However, it is unknown if the activation of TrkB in PV+ interneurons is important for other plasticity-related behaviors, especially for learning and memory. Here, using mice with heterozygous conditional TrkB deletion in PV+ interneurons (PV-TrkB hCKO) in IntelliCage and fear erasure paradigms, we show that chronic treatment with fluoxetine, a widely prescribed antidepressant drug that is known to promote the activation of TrkB, enhances behavioral flexibility in spatial and fear memory, largely depending on the expression of the TrkB receptor in PV+ interneurons. In addition, hippocampal long-term potentiation was enhanced by chronic treatment with fluoxetine in wild-type mice, but not in PV-TrkB hCKO mice. Transcriptomic analysis of PV+ interneurons after fluoxetine treatment indicated intrinsic changes in synaptic formation and downregulation of enzymes involved in perineuronal net formation. Consistently, immunohistochemistry has shown that the fluoxetine treatment alters PV expression and reduces PNNs in PV+ interneurons, and here we show that TrkB expression in PV+ interneurons is required for these effects. Together, our results provide molecular and network mechanisms for the induction of critical period-like plasticity in adulthood.Critical period-like plasticity (iPlasticity) can be reinstated in the adult brain by several interventions, including drugs and optogenetic modifications. We have demonstrated that a combination of iPlasticity with optimal training improves behaviors related to neuropsychiatric disorders. In this context, the activation of TrkB, a receptor for BDNF, in Parvalbumin-positive (PV Learning and memory dysfunction is a common neuropsychological symptom of neuropsychiatric and neurological diseases. It has been proposed that chronic treatment with antidepressants (ADs) improves impaired learning and memory in animal models –3 via in+) fast-spiking interneurons primarily targeting the perisomatic area of pyramidal neurons [+ fast-spiking basket cells exhibit plasticity by dynamically changing their states in response to recent experience: a state characterized by low PV expression in the PV+ interneurons is involved in plastic networks while a state with high PV-expression in PV cells promotes memory consolidation in the hippocampal CA3 region. This leads to a lower and a higher number of excitatory synaptic inputs onto PV interneurons, respectively, regulating experience-dependent network plasticity [The activation of the brain-derived neurotrophic factor (BDNF) and its receptor TrkB is a key factor in neuronal plasticity. The binding of BDNF to TrkB causes the autophosphorylation of TrkB and leads to the activation of intracellular signaling pathways involved in neuronal differentiation, survival, and growth, as well as synaptic plasticity in neurons , 10. Thi neurons –13. Donaasticity . Furtherasticity , an extrasticity , 17.+ interneurons is necessary and sufficient for iPlasticity in the visual cortex [+ interneurons combined with learning processes can improve learning and memory more generally.We have demonstrated that ADs induce a critical period-like plasticity in the adult brain (iPlasticity), which allows brain networks to better adapt to environmental stimuli, such as training or rehabilitation, and consequently ameliorate neuropsychiatric symptoms , 10, 18.l cortex . Therefo+ interneurons on reversal learning, we treated PV+ interneuron-specific heterozygous TrkB knockout (PV-TrkB hCKO) mice with fluoxetine in the fear extinction paradigm and in IntelliCage apparatus. We also examined the dependency of LTP on the expression of TrkB in PV+ interneurons by studying local field potential activity in the hippocampal CA1 of PV-TrkB hCKO mice. We then performed a transcriptomic analysis specifically for PV+ interneurons using translating ribosome affinity purification (TRAP) after chronic treatment with fluoxetine, and found intrinsic changes in PV+ interneurons, especially in genes related to the formation of PNNs. Finally, we immunohistologically confirmed the plastic changes in PV+ interneurons after chronic treatment with fluoxetine.In order to assess the effects of TrkB in PVDetails of material and methods are provided in the + interneurons , and littermate control mice that normally express TrkB  were produced by mating females from a heterozygous PV-specific Cre line [cre/wt; Pvalb-IRES-Cre, JAX: 008069, Jackson Laboratory) with males from a homozygous floxed TrkB mouse line (TrkBflx/flx) [cre/wt) to obtain the mice (2 months old) expressing GFP-L4 specifically in PV interneurons. The room temperature was kept at 23 ± 2°C, and all mice were kept in a room with a 12-h light/dark cycle (lights on at 6:00 a.m.) with access to food and water ad libitum. All experiments were carried out in accordance with the European Communities Council Directive 86/6609/EEC and the guidelines of the Society for Neuroscience and were approved by the County Administrative Board of Southern Finland (License number: ESAVI/38503/2019).Heterozygous mice with reduced expression of TrkB specifically in PVCre line  (PVcre/wflx/flx)    were croThe fear-conditioning paradigm was conducted following a protocol described previously .IntelliCage  is an automated device that allows housing, performance and measurements of specific tasks in a fully automated manner, removing the need for a human operator and operator-derived bias , 25. We Population spike-free field excitatory postsynaptic currents (fEPSPs) were recorded in an interface chamber using ACSF-filled electrodes (2–4 MΩ) positioned within the CA1 stratum radiatum . Notably3 until cutting on a vibratome . Free-floating sections (40 μm) were processed for fluorescence immunohistochemistry following a protocol described previously [Animals treated with control or fluoxetine were perfused transcardially with PBS followed by 4% PFA in PBS and the brains were isolated. The brains were post-fixed overnight and stored in PBS with 0.02% NaNeviously . The stahttps://fiji.sc/) [+ interneurons subgroups expressing low, intermediate low, intermediate high, and high PV according to the distribution pattern . PV/PNN was imaged in different sections containing CA3b region in the dorsal hippocampus (between −1.94 and −2.18 mm in the anterior-posterior axis relative to Bregma). The multilayer confocal images were stacked , and the fluorescence intensity of PV, PNN and TdTomato were analyzed with Fiji software  (iji.sc/) . We arbi+ interneuron (see above), after 2 weeks of treatment with 0.1% (w/v) saccharine in drinking water (control) or 0.1% (w/v) saccharine and 0.080% (w/v) fluoxetine in drinking water . The isolated hippocampi were stored at −80 °C until the TRAP experiments. After lysating the hippocampus, the tagged ribosomes were precipitated using magnetic beads covered with anti-GFP antibodies. Actively translated mRNA from tagged ribosomes co-precipitated and were sequenced using HiSeq2500  and SMART-Seq v4 Ultra Low Input RNA Kit  for making cDNA library. Primary statistical analysis was carried out with Negative Binomial Distribution with a significance limit of P < 0.05 and the pathway analysis was done from these significant genes using Fisher’s exact test in DAVID with a significance limit of P < 0.1.TRAP analysis was performed according to a previously published protocol . Brieflyt-test are shown in Supplementary Table P value < 0.05 was considered statistically significant.Biochemical and behavioral data were analyzed by two-way ANOVA, taking sessions, genotype, and treatment with fluoxetine, followed by post hoc test. All results of two-way ANOVA and + interneurons thereby reducing the proportion of PV+ interneurons expressing PNN [+ interneurons and would therefore be blunted in PV-TrkB hCKO mice  = 21.94, P < 0.0001; Genotype, F  = 4.049, P < 0.0452; post hoc, wild-type vs PV-TrkB hCKO in Trial 3, P = 0.0037)  = 1.988, P = 0.0288; Ext2, F  = 9.624, P < 0.0001), indicating that the freezing response decreased during the extinction training  = 34.34, P < 0.0001; Ext2, F  = 39.68, P < 0.0001)  = 0.8726, P < 0.0001), but not after day 1  = 0.8743, P = 0.5660). In PV-TrkB hCKO mice, fluoxetine treatment failed to significantly enhance extinction when compared to water-treated mice  = 3.866, P = 0.0502; Ext2, F  = 3.776, P = 0.0530)   = 14.05, P = 0.0003; Sidak’s post hoc test “water-treated vs Fluoxetine-treated” Trial 1, P = 0.0387)  = 4.585, P = 0.0370; Sidak’s post hoc test “water-treated vs Fluoxetine-treated” P = 0.0229)  = 1.876, P = 0.1739)  = 4.585, P = 0.0370, Sidak’s post hoc test “water-treated vs Fluoxetine-treated” for PV-TrkB hCKO P = 0.8618)  = 14.56, P = 0.0002; Sidak’s post hoc test “water-treated vs Fluoxetine-treated” Trial 1, P = 0.0159)  = 7.465, P = 0.0086; Sidak’s post hoc test “water-treated vs Fluoxetine-treated” P = 0.0122)  = 16.61, P < 0.0001, Sidak’s post hoc test “water-treated vs Fluoxetine-treated” Trial 1, P = 0.7996, Trial 2, P = 0.0195, Trial 3, P = 0.0243)  = 7.465, P = 0.0086; Sidak’s post hoc test “water-treated vs Fluoxetine-treated” P = 0.4948)  Fig. . The wiling Fig. . Moreove01) Fig. . Both co30) Fig. . Moreove08) Fig. , suggest87) Fig. , as well29) Fig. . The fre39) Fig. , and in 18) Fig. . In addi59) Fig.  (Treatme22) Fig. . Interes43) Fig. , but the48) Fig. . The absF  = 8,520, P < 0.0001; Treatment, F  = 1.021, P = 0.3133)  = 9.462, P < 0.0001; Treatment, F  = 16.37, P < 0.0001)  = 6.015, P < 0.0001; Genotype, F  = 30.09, P < 0.0001)  Fig. . The PV 33) Fig. , but int01) Fig. . The watF  = 4.212, P = 0.0002)  = 6.794, P = 0.0098)  = 3.550, P = 0.0013) , 02) Fig. , but the98) Fig. . These r13) Fig. . These rF  = 50.20, P < 0.0001). There was, however, no effect of fluoxetine treatment on LTP in hPV-TrkB CKO mice  = 0.2726, P = 0.6019)  Fig. . These r+ interneurons   (Supplementary Table P = 0.0268) and heparan biosynthesis  pathways  were significantly downregulated. These are associated with chondroitin sulfate proteoglycans, which are an integral part of PNNs [P = 0.0384) and glycerophospholipid metabolism  were downregulated. These pathways are involved in the regulation of lipid composition of the cellular membrane, which is highly related to AD effects [P = 0.0863), including G Protein Alpha Inhibiting Activity Polypeptide 3 (Gnai3), G protein subunit gamma 4, 8, and 13 , which are coupled with GABA type B receptor, and mediate slow and prolonged inhibitory action [A) receptors and influences the recycling of the receptor by inhibiting its degradation [A receptors are thought to be a plastic event resulting in the maintenance of the excitatory/inhibitory balance [In order to investigate gene expression in PV interneurons after chronic treatment with fluoxetine, we conducted a TRAP analysis, a system that allows the precipitation mRNA bound to ribosomes, to investigate ongoing protein translation specifically in PVons Fig.   