Tropical cyclones are among the major climatic disasters threatening human survival and development. They are also responsible in part for forest taxonomic composition and dynamics and may lead to catastrophic succession between ecosystems. In this study, we aimed to investigate the extensiveness and severity of the effect caused by Typhoon Hato among the three primary plant communities in Macau, China, including Guia Hill, Taipa Grande, and Ka Ho. The plants' damage was classified into seven categories, ranging from Degree 6, which represents the most severe damage, to Degree 0, which represents almost no damage. The impact of Typhoon Hato was evaluated at different levels, including sample plots, species, DBH, and community structure. Our results show that the sub‐climax community of Guia Hill was most disturbed, with the highest damage index (DI) of 55.28%. Similarly, the Ka Ho shoreline shrub community was also considerably influenced, with a DI of 48.14%. By contrast, the managed secondary forest around Taipa Grande was the least affected, with a DI of 32.66%. Additionally, from the tree layer perspective, the tall trees at Guia Hill canopy layer were directly affected by wind, while the dense understory layer suffered from severe secondary damage due to the fallen trees and branches. For Taipa Grande, the dominant species in the canopy layer were shorter and had less direct damage; the secondary damage was also small as a consequence. Ka Ho had more dwarfed and multibranched species surviving from the sea breeze since Ka Ho was close to the sea. The dense plant structure in Ka Ho protected plants from being easily broken by typhoons, but some twigs and leaves were lost. Some less damaged local species and easily recovered species found in this study could inform the selection of wind‐resistant species for the typhoon‐affected communities. The extensiveness and severity of the effect caused by Typhoon Hato among the three primary plant communities in Macau, China, including Guia Hill, Taipa Grande, and Ka Ho was researched in this study. The sub‐climax community of Guia Hill was most disturbed, the Ka Ho shoreline shrub community was also considerably influenced. By contrast, the managed secondary forest around Taipa Grande was the least affected. The representative pictures of overall views and close‐ups of three plots before and after super Typhoon Hato. Hato was a severe typhoon  and was the most damaging typhoon in terms of strength and economic losses since 1949   (Cadastre Bureau: http://www.dscc.gov.mo/zh‐hans/geo_statistic_web1.html) with a coastline of 76.70 km. It is located on the west coast of the Pearl River Estuary, between 113°31′33″–113°35′43″ E longitude and 22°06′39″–22°13′06″ N latitude. Macau has a subtropical oceanic climate with 2000 mm of annual average precipitation and a 22.6°C annual average temperature from 1981 to 2010. The rainfall is concentrated in spring and summer, with an average annual relative humidity of 78.8%. The typhoon season in Macau is from May to October, with July to September being the peak months. The terrain of Macau is high in the south and low in the north. The highest point is Coloane Alto on Coloane, with an elevation of 170.60 m. Two other main hills are Colina da Guia (90.00 m) on the Macau Peninsula and Taipa Grande (158.20 m) in Taipa   sample area (SA) of the Macau Peninsula, the shoreline shrub community at Ka Ho  on Coloane, and the managed secondary forest of Taipa Grande  on Taipa. For each community, the sample area was divided into 5 m × 5 m sample plots. There were 32 plots on Guia Hill and 16 plots in Ka Ho and Taipa Grande.To investigate the effects on forests caused by this extremely strong typhoon, three representative vegetation types in Macau were selected for study. These comprised the sub‐climax community of Guia Hill, representing, in total, an 800 m2. The slope of the sample area is south by southeast with a gradient of 50°. The upper canopy layer mainly consists of tall tree species like Cinnamomum burmannii, Triadica cochinchinensis, Litsea monopetala, and Ficus microcarpa. The sub‐canopy layer mainly includes medium‐ to small‐sized trees, such as Sterculia lanceolata, Mallotus paniculatus, Syzygium jambos, and Microcos paniculata. The understory is composed of dwarf shrubs and a few herbs. The main species are Psychotria asiatica, Desmos chinensis, F. hirta, Pteris semipinnata, and Alocasia odora. Several species of lianas are also present in the community of Guia Hill, such as Elaeagnus loureiroi, Parthenocissus dalzielii, and Vitis balansana.Guia Hill, situated in the middle part of mainland Macau, is the highest point of the peninsula. The vegetation of Guia Hill comprises a more than 100‐year‐old sub‐climax natural community Table . The Gui2, lower than that of Guia Hill. The slope is to the southeast with a 40° gradient. At Taipa Grande, the canopy is dominated by T. sebiferum, T. cochinchinensis, Schima superba, Homalium cochinchinense, and S. lanceolata. The sub‐canopy mainly consists of S. lanceolata, Schefflera heptaphylla, Ilex asprella, Archidendron lucidum, L. rotundifolia var. oblongifolia, and M. paniculatus. The lower layer mainly consists of P. asiatica, S. lanceolata, Adiantum flabellulatum, and Dicranopteris pedata. There are lianas such as Callerya nitida, Hedyotis hedyotidea, Gymnema sylvestre, Diploclisia glaucescens, and Mussaenda pubescens.Taipa Grande is near the highest point of the Taipa Peninsula, and the sample plot (Taipa Grande in short) is located at 22°09′37.15″ N latitude, 113°34′10.67″ E longitude, at 129 m above sea level. At Taipa Grande, the 50–60‐year‐old secondary forest was crafted by humans and is managed for ecological restoration of landfill sites Table . The tre2, the slope is to the south–south east, and the gradient is 55°. Trees within this plot are prostrate and intertwine with each other. The main tree species constituting the canopy are Acacia confusa and S. kwangtungense. The main sub‐canopy trees are L. rotundifolia var. oblongifolia, S. heptaphylla, Eurya nitida, and M. paniculatus. The understory plants mainly include P. asiatica and Liriope spicata. The main liana species are Embelia laeta, Gymnema sylvestre, Zanthoxylum nitidum, M. pubescens, and Hypserpa nitida. L. rotundifolia var. oblongifolia, and A. confusa, which occur throughout the whole community.The Ka Ho sample plot (Ka Ho in short) is located along the eastern shore of Coloane, at 22°07′45.90″ N latitude and 113°35′29.03″ E longitude, with elevation from sea level to 30 m. The vegetation is composed of ca. 50‐year‐old typical shoreline shrub community Table . The tre2.2Severe Typhoon Hato is the strongest typhoon to affect Macau since 1953. The typhoon Figure . Moreove2.3We measured DBH at 1.3 m from the bases of trees, and the trees with DBH ≥ 1 