC nanocomposite films was analyzed using a field emission scanning electron microscope , which was equipped with an energy-dispersive spectrometer for characterizing the chemical composition of TiNP/CNC nanocomposite films. XRD patterns were measured using an XRD spectrometer  with Cu Kα radiation operated at 40 kV and 40 mA in the 2θ range of 10°–80°.−1 region, and was performed using a FTIR spectrometer . The N2 adsorption–desorption experiments were performed using an automatic gas adsorption analyzer , with samples being degassed at 60 °C for 12 h to determine TiNP/CNC nanocomposite film porosity and specific surface area. Specific surface area was calculated using the Brunauer–Emmett–Teller (BET) equation and pore size distribution was calculated based on the Barrett–Joyner–Halenda (BJH) method.FTIR spectroscopy was used to identify functional groups in the TiNP/CNC nanocomposite films in the 4000–800 cm2 (33%), KCO3 (43%), MgNO3 (54%), KI (67%), NaCl (75%), KCl (84%), and KNO3 (97%). To achieve optimized sensing performance, six groups of TiNP concentrations  were used to fabricate the sensors, and the electrical signals were measured using a gas sensing measurement system .The characteristics of TiNP/CNC-based humidity sensors under various humidity conditions were determined by placing them in series of closed bottles with saturated solutions of different salts at room temperature. The various humidity conditions created by saturated solutions of different salts were as follows: LiCl (11%), MgCl0 is the initial resistance of the sensor at 11% RH and R is the final resistance at testing RH level.The response of the TiNP/CNC-based humidity sensor was calculated using Equation (1) as follows:2O) molecules on TiO2 surface, a representative (101) surface of anatase TiO2 thin film was considered [2 (101) surface was modeled by a periodic (2 × 2) slab with two O–Ti–O layers.To explore the adsorption behavior of water  surface has two types of atoms: the topmost atoms are five-fold coordinated Ti atoms  and two-fold coordinated O atoms (O2c), which have the highest probability of reacting with gases [6c) and three-fold coordinated O atoms (O3c).The anatase TiOth gases . The oth23p63d24s2, O 2s22p4, and H 1s, respectively. To calculate the adsorption energy, Eads of H2O molecules on anatase TiO2 (101) surface, the following expression was used:2O) surface, while E(slab) and E2O), (103), (004), (112), (200), (105), (211), and (204) crystal planes of anatase phase of TiO2, respectively (JCPDS-21-1272) [2 and CNCs were not altered after forming a composite.XRD was used to test the crystal structure of the 6% TiNP/CNC composite films. XRD spectra of TiNP/CNC composite films are illustrated in 21-1272) . Moreove21-1272) . Therefo−1 are ascribed to the -OH group absorption peaks, whereas those observed at 2894 cm−1 resulted from C–H stretching in methylene groups [−1. The broad absorption band observed in the 3100–3600 cm−1 region can be attributed to the stretching mode of the -OH group. The -OH group is hydrophilic, and can indicate the capacity of a sample to adsorb H2O molecules [The FTIR spectrum of 6% TiNP/CNC composite film is illustrated in e groups . The chaolecules .2 adsorption–desorption experiments were performed. The N2 adsorption–desorption isotherms and pore size distributions of pure CNC and of 6% TiO2/CNC composite films are illustrated in 2/g and 12.39 m2/g, respectively. The values indicated that the CNC films had considerably low porosity due to CNC aggregation. In contrast, 6% TiNP/CNC composite film exhibited relatively high surface areas, suggesting that TiO2 nanocrystals considerably hindered the stacking of CNC particles. The porous structure of TiO2 nanotubes also increases the specific surface area of the composite films. The 6% TiNP/CNC composite film exhibited type IV nitrogen adsorption isotherms when compared to pure CNCs , short response and recovery times, and high repeatability . TherefoThe humidity sensing capacity of TiNP/CNC composite films was investigated based on a RH range of 11–97%, which was achieved by using various saturated salt solutions at room temperature.2O molecules coming into contact with the composite film structure; in turn, response of the sensors decreases considerably. The highest TiNP/CNC-based humidity sensor response was observed when TiNP content was 6%.The dynamic response curve of the 6% TiNP/CNC-based humidity sensor under 11–97% RH conditions is illustrated in 2O molecules, which are one of the key indicators of the practical application value of a sensor.The response and recovery times for the 6% TiNP/CNC-based humidity sensors to 43% RH are 34 s and 18 s, respectively, as illustrated in 2. It can be seen in 2O molecules through hydrogen bonding; the interwoven network of CNC and multidimensional structure of TiNP gives more channels for H2O molecules.Repeatability and long-term stability are also key parameters of humidity sensors for practical sensing applications. The responses of the 6% TiNP/CNC-based humidity sensors are evaluated in five sensing cycles at 33%, 54%, and 75% RH a. The re2O molecules easily adsorbed on the TiO2/CNC composite film, and the interwoven network and TiNP structure give more channels for H2O molecules. To elucidate the humidity sensing mechanism of TiO2/CNC-based humidity sensors, the interaction between H2O molecules and anatase TiO2 (101) surface was investigated. The optimized geometries of H2O molecules adsorbed on anatase TiO2 (101) surfaces with four adsorption sites are illustrated in CNC is rich in hydroxyl groups, which makes H2O adsorption and surface relaxation, except those at the Ti6c site. The adsorption structure diagram of Ti6c revealed that one of