Fig. , and the of PNNs . Also, g effects . Furthery action . Huntingradation . Such mo balance .Fig. 4TR+ interneurons, such as synaptic formation and turnover of PNNs through the regulation of gene expression after fluoxetine treatment.Overall, our TRAP analysis provides insight into the observed phenomena of increased neural plasticity in PVF  = 1.757, P = 0.1934; Genotype, F  = 1.203, P = 0.2801; Fisher’s LSD post hoc test, control vs Flx: wild-type, P = 0.0138; CKO, P = 0.7571)  = 4.785, P = 0.0298; PV intensity, F  = 16.92, P < 0.0001; Treatment, F  = 3.103, P = 0.0796; Fisher’s LSD post hoc test, control vs Flx: Low, P = 0.6365; High, P = 0.0281)  = 1.326, P = 0.2520; PV intensity, F  = 28.88, P < 0.0001; Treatment, F  = 2.564, P = 0.1122; Fisher’s LSD post hoc test, control vs Flx: Low, P = 0.6722; High, P = 0.1075)  Fig. . Interes81) Fig. . However75) Fig. . These r+ interneurons promotes reversal learning in fear and spatial memory. We also showed that both the potentiation of LTP in Schaffer collaterals to CA1 synapses, and the shift in the configuration of the PV and PNN network to a more plastic state require TrkB expression in hippocampal PV+ interneurons. These alterations involve changes in the gene expression patterns related to GABAergic synapses and PNN formation.Here, we demonstrate that iPlasticity induced by pharmacological activation of TrkB in PVIt has previously been shown that heterozygous PV-hTrkB CKO male mice exhibit slightly impaired extinction consolidation but not fear acquisition/conditioning in the auditory fear paradigm . In thisWe show here that fluoxetine treatment decreases cued and contextual fear responses in wild-type mice as previously shown , but not+ interneurons plays a role in the basal level of spatial learning as well as in the reversal learning improvement promoted by fluoxetine. However, we cannot exclude the possibility of the TrkB-independent effects of fluoxetine, acting on spatial memory. For instance, serotonin receptor inhibitors might shed light on possible SSRI effects. More research is needed to elucidate the detailed mechanism of the effect of chronic fluoxetine treatment on spatial memory.To test whether fluoxetine treatment can improve behavioral flexibility or spatial reversal learning through TrkB receptors in PV interneurons, we used the IntelliCage system. IntelliCage is an automated setup that allows behavioral experiments without direct handling of mice except when changing cages and water bottles, which results in higher reproducibility and reliability . Previou+ interneurons, which was also observed in visual cortex plasticity [We found that fluoxetine treatment mainly affects the high PV-expressing PV interneurons, which have been demonstrated to be born earlier during embryonic development and to be responsible for memory formation of recent experiences , 36. Forasticity . These eHippocampal LTP is widely regarded as the cellular substrate underlying learning and memory, enabling plasticity processes to take place . PreviouIn addition to the genes related to the formation of PNNs, we found regulation of genes related to the composition of the cellular membrane, regulatory proteins of GABArgic receptors and genes related to axonal growth guidance.In a previous study from our lab, we observed that the optogenetic activation of TrkB in PV neurons modulates the expression of genes related to GABAergic signaling . This reAmong our data, we also found upregulation of Hap1, which is known to co-localize with TrkB and takes part in the reuptake of BDNF . LiteratMoreover, several studies show how the Trk family is involved in the development of thyroid cancer  and TrkBOverall, the observation of the upregulation of genes involved in TrkB signaling or in processes regulated by TrkB supports the notion that the BDNF/TrkB pathway has indeed been activated by the fluoxetine treatment.+ interneurons responding to environmental stimuli.All significant DE genes related to axon guidance were upregulated. For instance, Sem3D is a receptor of Sema3A, which is known to be localized in PNNs , and Srg+ interneurons [+ neurons in iPlasticity. Importantly, while the activation of TrkB in pyramidal neurons promotes their excitability, in PV+ neurons TrkB activation reduces excitability through the downregulation of Kv3-family potassium channels [+ interneurons does not counteract the concomitant TrkB activation in pyramidal neurons but synergizes with it by disinhibiting pyramidal neurons, thereby orchestrating an enhanced state of cortical plasticity that underlies iPlasticity. Our present data suggest that a similar kind of state of enhanced plasticity, involving reformation of GABAergic signaling and reduction in PNNs, is underlying the effects of fluoxetine on behavioral flexibility and reversal learning.We have recently shown that fluoxetine directly binds to the transmembrane domain of TrkB dimers and increases TrkB retention in the plasma membrane, thereby allosterically promoting BDNF signaling . Furtherrneurons . Taken tchannels . TherefoSupplementary noteSupplementary Table 2Supplementary Table 3Supplementary Table 4


The capacity to stop impending or ongoing actions contributes to executive control over behavior. Action-stopping, however, is difficult to directly quantify. It is therefore assayed via computational modeling of behavior in the stop signal task to estimate the latency of stopping  and, more recently, the reliability of stopping in terms of the distribution of SSRTs  and the frequency with which one outright fails to react to a stop signal . Critically, the validity of computational estimates remains unknown because we currently have no direct readouts of behavior against which to compare them. Here, we developed a method for providing single-trial behavioral readouts of SSRT and trigger failures. The method relies on an adaptation of the stop signal task in which participants respond by moving a computer mouse. In two online experiments, we used movement kinematics to quantify stopping performance , and then applied the standard Race Model and recent BEESTS model in order to examine the convergent validity of the methods. Overall, we demonstrate good correspondence between kinematics- and model-based estimates of stopping performance at the group and individual level. We conclude that the new method provides valid estimates of stopping performance that, unlike model-based estimates, can be read out at the level of single trials. Our approach might therefore be useful for interrogating single-trial neurophysiological correlates of stopping and for large-scale, online studies of behavioral stopping. Action-stopping is an important aspect of executive control, helping to ensure we behave appropriately in a given situation. For example, resisting the urge to swear when angry or reach for a doughnut when dieting. Stopping is typically studied with the stop signal task, in which participants prepare motor responses but are sometimes then cued to stop the impending response. Since successful stopping results in the omission of a response, behavioral stopping cannot be directly observed or quantified. Instead, stopping research relies on a computational model, the Race Model  shorter than Race Model estimates  using movement kinematics, the Race Model and the BEESTS model in order to examine the convergent validity of the methods.https://Prolific.co) and completed the experiments online. The experiments were approved by the UCSD Institutional Review Board.All participants were healthy, adult humans who provided informed consent and were compensated $6.50/hour. They were recruited via an online participant database (r = 0.5 as the minimum meaningful relationship of interest between the estimates of SSRT. We estimated that a sample size of ≥ 23 (difference score) and ≥ 26 (correlation) would be required to detect such effects with an alpha of 0.05 and power of 0.8. We therefore decided to recruit 40 participants, assuming an attrition rate of 25% after filtering data based on performance criteria (see below), to leave us with a final sample size of ~30. Note that in some cases we were predicting a null result , therefore we also computed Bayes factors to enable interpretation of null results, i.e., the strength of evidence for the null hypothesis .No previous study has directly contrasted kinematic- and model-based estimates of stopping performance. Therefore, for the first experiment, we chose a 25-ms difference between estimates of SSRT as the minimum meaningful difference of interest  and TF to enable comparisons at the group level with the same estimates produced by the Race Model and kinematic methods.We report the mean and 95% confidence interval of the population level mean parameters, focusing on the SSRT [μ + τ], SD-SSRT [√ from each performance metric . Under the assumption that our summary average for each individual reflected the ‘true’ value, we asked how many trials would be required to obtain values within an arbitrary ± 5 % of this true value. We ran the simulation 1000 times at each increment of x trials.In order to investigate how many trials are required to obtain reliable measures of stopping performance with our kinematic approach, we ran post hoc simulations on the data. We randomly sampled https://osf.io/d6a92/ ). The code used to run the tasks is also available.We provide the current data set and Matlab code used to perform the main behavioral analyses on the Open Science Framework . However, for output from the BEESTS model we report the posterior mean of the group-level data and the associated 95% credible intervals. Instead of using t tests when contrasting estimates from BEESTS with those produced by the other methods, we evaluated whether or not our Race Model and kinematic estimates fell within the credible intervals associated with the posterior distributions for each variable. This was to account for the fact that there is uncertainty in point estimates from the model  were computed for t tests and correlations, and were interpreted as follows: 1/10-1/3, substantial evidence for the null hypothesis (H0); 1/3-1, anecdotal evidence for H0; 1-3, anecdotal evidence for the alternative hypothesis (H1); 3-10, substantial evidence for H1; 10-30, strong evidence for H1; 30-100, very strong evidence for H1; and >100, extreme evidence for H1. Response times and single-trial SSRTK estimates were considered outliers and removed if they exceeded 1.5 times the inter-quartile range of the first and third quartiles, or if they were <100 ms.Analyses were performed in Matlab 2021a (The Mathworks Ltd.). Most data are reported as mean ± standard deviation and were analyzed using paired Overall, the results from experiments Performance on the primary go task was good, with overall very few omissions, missed targets, false alarms, and choice errors Table . As expeAlthough our primary aim here was to develop direct, single-trial measurements of stopping latency and trigger failures using movement kinematics, we begin by presenting the results of the Race Model analyses. The Race Model forms the backbone of most stopping research but is rarely used to study reaching movements or the stopping of ongoing, as opposed to planned, movements [though see . pStop was close to 50% for both staircases . Stopping latency estimates derived via the Race Model . This implies that the stopping of planned and ongoing actions relies on a common process, an idea that is strongly supported by the very high correlations between home staircase Race Model and kinematic estimates that we come to shortly.The staircasing procedure for the stopping aspect of the task worked well. The different stop performance criteria for the two staircases led to an expected greater SSD for the target versus home staircase , where amplitudes were measured as the maximum resultant displacement of the cursor relative to the point at which the cursor left the home pad.Here we assayed the stopping of ongoing movements shortly after their initiation, and therefore close in time to the planning stage evaluated when applying the Race Model to the same staircase. Despite the trials of interest being classified as failed stops according to the home staircase criterion, individuals still stopped before reaching the target in nearly every trial  of movement and during the early stages of movement execution (SSRTK-H) relies on heavily overlapping processes. Our kinematics-based measurement therefore appears to provide sensible readouts of the latency of stopping and does so on a single-trial basis. This is useful, because it therefore allows a direct estimate of the intra-individual variability in the latency of stopping across trials, which was ~60 ms in both experiments . Movement amplitudes tended to increase as a function of SSD too, consistent with predictions of the Race Model  but differed slightly in experiment t = 15.9, p < 0.001, BF10 > 100, mean difference 32 ±42 ms; Table The pattern of results here was very similar to that of the home staircase. As expected, movement amplitudes were smaller for successful stops of the target staircase compared to go trials . BEESTS produced similar SSRTs to those of the other methods  for each parameter in each data set , and ~240 trials for SD-SSRTK.The total number of stop trials required to obtain a precise estimate of SSRTWe developed a version of the stop signal task that required participants to respond by making reaching movements with a computer mouse and used movement kinematics to provide single-trial readouts of key performance metrics. Despite using a different mode of responding (reaching movements) to standard tasks and assaying the stopping of ongoing (as well as planned) movements, we found that behavior in the task was well explained by the standard Race Model and BEESTS model. Across the two experiments, we found