cm were tagged ; BR: rate of severe damage caused by Hato; degree of damages above Degree 4; DI: Wind damage index; SC: The highest damage grade (Degree 6).We calculated the average damage degree and index for communities or species using the following formulae to indicate typhoon effects  to analyze the difference.DRZ‐Tests through SPSS 22 to indicate the difference in Hato influence between sample plots. The number of plants of each damage degree in each sample plot was weighted before the analysis for an accurate result.The data of all tree damage degrees in Guia Hill, Taipa Grande, and Ka Ho were analyzed with chi‐square Tests and pairwise 2.5To further analyze the damage for plants in different size classes, trees and lianas were divided into eight classes based on DBH and then summarized in three tree layers as follows:n_DR) as followed:Furthermore, to find out the details of the severity of damage in each DBH class, we calculated the DBH damage rate  is a comprehensive quantitative index indicating the status and function of species in the community, using the following formula   Table , indicatts Table  showed tts Table .Our onsite field investigation revealed that the sub‐climax community of Guia Hill suffered the most serious disturbance from Hato Figure . The canAccording to the damage index, Guia Hill was the most affected sample plot. 581 individuals, or 92.08% of all the plants, were affected Table . The DG In Taipa Grande, there were only 22 individuals found in Degrees 4–6. Thus, the BR was 14.77%. We also recorded 18 plants (12.08%) as having no damage (Degree 0) Table .3.2Foster  demonstrT. cochinchinensis (5.7), M. paniculatus (4.4), and C. burmannii (4.1) , Claoxylon indicum (2.4), and S. jambos (3.0), meaning that in general these species were more wind resistant.In Guia Hill, the average damage degree varied with species. Three species had DG > 4.0, which means the whole population was seriously affected by the typhoon, including 1) Table . Not onlI. asprella, but with only a DG of 3.2. Four species had DG under 3.0 (Table The species in Taipa Grande was less injured compared with the species in GuiaHill. The most seriously damaged species was .0 Table .I. asprella) and 3.7 (A.a confusa)  Table .S. lanceolata incurred relatively light damage in all three plots, with 3.4 DG in Guia Hill, 2.8 in Ka Ho, and 1.8 in Taipa Grande  and D. chinensis  in Guia Hill, and P. asiatica  in Ka Ho were the dominant species. These species were also less affected by the typhoon. Although there were individuals in all damage degrees, their DGs were low since their total number of trees was relatively large. The field investigation also found they mainly suffered secondary damage from the fallen trees or branches from the upper canopy.Apart from the main tree species analyzed above, some canopy dominant tree species  and representative species in sub‐canopy and understory layers were not listed in Table 3.3The distribution of plants with different DBH in the tree layers is an important indicator reflecting the stability of community structure  in degree 6, followed by Taipa Grande with 5.37% (8 plants) and Ka Ho with 4.46% (12 plants) Table . We founTo better understand which DBH classes have more degree 6 plants in three plots, degree 6 plant distribution  and tree size distribution  are shown in Figure Guia Hill was the most affected plot since all DBH classes' DBH_DR ranged between 9.65% and 66.67%, which was higher than the max DBH_DR in Taipa Grande (8.33%)  had the highest three DBH_DR  Figure . In Ka HThese data also indicated some different characteristics of degree 6 plants' DBH in the three sample plots. Trees with large diameters (10 cm ≤ DBH ~≥ 30 cm) were more likely to be damaged in Guia Hill, while the seriously damaged trees in Ka Ho had smaller DBH, and serious damages in Taipa Grande were limited to DBH < 7.5 cm. If classifying the DBH classes into tree layers, we could conclude that severe damage to the canopy layer was mainly encountered in Guia Hill and, to a lesser extent, at Ka Ho, while the sub‐canopy layer was most affected in Taipa Grande.In conclusion, Guia Hill community structure was most impacted by Typhoon Hato since almost all DBH classes were damaged, and the large DBH trees were seriously damaged.3.4Our analysis of storm effects on the five most important species at each tree layer indicated some changes in community structure Table .C. burmannii decreased from 42.44 to 36.94 after Hato. Many large DBH individuals in C. burmannii died after the typhoon, reducing both the Breast Area (BA) and number of C. burmannii, leading to the lower IV. In contrast, the IV of T. cochinchinensis increased from 16.32 to 22.19 , with smaller BA individuals in the sub‐canopy or understory layer surviving. In general, the species' IVs and rank did not change much in these two layers in all three sample plots due to high species dominance. For example, in the lower two layers of Guia Hill, C. burmannii and S. lanceolata had a large number of individuals rank in the top 3. Therefore, after the storm effect, these two species IVs still rank in the top 3.Some species in the canopy layer had obvious changes in the following two special situations. One is that there were only a few individuals before the typhoon, and fewer individuals survived afterward, resulting in a significant decline of species' IVs and becoming associated species. One example would be Bridelia tomentosa had one individual in the sub‐canopy layer with DBH of 5.5 cm that died in the typhoon., leaving one individual with 2.1 cm DBH alive in the understory layer.Typhoon Hato also led to the decline or disappearance of some species in these two lower layers. In Guia Hill, 4Severe Typhoon Hato affected the local plant communities in Macau. Wind disturbance varied among three sample plots due to both biotic and abiotic factors. One of the biotic factors influencing differences in wind effects was forest age. Ni et al.  found thCommunity composition and structure also influenced storm effects. Strong winds selectively removed the oldest and tallest trees with large crowns in the forest ; data curation (lead); formal analysis (lead); funding acquisition (lead); investigation (lead); methodology (lead); project administration (lead); resources (lead); software (lead); supervision (lead); validation (lead); visualization (lead); writing – original draft (lead); writing – review and editing (lead). Wen Ye: Investigation ; writing – review and editing . Faguo Wang: Investigation . Fuwu Xing: Conceptualization (supporting). AJ Harris: Writing – review and editing (supporting). Lei Duan: Writing – review and editing (supporting). Hongfeng Chen: Writing – review and editing (supporting).The authors declare no conflicts of interest, including financial and nonfinancial interests.