the H atoms split from H2O and diffused to the adjacent bridging oxygen surface, indicating the formation of an O–H bond at the surface [ads), adsorption length (Da) (defined as the length between the gas molecules and the nearest atom on anatase TiO2 (101) surface), bond length (RO-H1 and RO-H2), bond angle (∠H–O–H), and band gap (Eg) are summarized in The atomic structures were almost identical after H surface . The ads2O molecules on the Ti5c site of TiO2 surface was −0.693 eV, which is consistent with the experimental results (−0.72 eV) of previous studies [2O and anatase TiO2 (101) surfaces were characterized by negative Eads values, which suggests that the exothermic process does not require additional energy, with the system becoming more stable after adsorption. Moreover, the adsorption energy value of H2O on the Ti5c site was higher than those on other sites. The adsorption energy of the O3c site was similar (−0.690 eV) to that of the Ti5c site; however, the adsorption bond length of the O3c site (2.896 Å) was significantly longer than that of the Ti5c site (2.313 Å). Shorter bond lengths observed between H2O molecules and anatase TiO2 (101) surfaces imply that there were stronger interactions between H2O molecules and TiO2 surface, and therefore a more stable adsorption conformation. Furthermore, when H2O adsorption at the Ti6c site causes a relatively large relaxation of the TiO2 structure, the Ti–O bond between the O atom below the TiO2 surface and the subsurface Ti atom is broken and the O atom position significantly shifts upward . The band gap of TiO2 decreased to 2.491 eV when H2O molecules were adsorbed on the Ti5c site.The calculated band gap values of TiOe system . The ban2O molecules on the Ti5c site of anatase TiO2 (101) surface at the initial stage. To further reveal the effect of each subsystem on molecular orbital density, projected density of states (PDOS) of TiO2 surface after adsorption of H2O molecules are shown in Therefore, response of the TiNP/CNC-based humidity sensor is stimulated by the chemical adsorption of H2O adsorption. New energy levels are formed in the energy band structure of TiO2 when H2O molecules are adsorbed. The chemisorption process of H2O molecules results in the formation of an adsorption complex ) [ complex b. The adion (3)) ,37.2H2O2O molecule is adsorbed through hydrogen bonding between two neighboring OH groups. The condensed topmost H2O molecules cannot move freely due to restrictions associated with the two hydrogen bonds [3O+) in the H2O layer becomes a challenge, which leads to low conductivity [Subsequently, another Hen bonds . During uctivity . The TiN2O molecules are ionized under an electrostatic field, in turn, generating numerous H3O+ as charge carriers with an increase in humidity levels (Equation (4)) [During the following stage, Hion (4)) .H3O+→H22O molecules on the physisorbed layer, which behaves like a normal liquid. Therefore, the hopping of protons through adjacent H3O+ increases conductivity [2O molecules with OH groups to form a physisorbed layer, where carriers can be transferred easily, in turn, decreasing resistance [2O molecules, which accelerates response speed [Afterwards, increasing humidity levels lead to adsorption of more Huctivity . A furthsistance . In addise speed . Therefo-) on CNC surface, more H2O molecules can be adsorbed on the surface. After the multi-level nanostructured TiO2 is compounded with CNC, spherical TiO2 particles form a porous structure with adjacent CNCs, which has good adsorption capacity and facilitates the formation of a continuous H2O layer, in addition to serving as a direct conduction pathway for the improvement of the sensing membrane conductivity. Tubular TiO2 also provides several adsorption channels for H2O molecules, which is conducive to the rapid adsorption and desorption of H2O molecules. Therefore, TiNP/CNC composite nanostructures enhance humidity sensing properties.Notably, due to the presence of certain hydrophilic functional groups  and 1-D residual nanotubes (average pore diameter of 200 nm) of TiO2O molecules. The band gap, adsorption energy, and PDOS of optimized TiO2 were calculated before and after adsorption of H2O molecules.The humidity sensing characteristics of the TiNP/CNC-based humidity sensors with various TiNP contents (1–12 wt%) were analyzed. The results revealed that the 6% TiNP/CNC-based humidity sensor exhibited remarkable sensing characteristics, including high response in a wide RH detection range (11–97% RH), rapid response and recovery time, as well as high reproducibility and stability. This highlights the advantages of using TiNP/CNC composite films in the fabrication of high quality humidity sensors at room temperature. Furthermore, DFT calculations were performed to determine the sensing mechanism of TiNP/CNC composite films based on interaction with H2O layer and act as a direct conduction pathway for the enhancement of humidity sensing performance of the flexible TiNP/CNC composite films. In addition, TiNP/CNC-based humidity sensors confer the following advantages: flexibility for application in wearable devices, non-toxic for human use, low cost, and simple manufacturing technology, in addition to degradability and non-polluting properties. Therefore, flexible humidity sensors fabricated from TiNP/CNC composite films could be a potential strategy for developing multifunctional humidity sensors due to their high response and wide RH detection range capacities.The nanostructure of the TiNP/CNC composite films enhanced the humidity sensing properties of the sensors. The interlaced CNC and multidimensional TiNP structures have superior adsorption properties, which facilitate the formation of a continuous H