that kinematically derived estimates of SSRT, SD-SSRT, and TF corresponded well with model estimates at the group and individual level, particularly SSRT which showed very strong correlations across the different methods. Overall, we conclude that our approach has good face and convergent validity and offers a range of practical benefits over computational methods of assaying action-stopping.The data were well described by both the Race Model and BEESTS, suggesting that stopping in our task and standard versions of the task share common underlying processes and principles. This is important because both models have typically been employed to study the stopping of planned saccades or finger movements [e.g.  is highly compatible with SSRT measured during the execution phase  [and see  movements can be interrupted during the planning and execution phases [see Fig. Our Race Model-based estimates of SSRT (~330 ms) were broadly consistent with that of previous work utilizing continuous measurement of movement kinetics/kinematics to directly quantify SSRT [~300 ms , and the opposite is true for the target staircase. Additionally, the Race Model only provides a good approximation of ‘true’ SSRT when model assumptions hold, SSRT is constant, go RTs are not heavily skewed, and the trigger failure rate is zero . In fact, most people did not have any trigger failures at all, meaning that most trigger failures at the group level were attributable to a sub-sample of individuals. We speculate that this heterogeneity may be of relevance to individual differences in real-world behavioral control, though this idea remains to be tested.Although BEESTS estimates of trigger failures did not overlap with our kinematic estimate, the absolute values were in fact very close (~0 and ~1%). The reason for the lack of overlap can be explained by two factors. First, the task design afforded relatively few trigger failures overall, particularly compared to the 4-18% trigger failures indicated by BEESTS in the standard button pressing version of the task  and different stop signals in experiment We note that our approach can only be used to estimate trigger failures at short stop signal delays . We assume that the proportion of trigger failures remains constant across all delays, but this may not be true. BEESTS also makes this assumption but, with sufficient data quality could be used to test this assumption.The present method permits precise quantification of stopping performance indices with seemingly few trials: SSRT and TF can be estimated with ~100 trials in total. Admittedly, our approach to estimating measurement precision was somewhat crude, using an arbitrary 5% criterion, and does not speak to the accuracy of the estimates (though this is supported by the similarity with BEESTS estimates). As a point of reference, standard applications of the Race Model require at least 200 trials to produce reliable estimates (Verbruggen et al., A further benefit is that the current method does not require any special equipment to collect data, such as a strain gauge [e.g., (Morein-Zamir et al., A potential downside of studying the stopping of ongoing movements is that biomechanical factors might play a bigger role in the absolute SSRT and SD-SSRT values than for the stopping of planned movements. For example, inertial movements might take some time to ‘brake’ and decelerate, and this could prolong the detection of the stop process in the movement kinematics. Approaches based on changes in acceleration might help to minimize any delay between the onset of the stop process at the level of the muscle and the kinematics. However, even these will be susceptible to factors such as the force-length and force-velocity profiles of the involved agonist-antagonist muscle pairs that undergo coordinated suppression/recruitment (Atsma et al., Biomechanical confounds are also present in simple button pressing tasks (Jana et al., We introduced a novel version of the stop signal task that provides direct readouts of stopping behavior. In addition to the standard performance metric, SSRT, the task also provides estimates of the variability of SSRT and the proportion of trigger failures – measures that are not possible using the standard Race Model approach. These additional descriptors of stopping performance, along with the inter-individual heterogeneity they exhibit, makes this task a good vehicle for future individual differences studies into the psychology of behavioral control. It is possible that although SSRT correlates only poorly with self-reported behavioral control in the literature, the standard deviation of SSRT and trigger failures might fare better. Finally, our approach offers some advantages over the use of model-based estimations of stopping performance since it requires fewer trials to obtain a reliable estimate and offers single-trial readouts of performance.


Idiopathic pulmonary fibrosis (IPF) is a progressive aging-related lung disease with a poor prognosis. Despite extensive research, the cause of IPF remains largely unknown and treatment strategies are limited. Proposed mechanisms of the pathogenesis of IPF are a combination of excessive accumulation of the extracellular matrix and dysfunctional lung tissue regeneration. Epithelial cell dysfunction, in addition to fibroblast activation, is considered a key process in the progression of IPF. Epithelial cells normally maintain homeostasis of the lung tissue through regulated proliferation, differentiation, cell death, and cellular senescence. However, various stresses can cause repetitive damage to lung epithelial cells, leading to dysfunctional regeneration and acquisition of profibrotic functions. The Hippo pathway is a central signaling pathway that maintains tissue homeostasis and plays an essential role in fundamental biological processes. Dysregulation of the Hippo pathway has been implicated in various diseases, including IPF. However, the role of the Hippo pathway in the pathogenesis of IPF remains unclear, particularly given the pathway’s opposing effects on the 2 key pathogenic mechanisms of IPF: epithelial cell dysfunction and fibroblast activation. A deeper understanding of the relationship between the Hippo pathway and the pathogenesis of IPF will pave the way for novel Hippo-targeted therapies.The online version contains supplementary material available at 10.1186/s41232-023-00295-1. Idiopathic pulmonary fibrosis (IPF) is a progressive aging-related lung disease of unknown etiology. Characteristics of IPF include excessive deposition of the extracellular matrix (ECM) and tissue remodeling of the lung, leading to respiratory failure , 2. The The pathogenesis of IPF has 2 critical components: the activation of fibroblasts and the dysfunction of lung epithelial cells . MyofibrThe Hippo pathway is a central signaling pathway for regulating the fundamental functions of epithelial cells, such as proliferation, differentiation, regeneration, and regulated cell death, and contributes to the maintenance of tissue homeostasis . LargelyDrosophila by screening for tumor suppressor genes [Drosophila disc large tumor suppressor [DlgA], and zona occludens 1 [ZO-1])–binding motif (TAZ); and a transcriptional enhancer associate domain (TEAD) family member. Both MST1/2 and LATS1/2 are serine-threonine kinases forming the Hippo kinase cascade. The cascade ends with LATS1/2 directly phosphorylating and inactivating YAP and TAZ (YAP/TAZ) [The Hippo pathway is an evolutionarily conserved signaling pathway, initially identified in or genes . Geneticor genes . Recent or genes . The Hipor genes  Fig. 1)DrosophilYAP/TAZ) . Both YAYAP/TAZ) . In addiYAP/TAZ) . ConversYAP/TAZ) .Fig. 1ThDysregulation of the Hippo pathway is believed to cause various diseases. Perhaps the most notable consequence of dysregulation is cancer progression because the pathway is an important regulator of cell proliferation and appropriate tissue growth. Indeed, in many types of cancer, overactivation of YAP/TAZ is observed and leads to aberrant cell growth evading contact inhibition, increased resistance to apoptosis, epithelial-mesenchymal transition, and the gain of cancer stem cell–like properties . InteresAnother important result of Hippo pathway dysregulation is fibrosis. The Hippo pathway is essential for mechanotransduction, which makes the pathway reasonable to have a significant effect on organ fibrosis or tissue stiffening diseases. Many studies have confirmed that the Hippo pathway is involved in fibroblast activation, the direct pathogenesis of fibrosis, in various organs, including the lung, liver, kidney, heart, and skin . AlthougSHH expression [The expression profile of the components of the Hippo pathway in the lung has been investigated. In adult mouse lungs, YAP mRNA expression level is much higher in basal cells than in ciliated cells or secretory cells . YAP is pression . In normpression . Converspression . On the pression .The pathogenesis of IPF is considered a combination of epithelial cell dysfunction and fibroblast activation. Although fibroblast activation leading to excessive accumulation of ECM is a clear and direct cause of IPF, dysfunctional epithelial cells play a critical role in the initiation and progression of IPF. In normal lungs, tissue homeostasis is maintained by the function of epithelial cells, including proliferation, differentiation, regeneration, cell death, and cellular senescence. These cellular functions are disturbed owing to repetitive cellular damage resulting from both intrinsic and extrinsic stresses, such as loss of proteostasis, endoplasmic reticulum stress, mitochondrial dysfunction, inflammatory cytokines, infection, and cigarette smoke. Dysfunctional epithelial cells are expected to be therapeutic targets for IPF, and their characteristics and pathogenic functions have been intensely investigated.Vβ6 integrin—which suggests their profibrotic functions [CDKN1A, CDKN2A, and MDM2 [Each cell type in the lungs of human patients with IPF has recently had, with the scRNA-seq technique, its transcriptional profiles revealed, and previously undescribed populations of epithelial cells have emerged as key players in the pathogenesis of IPF –19. Seveunctions , 18. In and MDM2 , 18. Anoand MDM2 –35. The In addition to basaloid cells, certain injury-induced populations of alveolar epithelial cells have been reported to appear in IPF lungs. Among them, AT2 cells isolated from IPF lungs have high expressions of cellular senescence-related genes and contribute to progressive pulmonary fibrosis , 16. Mor. The transitions of progenitor cells and differentiated secretory cells to each other are regulated by the YAP expression level. The loss of YAP in basal stem cells causes them to differentiate into secretory cells [SPDEF in the homeostatic state, and YAP/TAZ deletion can lead to goblet cell metaplasia, which is a hallmark of many chronic respiratory diseases including IPF [To maintain lung tissue, appropriate differentiation, cellular proliferation, and regeneration are essential. These cellular functions are closely related to each other and are regulated by the Hippo pathway Fig. . The trary cells . Conversry cells . YAP/TAZding IPF , 38. TheDuring normal turnover and tissue regeneration, important processes are the proliferation of AT2 cells and their differentiation into AT1 cells . InteresCellular senescence is an important biological process characterized by the loss of proliferative capacity. Cellular senescence is induced in response to tissue injury caused by repetitive DNA damage. Accumulation of senescent epithelial cells and myofibroblasts is a key mechanism of the pathogenesis of IPF , 43, 44.As a contradictory counterpart to cellular proliferation, regulated cell death plays a pivotal role in maintaining lung tissue homeostasis. Dysregulation of epithelial cell death led by numerous cellular stresses results in the pathogenesis of IPF , 52. ForFibrosis is composed of excessive deposition of ECM, resulting from a dysregulation of tissue repair , 60. ProPAI1, COL1A1, and CCN2, encoding profibrotic molecules [The proteins YAP/TAZ are believed to induce fibrosis in concert with TGF-β and Wnt signaling, which share a unique feature of regulating transcription via extracellular/cytosolic signaling and nuclear translocation . Signaliolecules . The actolecules .Known to regulate fetal