Worldwide research groups and funding bodies have highlighted the need for imaging biomarkers to predict osteoarthritis (OA) progression and treatment effectiveness. Changes in trabecular architecture, which can be detected with non‐destructive high‐resolution CT imaging, may reveal OA progression before apparent articular surface damage. Here, we analysed the tibial epiphyses of STR/Ort (OA‐prone) and CBA  mice at different ages to characterise the effects of mouse age and strain on multiple bony parameters. We isolated epiphyseal components using a semi‐automated method, and measured the total epiphyseal volume; cortical bone, trabecular bone and marrow space volumes; mean trabecular and cortical bone thicknesses; trabecular volume relative to cortical volume; trabecular volume relative to epiphyseal interior (trabecular BV/TV); and the trabecular degree of anisotropy. Using two‐way ANOVA (significance level ≤0.05), we confirmed that all of these parameters change significantly with age, and that the two strains were significantly different in cortical and trabecular bone volumes, and trabecular degree of anisotropy. STR/Ort mice had higher cortical and trabecular volumes and a lower degree of anisotropy. As the two mouse strains reflect markedly divergent OA predispositions, these parameters have potential as bioimaging markers to monitor OA susceptibility and progression. Additionally, significant age/strain interaction effects were identified for total epiphyseal volume, marrow space volume and trabecular BV/TV. These interactions confirm that the two mouse strains have different epiphyseal growth patterns throughout life, some of which emerge prior to OA onset. Our findings not only propose valuable imaging biomarkers of OA, but also provide insight into ageing 3D epiphyseal architecture bone profiles and skeletal biology underlying the onset and development of age‐related OA in STR/Ort mice. We analysed the tibial epiphyses of osteo‐arthritis prone  and healthy  mice to characterise the effects of mouse age and strain on multiple bony parameters. The two strains were significantly different in cortical and trabecular bone volumes, and trabecular degree of anisotropy; these parameters have potential as bioimaging markers to monitor OA susceptibility and progression. Additionally, the two mouse strains have different epiphyseal growth patterns throughout life, as revealed by significant age/strain interaction effects for total epiphyseal volume, marrow space volume and trabecular BV/TV. Thus, mouse strain represents OA predisposition, and age represents OA progression. All mice were killed using cervical dislocation or rising CO3), cortical volume (mm3), total epiphyseal volume (mm3), marrow space volume  and trabecular BV/TV ). Trabecular and cortical thicknesses (mm) were calculated in Fiji using the thickness plugin in BoneJ2 version 1.11.2  was then performed to clarify these relationships. For those parameters in which significantly different error variances were detected by initial Levene's test, a non‐parametric independent‐samples Kruskal–Wallis test with post hoc pairwise comparisons was performed instead of ANOVA, to identify any statistical differences between the groups shown in Table All parameters were subject to Levene's test in SPSS, with a null hypothesis of homogenous distribution of error variances. A two‐way multivariate ANOVA in SPSS was used to analyse all parameters with non‐significant outcomes, with the two fixed factors defined as mouse strain (either STR/Ort or CBA) and age group . 33.1Following confirmation by eye that semi‐automated segmentations of epiphyses and their components were successful Figure , two‐wayp = 0.369 for strain, p = 0.628 for strain/age interaction). For both strains, mouse trabecular volume relative to cortical volume declined steeply in co‐occurrence with skeletal maturation between 8–11 and 18–20 weeks of age (p < 0.001), and was then maintained in later life, a slight trend towards further decrease being non‐significant (p = 0.069). STR/Ort (OA) and CBA  mice appear indistinguishable from one another in this epiphyseal parameter throughout life , whilst STR/Ort mice only maintained their pre‐existing mean trabecular thickness between the same time points . This possible slight difference between the ageing trajectory of these strains means that it is possible for trabecular thickness to have some utility as a biomarker in future longitudinal OA studies, or cross‐sectional studies with an increased number of early‐life time points. Note that trabecular thickness was the only parameter that did not pass Levene's test; therefore, a Kruskal–Wallis test was used.STR/Ort and CBA mice were also statistically indistinguishable from each other in terms of trabecular thickness at the three age timepoints investigated Table , revealip = 0.040). Although this difference was consistent on average (Table p < 0.001), co‐occurring with the period of skeletal maturation) and no age/strain interaction  than in CBA  mice p = 0.00. Althoun Figure . Cortica3.3p‐values ≤0.001), and anisotropy was significantly greater in CBA than in STR/Ort mice (p = 0.002). The lack of statistical age‐interaction effect indicates that these could be reliable biomarkers by which to differentiate the strains, and thus assess OA probability, throughout life, so long as mouse age is known.STR/Ort mice differed significantly from CBA mice, with no detectable age‐strain interaction, in three probed epiphyseal parameters: cortical volume, trabecular volume, and trabecular degree of anisotropy Table . Corticap < 0.001) and 40+‐week‐old (p < 0.001) mice, the latter two age groups having no significant difference in cortical bone volume from one another , which in turn have near‐significantly greater trabecular volume than 40+‐week‐old mice (p = 0.052). Ageing, thus, is characterised in part by a reduction in the overall trabecular bone volume.Post hoc Tukey's tests Table  confirmep = 0.002), evidently differentiating the two strains most clearly at 18–20 weeks .Trabecular anisotropy was significantly greater in CBA than STR/Ort mice (3.4p = 0.012), epiphyseal marrow space volume  and trabecular BV/TV (p = 0.005). Interaction effects demonstrate that for these parameters, the two strains of mice differ from one another specifically with regard to the way in which these bony features change with age. Full pairwise simple main effects analyses using Fisher's LSD was performed to clarify these relationships.Mouse strain was also identified by ANOVA to have a significant effect in interaction with age for: total epiphyseal volume (p < 0.001), but any differences between the two strains were non‐significant at both 8–11 weeks (p = 0.841) and 40+ weeks (p = 0.416) of age , but this relationship was completely reversed by 18–20 weeks, by which point in the time series profiling the MSV of STR/Ort mice was significantly greater than those of CBA . STR/Ort mice had significantly greater trabecular BV/TV than CBA mice at 8–11 weeks of age (p < 0.001), with no data overlap  and 40+ weeks (p = 0.092)  Figure .4Our profiling of 3D epiphyseal architecture in CBA and STR/Ort mice with semi‐automated segmentation shows that: (i) while ageing affects all epiphyseal parameters, it is revealed as the exclusive factor in determining the ratio between trabecular and cortical bone volumes; (ii) despite exhibiting marked differences in magnitude between strains, the absolute volumes of the cortical and trabecular compartments, as well as anisotropy of the latter, display conserved age‐related behaviours