development, Wnt has recently been shown to be involved in the pathogenesis of IPF , 65. In s-coupled GPCR, shows antifibrotic activity by inhibiting YAP/TAZ and decreasing YAP-Smad interaction in human primary lung fibroblasts [In the pathogenesis of IPF, the Hippo pathway and TGF-β signaling synergistically cooperate to promote fibrosis. The fibrogenic cooperation of YAP and TGF-β is regulated by GPCR signaling. Three nonspecific GPCR ligands— lysophosphatidic acid (LPA), sphingosine-1-phosphate (S1P), and thrombin—have been shown to activate YAP/TAZ , 69. In roblasts . The conroblasts . The intMechanosignaling is a major mechanism of fibroblast activation. Mechanical stresses, such as ECM stiffness, and increased cell contractility activate fibroblasts through profibrotic signaling molecules, such as TGF-β and Rho-kinase (ROCK) –76. TypiLung fibrosis is closely related to the Hippo pathway, which is likely to be a therapeutic target. Because the pathogenesis of fibrosis consists of both the convergence of various signaling pathways in the fibroblasts and of complex intercellular communications, fibrosis might be difficult to fully control by modulating only a single pathway. However, being strongly involved in maintaining tissue homeostasis, the Hippo pathway is a promising candidate for a therapeutic target of fibrosis. Fibrosis is considered a result of an imbalance of cellular stress and normal tissue regeneration. The Hippo pathway plays a key role in both aberrant fibroblast activation and benign regeneration. A unique characteristic is that the Hippo pathway mediates mechanosignaling.Treatment targeting the Hippo pathway would certainly have effects on fibrosis. As mentioned earlier, YAP/TAZ signaling has both tissue homeostasis maintenance effects and profibrotic effects, which are apparently conflicting in the causative mechanisms of IPF. Interestingly, the profibrotic effect is at least partly induced by mechanosignaling, and the increased stiffness and YAP/TAZ activation form a positive feedback loop. A reasonable conclusion is that the tissue stiffness-YAP/TAZ feedback loop causes the fibrosis to be sustained and to progress. Therefore, targeting YAP/TAZ can be expected to break the feedback loop and to slow or even stop the fibrosis. To affect the transcriptional regulation by YAP/TAZ, possible therapeutic targets are as follows: upstream molecules that regulate YAP/TAZ activity, activation and nuclear translocation of YAP/TAZ, protein–protein interaction between YAP/TAZ and TEAD, protein-DNA interaction between the YAP/TAZ-TEAD complex and binding DNA, and downstream molecules regulated by YAP/TAZ transcription Fig. . The claTwo compounds, the active metabolite ACT-333679, which is a partial agonist of the IP receptor, and dihydrexidine (DHX), which is a full agonist of dopamine receptor D1 (DRD1), are both GPCR ligands and exert their antifibrotic effects by inhibiting YAP/TAZ nuclear translocation , 83. TheAnother possible novel drug modality is microRNAs (miRNAs). Overexpression of miR-15a alleviates fibrosis in a bleomycin-induced rodent model by reducing the expression of YAP . In thatA possible start of fibrosis is the loss of balance between cellular stress and cell-protective factors. Therefore, a potential preventive therapy is to support YAP/TAZ signaling and to reduce dysfunctional epithelial cells at an early stage. However, despite little evidence, YAP/TAZ show prosenescent effects in specific circumstances –91. The The key pathogenic mechanisms of IPF, a progressive fibrotic lung disease, are dysfunction of epithelial cells and activation of lung fibroblasts . The HipThe Hippo pathway is a potential therapeutic target of IPF, and several agents have been or are being developed. The main target has been the profibrotic effect of YAP/TAZ in fibroblasts; however, targeting the Hippo pathway to enhance its homeostatic effect at an early stage and to inhibit its fibrogenic effect at an advanced stage in epithelial cells might help prevent IPF from starting and advancing (see Additional file Additional file 1. Summary figure. The main points of this review are summarized in the figure. The Hippo pathway opposingly affects the 2 key mechanisms of IPF pathogenesis: dysfunction of epithelial cells and activation of fibroblasts. Excessive ECM accumulation upregulates YAP/TAZ in fibroblasts via mechanosignaling, leading to further activation of fibroblasts and progressive feature of IPF. Supporting the YAP/TAZ activity in epithelial cells at an early stage and suppressing the YAP/TAZ activity in fibroblasts at an advanced stage can be a potential Hippo-targeted therapeutic strategy for IPF.


Under a strain of 40%, the sensor still showed a sensitivity of 42.7 ± 3.1 mV/log [NH4+]. The proposed highly stretchable and robust electrochemical sweat sensor provides a new choice for wearable-device-based personal daily healthcare management beyond hospital-centric healthcare monitoring.Wearable electrochemical sensors have attracted tremendous attention and have been experiencing rapid growth in recent years. Sweat, one of the most suitable biological fluids for non-invasive monitoring, contains various chemical elements relating abundant information about human health conditions. In this work, a new type of non-invasive and highly stretchable potentiometric sweat sensor was developed based on all-solid-state ion-selective electrode (ISE) coupled with poly and polyurethane (PU). This highly stretchable composite of PDMS-PU allows the sensor to be robust, with the PDMS providing a flexible backbone and the PU enhancing the adhesion between the electrodes and the substrate. In addition, graphene–carbon nanotube (CNT) network 3D nanomaterials were introduced to modify the ion selective membrane (ISM) in order to increase the charge transfer activity of the ISEs, which also could minimize the formation of water layers on the electrode surface, as such nanomaterials are highly hydrophobic. As a result, the sensor demonstrated a wide detection range of NH Under a strain of 40%, the sensor still turned out to have a sensitivity of 42.7 ± 3.1 mV/log [NH4+]. We anticipated that this highly flexible and robust sensor could be applied as a personal daily healthcare management solution based on a wearable sensor.In this paper, graphene–carbon nanotube (CNT) network 3D nanomaterials have been introduced to modify the ion-selective membrane (ISM) to increase the charge transfer activity of ISEs. The introduction of the 3D Graphene–CNT network also minimized the formation of a water layer on the electrode surface, as such nanomaterials are highly hydrophobic, which also enhances the accuracy of ammonium detection and long-term stability. In addition, a highly stretchable composite material -polyurethane (PU)) was used as the substrate, in which PDMS provided a flexible backbone while PU enhanced the adhesion between the electrodes and the substrate. As a result, the proposed sensor turned out to have a high sensitivity of 59.6 ± 1.5 mV/log 3−/4− containing 0.1 M NH4Cl solution as redox probes. p) of the bare carbon electrode and the 2D Graphene and 3D Graphene–CNT deposited electrodes, repectively, at a sweep rate of 200 mV/s. The Graphene–CNT modified electrode responded to a higher current value than that of both the bare carbon electrode lectrode a and thelectrode b. Based 4Cl solution. The results of the EIS are illustrated as a Nyquist plot in ctR).The properties of electrodes with different types of modified surfaces were examined by EIS in 0.1 M NH4+, in which the ions move into the ISM phase and electrons travel through the Graphene–CNT layer to reach the electrode. The reduced diameter of the semicircle on the EIS of the 3D Graphene–CNT modified electrode with ISM also indicates a lower ctR compared with that of the bare carbon electrode with the ISM. Thus, a more effective ion-to-electron transduction has been demonstrated using high-surface-area carbon materials, e.g., 3D Graphene–CNT. The EIS results were also fitted and verified by the equivalent circuit model (See ctR—with regards to the 3D Graphene–CNT modified electrode with the ISM (~5.8 × 106 Ω)—was much reduced compared to that of the bare carbon electrode with the ISM (~1.47 × 107 Ω).In odel See , in whic4+ can “size-fit” the complex and combines with the chosen ionophore perfectly. On the contrary, the wall-like ionophores prevent the formation of both wrapping complexes of crown ether with small ions and complexes with large ions.An ISE exhibits selectivity because the chosen ionophore has a much larger free energy of complexation with the specific target ion than with other ions , as show4Cl solution with different concentrations from 10−6 M to 10−1 M, the sensor exhibited an almost ideal Nernstian response, with a sensitivity of 59.6 ± 1.5 mV/log [NH4+]  and an LOD lower than 10−6 M—as depicted in The potentiometric technique was employed to characterize the performance of the sweat sensor in ammonium ion detection. In this detection, both the working electrode and reference electrode were screen printed. The potential difference between the working and reference electrode was recorded using the Open Circuit Potential–Time (OCPT) technique using a CHI600D analyzer. In the tests using the NH4+ using the Graphene–CNT modified electrode, the sensors with the two types of electrodes (with and without the Graphene–CNT coating) were dipped into the same set of NH4Cl solutions for 50 s, at concentrations varying from 10−4 M to 0.1 M. The tests were conducted for two cycles of concentration variation. The potential signal was also recorded by the OCPT technique using the CHI600D analyzer. 4+ concentration. In addition, the potential signal was also not consistent between the two cycles of measurement. As shown in 4+ concentration, with reduced potential variation between cycles. This comparative experiment demonstrated that the sensor with the modified Graphene–CNT electrode achieved higher reversibility, which is one of the critical performance metrics of wearable sensors. Such enhanced reversibility was probably attributed to the hydrophobic properties of the 3D Graphene–CNT layer , in which the output signal looks very stable. 4+. The curves are generally linear, even under the highest strain level of 40% (Min R2 = 0.9108). Under a strain of 40%, the sensor still turned out to have a sensitivity of 42.7 ± 3.1 mV/log [NH4+]. The stretchability test provides evidence that the Graphene–CNT ion-selective membrane is structurally stronger than the bare carbon ion-selective membrane, which could still work stably under a 40% strain—indicating that leakage issues are not significant here.−6 M. Under a strain of 40%, the sensor still turned out to have a sensitivity of 42.7 ± 3.1 mV/log [NH4+]. Additional selectivity studies are critical for the proposed sensor for application in the detection of ammonium in real sweat samples. Thus, our future work is to conduct selectivity tests using samples made of a mixture of NH4+ and other ions in sweat, such as Na+, K+, etc. At the final stage, we shall conduct a test by recruiting volunteers to wear our sensor and detect the NH4+ in their sweat. If further integrated with a wireless communication system, this next-generation sensor could be applied as a key component of a wearable monitoring device used for daily healthcare management.A non-invasive and stretchable potentiometric sweat sensor was proposed to measure the ammonium concentration in human sweat. This wearable sensor was based on highly stretchable composite material substrate of PDMS and PU. With modifications of the 3D nanomaterials of the Graphene–CNT network on the ISEs, the charge transfer activity of the ISEs was enhanced and the water-layer issue was minimized, respectively. This highly stretchable and reliable sensor turned out to have a high sensitivity of 59.6 ± 1.5 mV/log and an LOD lower than 10


Teleogryllus oceanicus to either low or high‐humidity environments while holding temperature constant. We measured water loss and the expression of both pre‐ and postmating reproductive traits. Males exposed to a low‐humidity environment lost more water than males exposed to a high‐humidity environment. A male's cuticular hydrocarbon profile (CHC) did not affect the amount of water lost, and males did not adjust the composition of their CHC profiles in response to hydric conditions. Males exposed to a low‐humidity environment were less likely to produce courtship song or produced songs of low quality. Their spermatophores failed to evacuate and their ejaculates contained sperm of reduced viability. The detrimental effects of low‐humidity on male reproductive traits will compromise male fertility and population persistence. We argue that limits to insect fertility based on temperature alone are likely to underestimate the true effects of climate change on insect persistence and that the explicit incorporation of water regulation into our modeling will yield more accurate predictions of the effects of climate change on insect declines.Global declines in insect abundance are of significant concern. While there is evidence that climate change is contributing to insect declines, we know little of the direct mechanisms responsible for these declines. Male fertility is compromised by increasing temperatures, and the thermal limit to fertility has been implicated as an important factor in the response of insects to climate change. However, climate change is affecting both temperature and hydric conditions, and the effects of water availability on male fertility have rarely been considered. Here we exposed male crickets  The thermal limit to fertility has been implicated as a factor in the response of insects to climate change. However, climate change is also affecting hydric conditions. We demonstrate strong effects of hydration stress on mating behavior and male fertility that will adversely affect insect reproduction under climate change. The stock is reseeded annually with ~50 field‐collected individuals and is housed in a constant temperature room set at 26°C with a relative humidity ~50% and a 12 h:12 h light: dark photoperiod. Male nymphs at the eighth instar were collected from stock culture and held individually in plastic boxes (7 × 7 × 5 cm), provided with cat chow and water ad libitum. Crickets were monitored daily and used in experiments 7–10 days after the molt to adulthood.2.2n = 26). The mean annual 15.00 h relative humidity in Carnarvon, where these crickets were sourced, is 55% (range 52%–59%) . The experimental humidities thereby lie 28% below and 24% above the current normal humidities experienced by these insects in their natural population. Crickets were assigned haphazardly to one of the humidity treatments, and were maintained in their humidity treatments for 48 h before being assayed.The lids of individual cricket containers were removed and replaced with fly screen lids to facilitate free airflow between container and the external environment. Water bottles were removed, and crickets in their containers were placed into one of two humidity‐controlled environments. Crickets within their individual containers were placed inside 149‐L storage boxes, on a wire rack that rested above a tray containing a humidifying agent. Once crickets had been stacked inside these boxes, the boxes were sealed with plastic food wrap before securing the lid. High‐humidity was maintained using a saturated solution of potassium chloride. Low‐humidity was maintained using silicon dioxide (silica gel), which was dried in an oven at 60°C and changed every 2 days. Humidity within the storage boxes was recorded periodically throughout the course of the experiment, which lasted for 68 days: the high‐humidity treatment had a mean (±SE) relative humidity of 79.0 ± 0.2% (range 77%–83%) while the low‐humidity treatment had a mean humidity of 26.7 ± 0.3% (range 25%–31%) , once before and once after exposure to his respective humidity treatment. Thus, before being placed into their treatments, crickets were weighed and then sedated by placing them into a refrigerator at 4°C for 30 min. Crickets were then rubbed the full length of their bodies with a clean SPME fiber (Agilent) for a total of 2 min at a stroke rate of approximately 1 stroke per second; 60 strokes on the ventral surface and 60 strokes on the dorsal surface. The fiber was then emersed and agitated in 1 mL of Hexane (Merck) to dissolve the CHC sample. Samples of CHC were sealed in Chromacol vials (Agilent) and stored at −20°C until analysis. Fibers were washed twice before reuse, by emersion and agitation for 1 min in each of two beakers containing 50 mL of clean Hexane. Forty‐eight hours after being placed into their respective humidity treatments, crickets were reweighed and their CHCs resampled following the procedures outlined above. Crickets were then frozen, dried in an oven at 60°C for 12 h, and their dry weight and pronotum widths determined.Hexane extracts were transferred to new Chromacol vials containing 250 μL glass inserts with polymer feet , the samples dried and then reconstituted to 100 μL. We injected 1 μL of this sample via an Agilent 7693 Autosampler into a gas chromatograph and mass spectrometer (Agilent GCMS 7890B/5977E) operating in the split mode (ratio 2:1) and fitted with an Agilent VF‐WAXms column of 30 m × 0.25 mm internal diameter with 0.5 μm coating . Helium was used as the carrier gas at a flow rate of 1 mL/min. We optimized separation of the extract using a column temperature profile in which the analysis began at a temperature of 150°C for 1 min and rose to 250°C by 25°C/min for 16 min. Final temperature was set at 255°C for 8 min. The MS transfer line, the ion source, and the quad‐pole temperatures were 280, 230, and 150°C, respectively. The certified saturated alkanes standard  was used for concentration calibration and retention index calculation.Agilent Mass Hunter Software was used to acquire and analyze extracts. These were randomized and analyzed in two independent iterations. The mass spectra of unknown compounds were deconvoluted and identified by NIST MS Search 2.4 and AMDIS_32 with the NIST MS database 2020. The retention index was also used to help identify the compounds. Quantitation of metabolite features, peak detection, deconvolution, filtering, scaling, and integration were all processed by Mass Hunter Quantitative Analysis for GCMS (Ver. 7.045.7).x) was used because not all individuals contained every compound. This transformation has been used previously for hydrocarbon data in T. oceanicus  was placed into each male's container such that the container was divided into two equal‐sized sections. A sexually mature virgin female, 7–14 days post adult eclosion, was placed on the opposite side of the partition to the male who was able to contact the female through an array (5 × 10) of holes in the partition, 5 mm in diameter and spaced 5 mm apart. The pair was left for 2 h or until the male began to court the female, at which time his song was recorded. One minute after the onset of courtship singing, males were recorded for 2 min using a Marantz PMD660 Professional digital recorder via a Røde NTG2 condenser microphone. Sampling frequency was 48 Hz and sampling depth 16 bit. All recordings were made in an anechoic room at 26°C and were saved as WAV files.Songs were analyzed using Raven 1.6 (Cornell Laboratory of Ornithology). Recordings were filtered to remove noise at <3.5 and >6 kHz. The courtship song has two elements, a chirp followed by a trill . The evacuating tube was severed with scissors and the ejaculate was allowed to evacuate completely. If the ejaculate did not begin to evacuate within 20 min, the ampulla of the spermatophore was ruptured with forceps to release the ejaculate into the saline. The ejaculate was mixed gently with the saline on the slide. A 5 μL aliquot was then mixed with 5 μL of SYBR 14 (1 mM), diluted 1:50 with Beadle saline and the sample incubated in the dark for 10 min. Then 2 μL of propidium iodide (2.4 mM) was added and incubated in the dark for a further 10 min. The sample was then viewed using a fluorescence microscope, and the number of live (stained green with SYBR 14) and dead (stained red with propidium iodide) sperm in the first 500 sperm counted. The proportion of live sperm in the sample was used as a measure of ejaculate quality.Immediately after they had been recorded, or after 2 h of exposure to their allocated female if they had not produced courtship song, males were checked for the presence of a spermatophore in the genital pouch by gentle squeezing of the abdomen. Males produce a fresh spermatophore at the start of each scotophase Loher,  so that After checking for spermatophores, males were sedated for their second CHC sampling as described above before being frozen. All statistical analyses were conducted in JMP 15.33.129–C35 and represented a measure of the relative abundance of all CHC compounds (Table 31–C33) with both shorter (≤C31) and longer (C35) compounds . CHC profiles both before and after a male was exposed to its humidity treatment were available for 108 males. We putatively identified 29 hydrocarbon compounds in these CHC profiles that ranged in relative abundance from <0.01% to 26.7%  and a marginal within‐subject effect of sampling time . There was no significant interaction effect between treatment and sampling time . Males tended to have higher, though not significantly higher, scores on PC1 48 h after the first sampling  and a statistically significant within‐subject effect of sampling time . There was no significant interaction effect between treatment and sampling time . Males scored higher on PC2 48 h after the first CHC sample was taken  as might be expected. Humidity treatment had a significant effect on water loss  with males in the low‐humidity environment losing more water than those in the high‐humidity environment  and no interaction between PC1 and treatment . Quantitatively similar patterns were found when using PC2 in the model . Identical results were obtained when PC scores from the post‐treatment CHC sample were used.We examined water loss during a male's time spent in the different humidity treatments and the effect of a male's CHC profile on water loss, by entering the change in a male's wet weight over the 48 h period he spent in the humidity treatment, with treatment as the main effect and PC1 (from the pretreatment CHC sample) and dry weight as dependent variables. Heavier males (dry weight) lost more water . Of those surviving, 23 (48.9%) males from the low‐humidity treatment produced courtship song compared with 53 (96.4%) males from the high‐humidity treatment .Only 49 (89%) males survived to be assayed for courtship song in the low‐humidity treatment, while all 55 (100%) males survived in the high‐humidity treatment . Males from the low‐humidity treatment produced songs with positive scores on PC1 and negative scores on PC2, while the reverse was true for males from the high‐humidity treatment . Although there was no significant difference in spermatophore production, spermatophore function was affected by humidity treatment. Only 33 (70%) of the spermatophores produced by males from the low‐humidity treatment evacuated their ejaculate compared with 51 (93%) of the spermatophores produced by males from the high‐humidity treatment . Moreover, the viability of sperm in the ejaculates of males from the low‐humidity treatment was lower  also produced a spermatophore compared with all 55 (100%) males that survived the high‐humidity treatment  with one of two experimentally controlled humidities, either low (RH30%) or high (RH80%). Males from the low‐humidity treatment lost around 30% of their body weight due to water loss, some three times more than males from the high‐humidity treatment. Our manipulation of humidity was, therefore, successful in affecting differences in hydration stress between treatments. The composition of male CHC profiles did not change in response to hydration stress. Males from the low‐humidity treatment were significantly less likely to produce courtship song, and those that did, produced song of lower quality, having a lower duty cycle (sound per unit time) and shorter trill elements. Moreover, although males from the low‐humidity environment were equally likely to produce a spermatophore, their spermatophores were more frequently dysfunctional, failing to ejaculate their contents, and the ejaculate contained a lower proportion of live sperm. The effects of hydration stress on pre‐ and postmating reproductive traits that we have observed are expected to impact directly a male's ability to reproduce.We asked how male reproductive traits, including both premating courtship traits and postmating ejaculate traits, might be affected by hydration stress. Male T. oceanicus show strong mating preferences for males able to deliver energetically costly courtship songs, rejecting the courtship attempts of males delivering songs with a low duty cycle; songs that contain few sound pulses particularly in the trill element of the song, and sound pulses that are both short and delivered slowly are unattractive to females ; data curation ; formal analysis (lead); funding acquisition (lead); investigation (lead); methodology (lead); resources (lead); writing – original draft (lead); writing – review and editing . Maxine Lovegrove: Data curation ; investigation (supporting); writing – review and editing . Melissa Thomas: Data curation (supporting); investigation (supporting); methodology (supporting); writing – review and editing . Xin (Bob) Du: Data curation (supporting); investigation (supporting); methodology (supporting); writing – review and editing . Yonglin Ren: Investigation (supporting); resources (supporting); writing – review and editing .The authors declare no competing interests.