in both strains, and finally (iii) strain differences in the ageing‐related trajectories of total epiphyseal volume, MSV and trabecular ‘BV/TV’ may identify imaging biomarkers of OA. These data yield new perspectives on the potential to identify pre‐symptomatic OA via imaging, and confirm gross anatomical differences between the OA‐prone tibial epiphyses of STR/Ort mice and healthy CBA counterparts. Some differences are present from 8–11 to 18–20 weeks of age, representing pre‐and very early‐stage OA respectively. In one study, almost all male STR/Ort mice showed cartilage lesions on the medial tibial plateau from 16 weeks and 100% had severe OA by 15 months . Lower anisotropy means more three‐dimensionally symmetrical trabeculae. Significantly lower trabecular anisotropy values across all age groups in STR/Ort mice may suggest premature ageing in the trabecular arrangement. Indeed, the trabecular anisotropy values in STR/Ort mice even at 8–11 weeks are similar to the CBA anisotropy values at 40+ weeks  and was not significantly different between the two strains, whereas volume decreased with age and was higher in STR/Ort than CBA. The greater trabecular volume in STR/Ort than CBA mice is therefore not a consequence of greater trabecular thickness. There are several other changes in trabecular architecture that could increase trabecular volume; these changes remain to be investigated in the context of our findings.Our data touch upon the interaction between strain/age, which was confirmed as significant for total epiphyseal volume, MSV and trabecular BV/TV. This is intriguing as the effects of ageing are, crucially, different for STR/Ort and CBA mice. Although total epiphyseal volume was greater in STR/Ort than CBA, this was only transiently true at 18–20 weeks, indicating that this biomarker is only relevant during early OA. The total epiphyseal volume would therefore be a biomarker best used in combination with the aforementioned biomarkers of cortical and trabecular volume, and anisotropy which consistently differentiated STR/Ort OA, or OA‐prone, knees from healthy controls, but failed to specify any defined OA stage. Detecting a significantly higher total epiphyseal volume in addition to OA‐indications in these other biomarkers, then, could be highly useful as a biomarker specifically of early OA. Early‐OA biomarkers have perhaps the greatest potential for clinical impact, as cartilage lesions are impossible to detect radiographically and OA diagnoses conventionally rely on joint space narrowing (JSN) measurements which appear later. Early OA, prior to JSN, is also when targeted locomotory or pre‐emptive pharmaceutical treatments may prevent progression or even halt OA onset. Total epiphyseal volume is also easy to measure as it does not require the segmentation of trabecular and cortical bone necessary for other biomarkers.Trabecular BV/TV differentiates pre‐OA STR/Ort vs age‐matched healthy CBA even earlier . OA patients had higher trabecular BV/TV than healthy subjects in the superior (not inferior) part of the femoral head  and OA. Renault et al.  analysedlower BV/TV with OA (vs controls) in cores of the proximal 6 mm of human tibiae. These discrepancies may rely on methodological differences; we find that age and strain affect trabecular BV/TV which may also explain these differing results.Cox et al.  found thlower in STR/Ort than CBA mice at 3 months with only CBA mice exhibiting further increases with age. These studies also found BV/TV to be higher in femoral epiphysis in STR/Ort relative to CBA mice, with Stok et al. noting that the greater trabecular number in CBA could explain the higher BV/TV tibial epiphysis , then decreased significantly between 18–20 and 40 + weeks . The differences between our study and Stok et al.  may also be due to low power. Additionally, differences between CBA and STR/Ort mice at young ages (e.g. MSV) could feasibly be strain differences unrelated to OA. Research in female STR/Ort (less OA predisposed) may help tease out these relationships, and further studies with larger datasets, or literature meta‐analyses, can build on our study in the future. It is worth noting here that in humans, females are more predisposed to knee OA, especially after the age of 55, when their sex hormones decrease  mice had higher cortical and trabecular volumes and lower anisotropy. OA and normal mice also differed in how total epiphyseal volume, MSV and trabecular BV/TV changed during growth . These parameters provide promising biomarkers for early diagnosis of OA, and for monitoring OA progression and efficacy of treatments. We also characterised the profile of normal skeletal changes in healthy mouse epiphyses that can serve as an important baseline for studies investigating the effects of various pathologies on skeletal structure and growth.E.C.H. designed the study, with contributions from A.A.F., L.A.E.E. and A.A.P; E.C.H. and L.A.E.E. wrote the manuscript and made the figures. L.A.E.E. conducted the statistical analysis. E.C.H., A.A.F. and L.A.E.E. segmented the scans and calculated the 3D metrics. B.J. provided scans for the STR/Ort and CBA mice. E.C.H., A.A.F., L.A.E.E., B.J. and A.A.P. all edited and revised the manuscript and approved submission of this manuscript.This work was funded by the OA Tech+ Network (grant EP/N027264/1) and the Anatomical Society (grant SSD 011018SEAL – v1‐011217) and a Medical Research Council ImagingBioPro Network Proof of Concept Award (MR/R025673/1).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The incidence of persistent AF could be better controlled in the CBA group than in the AAD . CBA could improve the quality of life (QoL) of patients better than AAD . CBA can reduce hospitalization rate significantly than AAD at 36‐month follow‐up . Compared to AAD, CBA as first‐line therapy could reduce the recurrence rate of atrial arrhythmia and incidence of persistent AF and improve QoL in PAF patients with lower incidences of hospitalization.Cryoballoon ablation (CBA) is an effective treatment for drug‐refractory atrial fibrillation (AF) patients. Whether CBA as a first‐line treatment is superior in the rhythm control of AF than antiarrhythmic drugs (AAD) remains unclear. CBA is superior to AAD as initial therapy for rhythm control of paroxysmal atrial fibrillation (PAF). A comprehensive database search was performed in PubMed, Embase, Cochrane, and Web of Science from inception to March 22, 2023. Treatment efficacy was pooled using risk ratio (RR) and standardized mean difference (SMD) with a 95% confidence interval (CI). This study was registered with Prospero (CRD42023401596). Five randomized‐controlled trials involving 923 patients and an observational study were included in this study. The CBA group had a significantly lower overall recurrence rate than the AAD group (CBA vs. AAD: RR = 0.59, 95% CI = 0.49–0.71,  Short‐term and long‐term effects of cryoballoon ablation versus antiarrhythmic drug therapy as first‐line treatment for paroxysmal atrial fibrillation. L. and Y. L.) independently by systematically searching Embase, PubMed, Cochrane Library, and Web of Science from inception to March 22, 2023, using the terms “atrial fibrillation” and “cryoballoon ablation.”2.2(1)Study types included RCTs and observational studies;(2)study participants were adults with PAF, who had a structurally normal heart and had not received daily AAD therapy;(3)interventions included CBA or AAD therapy with at least 12 months of follow‐up; and(4)studies reported at least one of the following outcomes: efficacy, safety, recurrence rate, QoL, and/or the incidence of persistent AF.Included studies met the following criteria:2.3(1)Including participants under the age of 18 or animals;(2)focusing on treatment for persistent or drug‐refractory AF patients;(3)comparing the efficacy and safety between CBA and RFA as the initial therapy; and(4)lacking full texts, such as abstracts from conferences or review articles, or letters to editors.Excluded studies met the following criteria:2.4Data were extracted by two independent investigators (Z. L. and Y. L.), and consistency in the extracted data was verified by a third investigator (H. W.). Any disagreement was resolved through discussion and consensus. The extracted data were verified by the reviewers and duplicates were removed using Endnote X9 . A structured data collection form was used to record the baseline characteristics of the studies including name, year of publication, study design type, and demographics. Additionally, left atrial diameter, left ventricular ejection fraction, and underlying diseases associated with side effects were also recorded, including hypertension, coronary artery disease, previous stroke, and transient ischemic attack. Various outcomes including the recurrence rate, QoL, SAEs, hospitalization, and intervention‐related side effects were also extracted. The quality of RCTs was assessed using the Cochrane risk‐of‐bias tool for randomized trials version 2 (ROB 2)2.52.5.1The primary outcome of this meta‐analysis was the rhythm control of PAF, which included the recurrence rate of any symptomatic and asymptomatic atrial tachyarrhythmias at 1‐year follow‐up and the incidence of persistent AF at 3‐year follow‐up.2.5.2The secondary outcomes included QoL measured by the Atrial Fibrillation Effect on Quality‐of‐Life (AFEQT) questionnaire, hospitalization, intervention‐related side effects, and SAEs. Atrial arrhythmia recurrence was defined as AF, atrial tachycardia, or atrial flutter for ≥30 seconds during ambulatory monitoring or for ≥10 seconds on a 12‐lead electrocardiogram (ECG).2.6I2 < 50% or p ≥ .05)I2 > 50% or p < .05). The sensitivity analysis was performed to determine the robustness of the results. Outcomes of interest were analyzed using StataSE 15. A p < .05 was considered statistically significant.The efficacy of treatment was evaluated using risk ratio (RR), standardized mean difference (SMD), and 95% confidence interval (CI). A fixed effects model was used when heterogeneity was low (33.1n = 1599), Embase (n = 4089), Cochrane Library (n = 447), and Web of Science (n = 2540), and 3605 duplicates were removed. After screening the title and abstract, the full texts of the remaining records were reviewed to select studies that met the eligibility criteria. A final total of 5 eligible RCTs involving 928 participants (467 patients in the first‐line CBA group and 461 patients in the AAD group) were included in this meta‐analysisA total of 8675 potential records were retrieved from PubMed n = 599, Emba3.2n = 2), China (n = 1), the United States (n = 1), and Germany (n = 1). There were 467 patients in the CBA group and 461 patients in the AAD group. It is worth noting that PROGRESSIVE‐AF included the same patients as EARLY‐AF, but the former focused on the incidence of persistent AF after intervention, which is in accordance with the Chinese study.The baseline characteristics of the five included RCTs are listed in Supporting Information: Table 3.3We used the risk of bias tool RoB 2 (revised version 2019)3.43.4.1n = 724) reported the recurrence rate of any atrial tachyarrhythmia  at the 1‐year follow‐up. The CBA group had a significantly lower overall recurrence rate compared with the AAD group  .Three RCTsn = 507) reported the incidence of persistent AF at 3 years. A significantly lower incidence of persistent AF was observed in the CBA group than in the AAD group  .Two RCTs3.4.2n = 724) evaluated the QoL of PAF patients using the AFEQT questionnaire.p < .05) .Three RCTs n = 72 evaluatep < .05) p < .05)SAEs were assessed for each patient in all five RCTs.Other adverse events affecting the heart, lungs, and gastrointestinal tract were also reported in the RCTs. Meta‐analysis showed that the CBA group had a significantly lower incidence of heart adverse events at the 1‐year follow‐up . However, the subsequent DECAAF II trial found no significant difference in recurrence rate between the CA and CA plus magnetic resonance imaging‐guided fibrosis ablation groups,AF is a progressive disease that can be life‐threatening. Left atrial fibrosis has been shown to play a key role in the pathophysiology of AF,To the best of our knowledge, this is the first systematic review and meta‐analysis to comprehensively evaluate the short‐term and long‐term effects of first‐line CBA on PAF rhythm control. It is important because 3%–5% of patients who had successful CA eventually relapsed in a year.Our work also indicated that the incidences of SAEs were similar between the CBA and AAD groups at both the 1‐ and‐3‐year follow‐up, which further confirmed the safety of first‐line CBA. This outcome is also consistent with the FREEZE study,4.1There are several limitations in this systematic review and meta‐analysis. First, despite our systematic and comprehensive review, only a few studies were included in our meta‐analysis and hence publication bias could not be assessed. Second, all RCTs compared the efficacy of surgical and pharmaceutical treatments, rendering blinding impractical. This may result in an overestimation of the benefits of ablation. However, all experiments were conducted with concealed allocation to ensure rigorous research quality. Third, using intermittent ECG monitoring or symptoms in Ding's trial may have underestimated the incidence of persistent AF in long‐term follow‐up. Last, the rate of loss to follow‐up is also an inevitable problem that will need to be tackled in subsequent trials. Nonetheless, further high‐quality studies are needed to address these limitations and verify our findings.5Compared to AADs, first‐line CBA can effectively prevent atrial arrhythmia recurrence and lower the incidence of persistent AF. Furthermore, this strategy can improve QoL and reduce hospitalization rates without increasing the incidences of SAEs in PAF patients. Although our research has certain limitations, our findings provide valuable insights into new treatment strategies for PAF.Cao Zou: Conceptualization. Zirui Liu: Methodology, formal analysis, and investigation. Zirui Liu, Zhengkai Yang, Yu Lu, and Haocheng Wang: Writing—original draft preparation. Zou Cao and Zirui Liu: Writing review and editing. Cao Zou: Resources and supervision. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.The authors declare no conflict of interest.Figure S1: Risk of bias. (A)Risk of bias domains of included RCTs (traffic light plot). (B) Risk of bias summary.Click here for additional data file.Figure S2: Forest plot of the incidence of SAE at 1 year.Click here for additional data file.Figure S3: Forest plot of the incidence of SAE at 3 years.Click here for additional data file.Figure S4: Sensitive analysis of the recurrence of atrial tachyarrhythmias at 1 year.Click here for additional data file.Figure S5: Sensitive analysis of the incidence of persistent AF at 3 years.Click here for additional data file.Figure S6: Sensitive analysis of the QoL of PAF patients at 3 years.Click here for additional data file.Figure S7: Sensitive analysis of the 3‐year cumulative hospitalization of PAF patients.Click here for additional data file.Supporting information.Click here for additional data file.