ChatGPT has shown impressive performance in national medical licensing examinations, such as the United States Medical Licensing Examination (USMLE), even passing it with expert-level performance. However, there is a lack of research on its performance in low-income countries’ national licensing medical examinations. In Peru, where almost one out of three examinees fails the national licensing medical examination, ChatGPT has the potential to enhance medical education.We aimed to assess the accuracy of ChatGPT using GPT-3.5 and GPT-4 on the Peruvian National Licensing Medical Examination . Additionally, we sought to identify factors associated with incorrect answers provided by ChatGPT.We used the ENAM 2022 data set, which consisted of 180 multiple-choice questions, to evaluate the performance of ChatGPT. Various prompts were used, and accuracy was evaluated. The performance of ChatGPT was compared to that of a sample of 1025 examinees. Factors such as question type, Peruvian-specific knowledge, discrimination, difficulty, quality of questions, and subject were analyzed to determine their influence on incorrect answers. Questions that received incorrect answers underwent a three-step process involving different prompts to explore the potential impact of adding roles and context on ChatGPT’s accuracy.GPT-4 achieved an accuracy of 86% on the ENAM, followed by GPT-3.5 with 77%. The accuracy obtained by the 1025 examinees was 55%. There was a fair agreement (κ=0.38) between GPT-3.5 and GPT-4. Moderate-to-high-difficulty questions were associated with incorrect answers in the crude and adjusted model for GPT-3.5  and GPT-4 . After reinputting questions that received incorrect answers, GPT-3.5 went from 41 (100%) to 12 (29%) incorrect answers, and GPT-4 from 25 (100%) to 4 (16%).Our study found that ChatGPT (GPT-3.5 and GPT-4) can achieve expert-level performance on the ENAM, outperforming most of our examinees. We found fair agreement between both GPT-3.5 and GPT-4. Incorrect answers were associated with the difficulty of questions, which may resemble human performance. Furthermore, by reinputting questions that initially received incorrect answers with different prompts containing additional roles and context, ChatGPT achieved improved accuracy. ChatGPT (OpenAI), a large language model (LLM) trained with over 175 billion parameters, has gained growing attention owing to its performance in different tasks, including mathematics, economics, and medicine  to passiIn the Peruvian context, low-quality medical education is evidenced by high failure rates 42.8%) in the Peruvian NLME  % in the , leadersENAM is a professional requirement for Peruvian medical doctors and international physicians who aspire to practice medicine within Peruvian borders. Since its introduction in 2003 by the Peruvian Society of Medical Schools, this examination has served as a key evaluation of doctors’ readiness to practice medicine in the country . ENAM isThe passing score on the ENAM is 10.5 on a vigesimal scale (95/180). Over the years, the examination has gained even more significance owing to the regulatory measures that have made it a critical element in the selection process for Rural Service positions . AdditioBearing this in mind, we hypothesized that if ChatGPT can pass the ENAM, it may be used as a medical tutor to enhance medical students’ experience. Thus, in this study, we aimed to assess the accuracy of ChatGPT (GPT-3.5 and GPT-4) on the ENAM and identify factors associated with incorrect answers provided by ChatGPT.Our primary data source was the 2022 ENAM question set obtained directly from the official website of the Peruvian Society of Medical Schools (ASPEFAM) . The datThe 2022 data set was chosen for two main reasons: first, the ENAM blueprint ensures that each examination evaluates the same construct, thereby allowing a single year’s data to be representative; second, since ChatGPT’s training information only covers knowledge up to September 2021, the 2022 data set assures that the selected questions were not part of the model’s training data. Therefore, we assert that our data set selection strategy offers a degree of generalizability to the ENAM. The ENAM 2022 data set is available in We carefully collected the exam questions and divided them into four parts: (1) stem, the main problem or story ; (2) lead-in, the question asked ; (3) response options, the different answers provided for each question; and (4) the correct answer, as given by the exam creators .Two ChatGPT versions were used, namely, GPT-3.5 and GPT-4. Our approach involved the development of three distinct prompts to guide the artificial intelligence (AI) response. To create these prompts, two authors (JAF-C and JG-A) engaged in discussions to ensure they accurately represented the cognitive processes an examinee would typically use when answering a multiple-choice question. After reaching a consensus, we designed a three-step prompt that, to the best of our understanding, mimics this thought process effectively.The prompt was, “Analyze the following question, determine what is being assessed, and provide the correct answer/explanation.” With this prompt, we followed the same process as Kung et al , inputtiOpen-ended prompt: We removed response options, thus providing only the stem and lead-in with the prompt.Multiple-choice question with no justification: We provided the whole question with a stem, lead-in, and response options. In the prompt, we asked only to provide the correct answer with no further explanation.Multiple-choice question with justification: We provided the whole question with stem, lead-in, and response options. In the prompt, we asked for a lengthy explanation.Five of us  entered the questions into ChatGPT. Students received training on how to use ChatGPT through a prerecorded video, and their proficiency was assessed to ensure consistency in the application of prompts. A new chat session was initiated for each question to eliminate any potential memory retention bias. In situations where ChatGPT initially failed to deliver a clear response, we reattempted the question up to three times. The responses were then transferred to a structured Google Spreadsheet for further examination. The first (GPT-3.5) data extraction process was conducted between March 15 and 20, 2023, and the second (GPT-4) was conducted on May 5, 2023.On May 20, 2023, we conducted a second run, which incorporated three prompts following incorrect answers in GPT-3.5 and GPT-4. After providing the question and lead-in without instructions, if an incorrect answer was provided, we asked, “Are you sure? Pretend to be a junior doctor with expertise in clinical practice and exam solving and retry.” If an incorrect answer was provided, the following final prompt was provided: “Are you sure? Re-assess the question and pretend to be a Peruvian junior doctor with expertise in clinical practice and exam solving and retry.”Additionally, we obtained the results of 1025 examinees who took the ENAM as a progress test in a national preparation course. The examinees comprised final-year medical students and medical doctors preparing to undertake the ENAM in 2023. Using this data set, we analyzed questions using classical test theory to calculate the difficulty and discrimination index using the psychometrics package in RStudio . The difficulty index was calculated as a quantitative assessment of the proportion of examinees answering each question correctly, estimating the individual question’s difficulty level. The discrimination index refers to the question’s capacity to differentiate between high and low performers on the overall test . These tThe outcome was the performance of ChatGPT (GPT-3.5 and GPT-4) on the ENAM measured as correct or incorrect answers. We classified answers as correct if the answer provided by both versions matched the official answers provided by ASPEFAM.Independent variables were as follows: (1) type of objective, which was categorized as recall, whenever a question only required factual knowledge, or application, whenever a question required application of knowledge through clinical, therapeutic, communication, or professional decision-making; (2) Peruvian-specific knowledge ; (3) discrimination index; (4) difficulty index; (5) quality of questions; and (6) subject, which was categorized into basic sciences, internal medicine, surgery, obstetrics and gynecology, pediatrics, emergency medicine and critical care, and public health by two physicians with experience in assessing and preparing candidates for the ENAM. Both the discrimination and difficulty indices were calculated using classic test theory for the sample of 1025 examinees. For the discrimination index, we considered the question to provide good discrimination if the index was ≥0.25. For difficulty, questions were classified as hard (<0.30), moderate (0.30-0.70), or easy (>0.70). The quality of questions was measured by JAF-C and JG-A using a 5-point Likert scale with the question, “What is the quality of this question?”. Using this approach, we estimated the overall quality of the questions including the stem, lead-in, and response options using a tool based on the National Board of Medical Examinees’ item writing flaws .We downloaded the data as Microsoft Excel files and exported the data to RStudio for analysis.For descriptive analyses, we used absolute and relative frequencies for categorical variables and measures of central tendency and dispersion for numerical variables.To compare the agreement between GPT-3.5 and GPT-4, we used Cohen κ. To evaluate factors associated with incorrect answers from GPT-3.5 and GPT-4, we used a logistic regression model to calculate the odds ratio (OR) and 95% CI.We used the variance inflation factor (VIF) and Hosmer-Lemeshow test for goodness of fit to assess multicollinearity among predictors. All variables of interest were entered into the multivariable model, and this process was conducted for GPT-3.5 and GPT-4. The predictive accuracy of each version of ChatGPT was assessed using the receiver operating characteristic (ROC), from which we calculated the area under the curve (AUC). The data set and the RStudio script are available in This study adhered to the Helsinki Declaration. No humans were involved during the study. Therefore, evaluation by the ethics committee was not considered necessary.The performance of GPT-4 was 86% (155/180). GPT-3.5 scored 77% (139/180), 73% (133/180), and 60.5% (109/180) for multiple-choice questions with justification, multiple-choice questions with no justification, and open-ended prompts, respectively. The historical performance of the examinees was 54% (97.5/180), and the examinees’ performance in our data set was 55% (99/180). Additionally, we calculated that 7.7% (79/1025) and 22.53% (231/1025) of examinees scored better than GPT-3.5 and GPT-4, respectively, as shown in As shown in We used Cohen κ to assess the agreement between GPT-3.5 and GPT-4; the overall agreement was κ=0.38 . The agrWhen analyzing the odds for incorrect answers on GPT-3.5 and GPT-4, we found that high- and moderate-difficulty questions presented higher odds for incorrect answers in the adjusted model both for GPT-3.5  and GPT-4 , and