L‐phenylalanine ammonia lyase (PAL) and L‐tyrosine ammonia lyase (TAL), as well as a higher level of ShA, free and cell wall‐bound p‐hydroxybenzoic acid, free homovanillic acid, free sinapic acid, and cell wall‐bound syringic acid can be considered biochemical indicators of the dominance of the rye genome.The aim was to identify subgenome‐related specific responses in two types of triticale, that is, of the wheat‐dominated genome (WDG) and rye‐dominated genome (RDG), to water stress induced in the early phase (tillering) of plant growth. Higher activity of the primary metabolism of carbohydrates is a feature of the WDG type, while the dominance of the rye genome is associated with a higher activity of the secondary metabolism of phenolic compounds in the RDG type. The study analyzed carbohydrates and key enzymes of their synthesis, free phenolic compounds and carbohydrate‐related components of the cell wall, monolignols, and shikimic acid (ShA), which is a key link between the primary and secondary metabolism of phenolic compounds. Under water stress, dominance of the wheat genome in the WDG type was manifested by an increased accumulation of the large subunit of Rubisco and sucrose phosphate synthase and a higher content of raffinose and stachyose compared with the RDG type. In dehydrated RDG plants, higher activity of  Typical responses of the latter to water stress are an increase in the content of cell wall‐bound phenolics or the activity of L‐phenylalanine ammonia lyase (PAL) and L‐tyrosine ammonia lyase  in triticale adaptation to water stress in the generative phase is responsible for; for example, higher activity of the photosynthetic apparatus, accumulation of proteins related to photosynthetic fixation of COSo far, the activity of individual triticale genomes under water stress was mainly analyzed during the phase of generative growth, and clear and specific responses of WDG and RDG type due to the activity of either the wheat or the rye genome were confirmed. In addition, specific features and responses resulting from the interplay between these two genomes were identified , which is a key link between the primary and secondary metabolism of phenolic compounds. The intensity of water stress was analyzed by measuring the water potential in leaf cells, the level of abscisic acid (ABA), and stomatal conductance.22.1The study involved two lines of triticale doubled haploid (DH), Hewo  and Magnat (DANKO Plant Breeders Ltd.). Our previous studies showed that under water stress, the DH line Hewo has a WDG type, and the DH Magnat line has a RDG type  and the PPFD was about 200–250 μmol m−2 s−1 . The plants were irrigated with Hoagland's nutrient solution once per week . Following emergence (one leaf stage), the plants were vernalized for 8 weeks at 4°C, photosynthetic photon flux density (PPFD) 150 μmol m2.3Soil drought was applied to both triticale types at the stage of four leaves (beginning of tillering). By withholding watering, the water content in the pots was gradually reduced to about 30% for 7 days and kept at this level for the next 14 days . Water content in the control pots was maintained at about 75%. Soil moisture in the pots was inspected daily, between 8.00 a.m. and 10.00 a.m., using a gravimetric method and taking into account plant weight (soil water content in the pots was immediately restored to 30%—drought or 75%—control).2.4The measurements were performed on Day 21 of the soil drought. For the analysis, we collected a fully expanded leaf, that is, the fifth one from the bottom. For biochemical and molecular measurements, leaf samples were collected and immediately frozen in liquid nitrogen, lyophilized, and then powdered using stainless steel beads .2.4.1The leaf water potential was analyzed with an HR‐33 T dew point microvoltmeter . Leaf discs (5 mm in diameter) were cut, placed inside the thermocouple psychrometer chamber (C‐52) and allowed to reach temperature and water vapor equilibrium for 60 min before measurements were made by the dew point method.2.4.2Stomatal conductance was recorded using an SC‐1 leaf porometer (Decagon Devices Inc.) on a fully expanded mature leaf.2.4.3ABA content was assessed according to the procedure described by Hura, Dziurka, et al. . Ultrahi2.4.42  and sucrose phosphate synthase , a protein associated with carbohydrate metabolism. Protein extraction, electrophoresis, western blot, and gel analysis were carried out as described in our previous paper  of the fructans/oligofructans was calculated using the following formula: DPav = 1 + (Ff/Gf), where Ff and Gf are molar concentrations of fructose and glucose released after enzymatic treatment of fructans/oligofructans  at 0.5 mL min‐1 and 40°C. [2H5]benzoic acid (D‐BeA) was used as ISTD . Further technical details are given in Table ShA was estimated in the same extract as monolignols, excluding SPE sample cleanup. After solvent exchange to 2% MeOH, a clear centrifuged solution was evaporated and resuspended in 100 μL acetonitrile (ACN). The content of ShA was measured utilizing UHPLC‐tandem MS (MS/MS) in negative ion mode MRM (multiple reaction monitoring). Chromatographic separation was done on Poroshell 120 HILIC‐Z 2.1 × 100 mm, 2.7 μm column (Agilent Technologies), in gradient mode  were measured employing UHPLC‐MS/MS (Agilent Technologies) in positive ion mode MRM. An Ascentis Express RP‐Amide analytical column  in a linear gradient of 3% ACN in H2O versus ACN with 0.01% (vol/vol) HCOOH was used. Further technical details are given in Table The samples were extracted as described for phytohormones . A two‐way analysis of variance (ANOVA) was performed to evaluate the effect of drought and cultivar on each tested parameter. Duncan's multiple range test at the 0.05 probability level was chosen to determine the significance of differences among treatment means.3w), stomatal conductivity (sg) and the content of ABA in WDG and RDG triticale exposed to water stress (WS). At a similar level of cell dehydration, ABA content was significantly higher in the WDG type (3827.5 pg mg−1 DW) than in the RDG type (2188.1 pg mg−1 DW). Under optimal growth conditions, both types showed a similar cell hydration level and ABA content. Stomatal conductivity (gs) under water stress conditions was approximately two times lower in WDG than in RDG plants  and TAL (165% of control) under water stress was only observed for RDG triticale. Water stress did not affect PAL or TAL activity in the WDG type , caffeic acid , syringic acid , and cinnamic acid , 3,4‐dihydroxybenzoic acid and salicylic acid (similar content in WDG and RDG both control and stress‐exposed plants), rosmarinic acid , and cinnamic acid  were detected for gallic acid , p‐hydroxybenzoic acid , vanillic acid , homovanillic acid , p‐coumaric acid  and cinnamic acid   and gallic acid  or stress (WS) exposed WDG and RDG plants Table . In contp‐coumaryl alcohol (63% of control) in the WDG type under water stress . Under optimal growth conditions (C), WDG and RDG plants significantly differed in their content of coniferyl alcohol (significantly higher content in RDG than in WDG) and p‐coumaryl alcohol (significantly lower content in RDG than in WDG), while under water stress, the content of these three monolignols was significantly lower in RDG than in WDG type  and a significant decrease in e Figure .4w), resulted in greater increase in ABA and considerable limitation of stomatal conductance in WDG plants as compared with RDG ones  in the WDG type under water stress accompanied by accumulation of proteins responsible for carbohydrate synthesis, RbcL, and SPS, may be due to intense consumption of carbohydrates during water stress  indicates that the synthesis of low‐molecular weight fructans and oligofructans dominates in the leaves of both triticale types, irrespective of growth conditions Table . This inShA is an important intermediate in the biosynthesis of secondary metabolites, such as phenolic compounds, involved in plant adaptation to water stress : Tomasz Hura. Writing (review and editing): Tomasz Hura, Katarzyna Hura, Kinga Dziurka, Agnieszka Ostrowska, and Karolina Urban. Funding acquisition and project administration: Tomasz Hura.Table S1. Multiple reactions monitoring (MRM) transitions for the analyzed monolignols  and shikimic acid (ShA): positive ion mode (+ESI), capillary voltage 4 kV, gas temperature 350 °C, gas flow 12 l/min and nebulizer pressure 35 psi. MassHunter software was used to control the LC–MS/MS system and in data analysis. For MRM parameters optimization MassHunter Optimizer was used. D‐BeA ‐ [2H5]benzoic acid.Click here for additional data file.