low-quality questions were associated with correct answers in the GPT-3.5 adjusted model , as shown in Finally, we reinput prompts for incorrect answers following a three-step process, as shown in Here we showed that ChatGPT (GPT-3.5 and GPT-4) can pass the ENAM with expert-level performance. Furthermore, GPT-4 surpassed almost 90% of examinees in our data set with an accuracy of 86.1%, and GPT-3.5 surpassed 80% of examinees with an accuracy of 77.2%. These results are in concordance with the findings of Nori et al . HoweverWhen analyzing differences between the two versions, GPT-4 outperformed GPT-3.5 in almost all areas; however, we observed fair agreement between versions. The agreement was higher for high-difficulty questions, for which both versions failed all questions, and low-difficulty questions, for which both versions answered all questions correctly. These results suggest that the improvement in performance from GPT-3.5 to GPT-4 is due to enhanced reasoning rather than randomness .Although previous studies reported the likelihood of lower accuracy in GPT-3.5 for higher-order problem-solving , we founTo our knowledge, this is the first study to assess the agreement between GPT-3.5 and GPT-4 in the context of medical education and to examine factors linked to incorrect answers. We demonstrated that reformulating incorrect answers by varying prompts and changing roles and contexts improved the accuracy of ChatGPT.However, certain limitations of this study should be considered when interpreting our results. First, our study was confined to the Peruvian medical education system and involved a relatively limited number of questions. Therefore, the results may not be generalizable to other educational settings or a wider range of questions. We recommend future research with larger sample sizes, more diverse examinations, broader question sets, and different factors to identify reasons for wrong answers, such as the date of the questions.Second, while GPT-4 exhibited expert-level performance on the ENAM, this finding must be cautiously interpreted. The competencies required by a medical professional, as defined by frameworks such as CanMEDS or the Accreditation Council for Graduate Medical Education core competencies, extend beyond the confines of a licensing examination. These examinations assess knowledge and its application under controlled conditions, which may differ substantially from real-world clinical scenarios. Furthermore, more valid assessment tools, such as entrustable professional activities, represent the gold standard in medical education. Consequently, despite GPT-4’s promising performance, it is premature to suggest that it could replace human doctors. We encourage additional research to assess the potential use of ChatGPT in different roles or as a supportive tool for medical practitioners.Finally, our study did not evaluate the use of “mega-prompts”—large, intricate prompts detailing specific roles, contexts, and tasks, which might elicit more sophisticated and targeted responses—or other novel methods, such as chain-of-thought prompts  or threeThis study has several implications for both medical education and research on ChatGPT and AI. First, we demonstrated that ChatGPT can pass the ENAM with expert-level performance, surpassing 9 out of 10 examinees. Although our sample does not represent the real score in the ENAM, a previous study [Second, we found that incorrect answers provided by ChatGPT using GPT-3.5 and GPT-4 were associated with question difficulty, which opens further research directions to identify reasons for why ChatGPT fails some questions and inform new directions to understand the behavior of LLMs. Also, to our knowledge, this study is the first to apply psychometrics to ChatGPT, and further studies could explore different theories, such as cognitive diagnostic modeling or other diagnostic classification models with larger data sets, searching for a more in-depth understanding of the reasoning process of ChatGPT.Third, by reinputting incorrectly answered questions and adjusting prompts with more complexity , we found that ChatGPT may perform better. This requires further research on prompt engineering in medical education with tailored prompts for specific tasks, such as the development of assessment tools, curriculum development, communication with patients, or tutoring students. Additionally, tailored LLMs trained with specific and curated medical knowledge are needed for these different applications.Finally, despite the outstanding performance of ChatGPT in the ENAM, as previously stated by Thirunavukarasu , practicOur study found that ChatGPT (GPT-3.5 and GPT-4) can achieve expert-level performance on the ENAM, outperforming most of our examinees. We found fair agreement between both versions. There was an association between high-to-moderate-difficulty questions and wrong answers in both versions of ChatGPT. Furthermore, we observed enhanced performance by reinputting new prompts for incorrectly answered questions and adding roles and context for ChatGPT. Despite the outstanding performance of ChatGPT, we note that being a doctor goes beyond passing a licensing examination.


The study aimed to investigate comorbidities, major adverse respiratory events, and mortality in patients with idiopathic pulmonary fibrosis (IPF). We established an IPF cohort and a comparative cohort matched for sex, age, and the date of IPF diagnosis. We recorded the most frequent comorbidities, the proportions, and time durations to the episode of major adverse respiratory events and death. Both cohorts were followed up to the end of 2016. We included 921 patients in the IPF cohort and 3,677 individuals in the comparative cohort. Comorbidities associated with IPF included pulmonary hypertension, chronic obstructive pulmonary disease, heart failure, asthma, and gastroesophageal reflux disease. The IPF cohort was more likely to have pneumonia (47.6 vs 12.0%), acute respiratory failure (17.8 vs 4.30%), chronic respiratory failure (4.23 vs 0.63%), and death (36.3 vs 15.0%) than the comparative cohort. The time durations to the first episode of pneumonia, acute respiratory failure, chronic respiratory failure, and death were 2.09 ± 2.98, 3.12 ± 3.62, 3.20 ± 4.03, and 3.27 ± 3.03 years in the IPF cohort. In conclusion, patients with IPF had significant comorbidities, particularly pulmonary and cardiovascular comorbidities. The duration from diagnosis to the major adverse respiratory events or death was short. The incidence has been reported to be 5–15 per 100,000 per year and appears to be increasing  owing toAlthough IPF is a disease of unknown etiology, previous studies have identified several associated factors. Genetics, smoking, environmental exposure, microbes, and comorbidities may be associated with the development of IPF. Respiratory and cardiovascular comorbidities may play a major role in the development of IPF –5. Howev22.1We utilized the Longitudinal Health Insurance Database, a subset of the National Health Insurance Research Database in Taiwan, which recruited 2,000,000 persons and contained their detailed medical information between 1996 and 2016. The database was managed and updated by the Ministry of Health and Welfare of Taiwan.Institutional review board statement: The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of China Medical University Hospital (CMUH-107-REC2-181).Informed consent statement: Informed consent was waived due to the data extracted from the National Health Insurance Database, which provides only comprehensive de-identified healthcare information.2.2We established an IPF cohort that included adult patients newly diagnosed with IPF  between 2000 and 2016. Similar methods have been reported elsewhere . The bas2.3t-test was applied to compare mean age in the two cohorts. The estimation of cumulative incidence of death in IPF and comparison cohorts was performed by the Kaplan–Meier method. A log-rank test was utilized to determine the significance. Univariable and multivariable logistic regression analyses were adopted to calculate the crude and adjusted odds ratios (ORs) and 95% confidence intervals (CIs). Furthermore, the Chi-squared test was used to compare the proportions of major adverse respiratory events and death and the Student’s t-test to compare time durations to the first episode of major adverse respiratory events and death between the IPF cohort and the control during the follow-up period. Data analysis for this study was conducted using the SAS statistical software . Statistical significance in this study was set as a p-value <0.05.Chi-squared test was used to examine the proportion distribution of age group, gender, and comorbidity; student’s 3p < 0.001), asthma , pulmonary hypertension , and pulmonary embolism , and non-respiratory comorbidities, including ischemic heart disease , gastroesophageal reflux disease , chronic liver disease , heart failure , and atrial fibrillation , than the comparative cohort.We included 921 patients in the IPF cohort and 3,677 individuals in the comparative cohort during the study period . The meaSimple logistic regression analysis revealed significant comorbidities associated with IPF development, including pulmonary hypertension , COPD , asthma , pulmonary embolism , heart failure , GERD , atrial fibrillation , ischemic heart disease , and chronic liver disease  . Moreovep < 0.001), acute respiratory failure , chronic respiratory failure , and death  than the comparative cohort. The time durations of pneumonia, acute respiratory failure, chronic respiratory failure, and death from the index date to the first episode were 2.09 ± 2.98, 3.12 ± 3.62, 3.20 ± 4.03, and 3.27 ± 3.03 years in the IPF cohort and 4.03 ± 3.90, 4.29 ± 4.09, 4.31 ± 4.11, and 4.33 ± 4.11 years in the comparative cohort, respectively. The IPF cohort demonstrated significantly shorter time durations from baseline to the first episode of pneumonia (p < 0.001), acute respiratory failure (p = 0.007), and death (p < 0.001) than the comparative cohort. The cumulative incidence of mortality was significantly higher in the IPF cohort than in the comparative cohort during the follow-up period  and completed the follow-up. We also set up a well-matched cohort  for comparison. We reported the IPF demographic data  and identified several important baseline comorbidities and showed their proportions. In addition, we identified the higher risk of major adverse respiratory events and death in the IPF cohort compared with the comparative cohort. Further, we found the time durations to the first episode of pneumonia, acute respiratory failure, and death were significantly shorter in the IPF cohort than in the comparative cohort. These results will serve as a good epidemiological reference.In the present study, we established a large-scale IPF cohort . They reported the top ten comorbidities of IPF, including hypertension (53.0%), diabetes mellitus 24.0%), hyperlipidemia (23.5%), coronary heart disease (23.3%), GERD (21.1%), COPD (16.8%), osteoporosis (13.5%), prostatic hyperplasia (12.1%), pulmonary hypertension (11.7%), and arrhythmia (10.6%). In addition, they reported that 5-year survival was 53.7% in patients without comorbidity, whereas it was 48.4, 47.0, 43.8, and 41.1% in patients with 1, 2, 3, and ≥4 comorbidities, respectively [.0%, hypeIPF carries a poor prognosis, with a median survival of 3.8 years among those aged >65 years in the United States . Most paThe strength of this study was the establishment of a large-scale IPF cohort with completed follow-up. A well-matched cohort for comparison was another highlight. Moreover, this study was able to reflect a “real world” scenario, in which IPF, all comorbidities, and the major adverse respiratory events were directly diagnosed during medical consultation. Some limitations of our findings should be considered ,11. Firs5Patients with IPF had several important comorbidities, particularly pulmonary and cardiovascular comorbidities. The duration from diagnosis to the major adverse respiratory events or death was short. Therefore, it is necessary to provide more medical resources to slow down the progression of IPF. In addition, this study may lack more detailed and patient-specific data to comprehensively understand the disease and its associations. We hope that future research can address this limitation.