Episodic future thinking (EFT) is the ability to subjectively pre-experience a specific future event. Future-oriented cognition in young children positively predicts physical health and financial status later in life. Can EFT be improved in children, even temporarily? Developmental research emphasizes the importance of thinking about one’s own near future to enhance EFT, whereas research in adults suggests benefits reside in constructing a richly detailed event. We bridged the two perspectives to examine whether a procedure, the “episodic specificity induction” (ESI), could be adapted to encourage an episodic mode of thinking in children, benefitting performance on a variety of subsequent EFT tasks. The present study implemented a child-friendly ESI in which children mentally simulated a future event and were probed for specific details about it. We randomly assigned 66 children aged 6 and 7 years to one of two conditions: (1) ESI, in which children imagined “having breakfast tomorrow” in detail, describing surroundings, people, and actions, or (2) a Control condition , in which children simply viewed and described a picture of another child having breakfast. Children then completed a series of future thinking tasks assessing prospective memory, recollection/imagination of events, delay of gratification, and planning. Our ESI was successful in promoting the construction of a detailed event, and subsequently increasing the number of details of recollected and imagined events on an outcome task as compared to a control condition. Nonetheless, the effect of ESI was smaller than expected – a finding that fits with recent work suggesting that such interventions may be too cognitively taxing for young children and/or that benefits may hinge on further development in episodic processes. We discuss possible modifications to the induction and implications for EFT amelioration in young children. Indeed,prompt children’s talk about the future in turn benefit a host of future-oriented behaviors, including episodic simulation, prospective memory, anticipation of future needs, and delay of gratification? On the one hand, developmental research suggests that thinking about one’s own future self – even in general terms – can improve EFT in children  benefits EFT in children.Future thinking is also highly adaptative for children. Might prospection also be ameliorated, even on a temporary basis, in this population? Most of the work on children’s EFT has attempted to measure the ability, identify developmental milestones, and understand its relation to other cognitive abilities  and opportunities  of future thinking interventions. With age, children sharpen their ability to describe plausible future events , envisioOne of the most common assessments of EFT in adults, also used in children, entails prompting a mental simulation and verbal description of a specific event that could plausibly happen in the future . The verSeveral tasks assess EFT development in childhood while attempting to avoid its conflation with language by minimally relying on verbal responses . We recruited a somewhat older sample than 2.2.1.M age = 83.00 months, SD = 6.62, range = 72.41–95.51 months, 33 boys and 37 girls; 3 parents did not report the date of birth). We excluded data from four participants because: (1) two children wished to end the induction before its completion (one in each condition), (2) one was ineligible due to age, and (3) one had already participated during the piloting stage. After exclusion, the sample was composed of 34 children randomized to the control condition and 32 children randomized to the ESI condition. An independent sample t-test showed that the two groups did not differ on age in months, t(62) = 0.22, p = 0.829, Hedges’ g = 0.05, CI 95% , and a Chi-square test with continuity correction found no significant difference in gender, Χ2  = 3.12, p = 0.077, φ = 0.22. All children were fluent in English  which suggested a sample size of N = 66. Children were recruited through email from participant databases associated with University of Ottawa’s Child Development Laboratories and tested online on Zoom (zoom.us). Children received a $10 gift card for their participation. The ethical aspects of the study were reviewed by the Health Sciences and Sciences Research Ethics Board of the University of Ottawa (file # H-02-19-2769). Many constraints arose from aiming to offer a safe and positive research opportunity to children who completed the study from their home during the COVID-19 pandemic (from December 2020 to May 2021). Even though online administration introduced challenges , it was closer to the optimal format of an ESI activity in children. That is, we endeavored to develop an ESI that could be administered with minimal training, at home or in school settings.Our sample size was determined through an ower 3.1  analysis2.2.Two female researchers collected data remotely over the Zoom video-conferencing platform. We obtained verbal consent from the parents and verbal assent from children. We asked parents to remain available throughout the session to assist with any technological issues, while emphasizing the importance of not interacting with their child. We also specified that the session should take place away from the kitchen or dining room to minimize risks of “breakfast” cues being immediately available in the environment. This was possible for most but not all children, due to constraints such as space configuration or family members using other rooms. After the ESI or control condition (described next), the outcome tasks unfolded in this order: Instructions for the Prospective memory task, the Recollection/Imagination task, Delay of gratification task, Picture-book task and, if necessary, a Prospective memory reminder. The order was fixed to introduce enough time between instructions for the Prospective memory task and the end of the session, and between the Delay of gratification task and receipt of the “delayed” reward if chosen by the child. Further, the Recollection/Imagination task was always administered second, because a similar outcome task directly followed the adult ESI in 2.2.1.We randomly assigned children to an ESI or control condition. Both conditions were designed to last approximately 10 min.2.2.1.1.Imagination specificity induction. Our version began with the researcher telling children that they were going to play an imagination game and that they were going to imagine that they would be “having breakfast tomorrow.” We specified the event, rather than allowing children to freely select one, because know best what could happen,” and that they could say “whatever comes to your head.” These instructions were important to ensure that children felt comfortable sharing anything that they were imagining. Children were then informed that closing their eyes helps to imagine and the researcher demonstrated briefly how to imagine something with your eyes closed . The researcher then showed the children a picture of herself with a thought bubble representing the scene.Our ESI was based on Tell me where you will be.”), (2) people , and (3) actions  surroundings , what color is it?”; “Is there anything on the walls?”). If children did not respond to a question, they were reminded to close their eyes (if they had opened them), and that they could imagine anything. The initial question was then repeated. A decision tree was used during questioning so that the researcher knew when to move on to the next question, and to ensure that the ESI was administered in a standardized way . They were then presented with a picture of a child having breakfast and were asked questions about the picture. As in the ESI, questions were about surroundings , people , and actions , how clear did it look in your head?” (for the ESI condition), and “When you looked at the picture, how clearly could you see ?” (for the control condition). Children could choose from one of six options, each corresponding to an image , the researcher showed children the Visual Clarity Scale see  and aske2.2.2.2.2.2.1.I would love to see this, but I have a very bad memory. I will likely forget. Can you remember to get this (object) after we finish all our games?” The researcher