Suspended graphene film is of great significance for building high-performance electrical devices. However, fabricating large-area suspended graphene film with good mechanical properties is still a challenge, especially for the chemical vapor deposition (CVD)-grown graphene films. In this work, the mechanical properties of suspended CVD-grown graphene film are investigated systematically for the first time. It is found that monolayer graphene film is hard to maintain on circular holes with a diameter of tens of micrometers, which can be improved greatly by increasing the layer of graphene films. The mechanical properties of CVD-grown multilayer graphene films suspended on a circular hole with a diameter of 70 µm can be increased by 20%, and multilayer graphene films prepared by layer-layer stacking process can be increased by up to 400% for the same size. The corresponding mechanism was also discussed in detail, which might pave the way for building high-performance electrical devices based on high-strength suspended graphene film. HowevET, etc. . SuspendET, etc. ,10,11. AET, etc. ,13,14. FET, etc. .Up to now, most studies on suspended graphene are realized via pristine graphene prepared by the mechanical exfoliation method . AlthougIn this work, various suspended graphene films prepared by the CVD method were successfully obtained, including CVD-grown monolayer and multilayer graphene films, and stacked multilayer graphene films. By systematically studying the mechanical properties of as-prepared graphene film, the optimal suspended graphene film is selected and the corresponding mechanism is discussed. The result is very useful for further building high-performance suspended graphene-based electrical devices.2 and 500 sccm Ar at low pressure of 400 Pa. After reaching 1035 °C for 10 min, 30 sccm CH4 was introduced for 15 min to grow monolayer graphene on the surface of the Cu foil. Finally, the Cu foil with graphene on the surface was cooled to room temperature in an atmosphere of 10 sccm H2 and 500 sccm Ar. By increasing the growing pressure to 2000 Pa and extending the growth time to 20 min, three-layer graphene film was prepared. Six-layer graphene was obtained by introducing 20 sccm CH4 and 100 sccm H2 under atmospheric pressure for 10 min.The graphene films are synthesized by the CVD method. Cu foil  was heated to 1035 °C in an atmosphere of 100 standard cubic centimeters per minute (sccm) H2 Cu-based graphene is laminated to the sticky side of the thermal release tape and the other side of the Cu-based graphene was removed using plasma. The samples were then etched in ammonium persulphate ((NH4)2S2O8) solution (0.5 mol/L) to etch the Cu. When the etching process was completed, the sample was slowly immersed in deionized water to remove any residual etchant and finally blown dry with nitrogen gas. The obtained thermal release tape/graphene was laminated onto the new Cu-based graphene to form a thermal release tape/graphene/graphene/Cu structure, and the sample was then heated to 140 °C to make the thermal release tape lose its adhesion, at which point a graphene/graphene/Cu structure was formed, thus completing the stacking of the two graphene films (A 4′4cmne films . The lamThe ultraviolet (UV) lithography process was used to fabricate circular hole arrays with different diameters on Cu foil with graphene films on top. The Cu foil with graphene films on top was firstly spin-coated with a photoresist. After spin coating, the first soft baking was carried out on the dryer to volatilize the solvent and enhance the adhesion between the photoresist and the substrate. The first exposure was carried out after the first soft baking sample cooled down. After the exposure was complete, a second baking was carried out to allow the first exposure area to form a mask, and then the second exposure was carried out this time without a mask plate . At this4)2S2O8 solution (0.5 mol/L) to etch the Cu. When the Cu in the hole array was etched, the sample was placed in deionized water to remove any residual etchant. After natural drying, the sample was placed positively down in acetone to remove photoresist from the graphene surface , cold field emission scanning electron microscopy , polarized optical microscopy , scanning probe microscope .The optical image of the suspended graphene array is shown in ID2G/I) peak ratio of ~2 which demonstrates the nature of monolayer graphene, and the D peak on this curve is negligible, which demonstrates that the corresponding graphene film is low-defect monolayer graphene [ID2G/I peak ratio of ~0.5, which demonstrates the characteristics of multilayer graphene [ID2G/I. These results show that the prepared graphene films have a uniform number of layers, meanwhile, To further characterize the structure of as-prepared suspended graphene film, Raman spectra are measured in detail as shown in graphene ,35,36,37graphene . By furtgraphene ,40. Even−1, moisture and oxygen in the storage environment, as well as doping of the etchant, resulted in a slight shift from the typical G peak of other reported monolayer graphene [−1 without any shift. In comparison, the Raman 2D peak of the CVD-grown 6L graphene film is located at 2696 cm−1 while the D peak is located at 1580 cm−1. The 2D peak position is shifted by 16 cm−1 towards the higher wave number, which is due to the splitting of the electronic band structure of multilayer graphene. It allows the 2D peaks to be fitted as the superposition of multiple Lorentz peaks, while the half-peak width of the 2D peaks gradually increases and shifts towards higher wave numbers as the number of layers increases [−1 towards the lower wave number, and the 2D peak level is shifted by 19 cm−1 towards the lower wave number [graphene ,42. Simincreases ,44. Howee number . This phe number . TherefoThe size of the crystal domains can significantly affect the survival rate of suspended graphene . The aveOn the other hand, stacking multilayer graphene is another method to improve the survival rate. In order to quantitatively study the mechanical properties of suspended graphene film, nanoindentation experiments were carried out on various suspended graphene films with different layers using atomic force microscopy (AFM) ,49. The The summarized results show that the maximum rupture force for a CVD-grown 3L suspended graphene with a diameter of 50 µm reaches 2.46 µN, while the one drops to 1.1 µN when the hole diameter is increased to 200 µm, as shown in Furthermore, it can be observed in The nanoindentation tests are also performed on the suspended stacked graphene film, as shown in To further understand the phenomena observed above, the architecture of CVD-grown graphene film and the corresponding stacked multilayer graphene film is studied in detail. The SEM image in The graphene on the uneven part of the Cu foil will become folds after the etching process. These new folds are stacked on the surface of the film at the same time. When the load is loaded onto a suspended CVD-grown graphene film, the fold would be stretched and the film is then stretched. When the load is removed, the folds are in a post-stretch state, which could not recover its original tension and hence greatly affects the application of graphene film in devices.Folds existing in the stacked graphene film can greatly enhance the mechanical properties of the entire film. During the stacking graphene layers process, the folds on each layer are also maintained. Folds in different layers can intersect each other to form a network. A network of fork-like folds can be clearly observed on the suspended stacked 4L graphene film in In contrast, polycrystalline multilayer graphene films prepared by CVD have line defects and point defects distributed throughout the whole surface. When a load is applied to the film, cracks are easily initialized at the defect. Once a crack has formed, it can expand rapidly and lead to the break of the entire film. As illustrated in In summary, suspended graphene film prepared by the CVD method, including CVD-grown monolayer and multilayer graphene film and stacked multilayer graphene film, were successfully prepared. As shown in the nanoindentation test results, at 70 µm diameter, CVD-grown multilayer graphene films only improve mechanical properties by 20%, while stacked multilayer suspended graphene films can improve them by 400%. Compared to the CVD-grown multilayer graphene directly, layer-by-layer stacking can compensate for defects in monolayer graphene and greatly reduce the probability of defect-induced rupture of suspended graphene. In addition, the inevitable folds generated from the folded dendritic network when stacking CVD-grown graphene film can further enhance the mechanical properties of the stacked suspended graphene. This work significantly improves the mechanical properties of CVD-grown suspended graphene film, which is important for expanding the electrical applications of CVD-grown suspended graphene film.