then reiterated the instructions  and asked the child to repeat the instructions back to them to ensure they understood. If the child could not remember the task, the researcher reminded them that they must get their favorite thing at the end of the games.The instructions for this task were administered immediately after either the ESI or the Control condition , near future 0.77, 95% , and distant future 0.81, 95% . One of the 33 participants only completed the near future trial.The Recollection/Imagination responses were transcribed following guidelines in n = 11; ESI: n = 9) for each trial: near past , near future , and distant future  for the Recollection/Imagination task.Additionally, responses to the Recollection/Imagination task were scored using the adapted Autobiographical Interview procedure . The AI We used a similar AI scoring procedure to examine the amount of details elicited during the manipulations . The scoring of ESI was similar to regular AI procedure as described above: Details about the event of having breakfast tomorrow were categorized as internal details . Other details were categorized separately as “external details”; these included semantic details , metacognitive statements, repetitions, and details about another event than tomorrow’s breakfast. The main adaptation concerned the Control condition: As in 2.2.2.3.This task, adapted for online administration using Gorilla.sc  was mean2.2.2.4.tomorrow.” Each photograph was meant to evoke thoughts about a specific physiological state . With the photograph of the scene on the screen, the audio recording stated, “You can only bring one of these things there.” And asked “Which one do you need to bring to this place?” The image for each response appeared one by one with concurrent audio recorded object labels. The correct item  was the one that best addressed the physiological state in question . The remaining three choices included an option that was semantically associated with the best option  – but that was suboptimal – and two options that were semantically associated with the scene, but inappropriate and incorrect . Trial order was randomized and item presentation order was pseudo-randomized with the Latin Square design. After selecting an item, the selected response option appeared on the screen and an audio recording asked children to explain their choice.This task was intended to examine children’s ability to anticipate a future physiological state . The tasFor the item choice measure, children were given a score of 0 if they chose an incorrect item and a score of 1 for the correct item on each trial, for a total score between 0 and 4. For the explanation measure, answers were transcribed and coded to determine whether children referred to the relevant future physiological state to justify their choice. Using an adaptation of Two coders independently scored the responses on each trial for all of the children. One child opted to skip this task and was not included in any of the Picture-book task analyses. We obtained an ICC value of 0.91, 95% CI  for the mean score obtained by children on the verbal measure based on single rating, consistency, two-way model, indicating excellent inter-rater reliability. The scores of one of these two coders were included in the analyses. The selected scorer was unaware of the participants’ condition.2.3.zt-tests) or a robust alternative . Specifically, we used the ‘stats’ v. 4.2.2 package to conduct t-tests and Mann–Whitney tests, ‘rstatix’ package  lasted longer than the Control manipulation  from the beginning of the instructions to the end of the manipulation, t(64) = −10.44, p < 0.001, Hedges’ g = −2.54, CI 95% .The ESI manipulation  than the Control manipulation , W = 294, p = 0.001, r = 0.40. The greater amount of information in the ESI  than Control manipulation  is also supported by the participants’ number of words .Participants generated more internal (“on task”) details during the ESI manipulation  than in the ESI condition , W = 730.5, p = 0.015, r = 0.30. A mean rating of approximately 5 (out of 6) suggests children could visualize events that were “very clear.” The high number of internal details (reported above) and the high vividness rating combined strongly suggest that the ESI was successful. Indeed, one child even spontaneously justified the ratings saying the image was “a 3D one, where it’s really me at the table eating my pancakes.”As expected given the nature of both tasks , a Mann–Whitney test showed a significantly higher mean visual clarity rating in the Control condition  and Control  conditions did not significantly differ in their ratings of their surroundings, W = 670.00, p = 0.059, r = 0.23, or actions, W = 672.00 p = 0.083, r = 0.21  was significantly lower than the Control condition , W = 709.50, p = 0.023, r = 0.28  the gap in visual clarity ratings between the conditions. Children in the ESI , F =1.14, p = 0.290, η2G = 0.01, and the Condition’s interaction with Time perspective, F = 0.25, p = 0.780, η2G < 0.01, were not significant. The specificity of the event depended on the temporal orientation, F = 11.83, p < 0.001, η2G = 0.08. Children’s event descriptions were more specific and detailed in the near past  than near future , p adj. < 0.001, Hedges’ g = −0.50, CI 95% , and near past than distant future , p adj. < 0.001, Hedges’ g = −0.56, CI 95% . The near and distant future, however, had similar episodicity scores, p adj. = 0.453, Hedges’ g = −0.10, CI 95% .We tested the effect of Condition  and Time perspective  on the episodicity score  using a mixed ANOVA. The effect of Condition  than the Control condition , F = 6.81, p = 0.011, η2G = 0.05  = 11.26, p < 0.001, η2G = 0.09. Children generated fewer details for events in the near future  than the near past , t(61) = 4.06, p adj. <0.001, Hedges’ g = 0.51, CI 95% , and for events in the distant future  than the near past, t(61) = 3.68, p adj. = 0.001, Hedges’ g = 0.46, CI 95% . The near and distant future trials did not differ, t(61) = −0.30, p = 0.763, Hedges’ g = −0.04, CI 95% . The interaction between Condition and Time perspective was not significant, F = 0.35, p = 0.702, η2G < 0.01. Nevertheless, we conducted additional planned comparisons to examine the hypothesis that the ESI would have a particularly strong effect on the “near future” trial. In agreement with the main effect of Condition, means were in the direction of children narrating a more detailed specific event following the ESI than Control condition for each of the trials, but the difference was only significant for the distant future trial . A mixed ANOVA showed that children produced a larger number of internal details in total for recalled and imagined events following the ESI  = 2.78, p = 0.105 . All significant results above were detected here too , but the main effect of Condition was only approaching significance, Although we had also pre-registered a coherence analysis based on 3.2.2.log10 transformed as in analysis: M = 1.16, SE = 0.02; untransformed: M = 15.20 min, SE = 0.81 min, N = 32) and Control  conditions did not differ in the delay between receiving the Prospective memory instructions and the reminder , t(64) = 0.67, p = 0.505, Hedges’ g = 0.16, CI 95% .Children in the ESI  = 3.29, p = 0.193, Cramer’s V = 0.29  = 0.06, p = 0.806, φ = 0.03. The ESI  and Control  conditions did not differ on the Delay of gratification task, W = 547.50, p = 0.606, r = 0.07  and Control condition  preferred dog and cat versions of the task similarly, 0.07 see .3.2.4.3.2.4.1.Mdn = 4.00, M = 3.41, SE = 0.13, N = 32) and the Control condition  in terms of the number of correct items selected on the Picture-book task, W = 432.50, p = 0.175, r = 0.17  and Control  conditions, W = 572.50, p = 0.560, r = 0.07.Children’s verbal explanations for their item choices were scored based on whether they contained a future term and referenced a future physiological state. A Mann–Whitney test showed no significant difference between children’s explanations in the ESI  performance on a Delay of gratification task in children aged 4–5 years  was successful in facilitating the imagination of a richly detailed future event, as reflected in the number of internal details during the induction and subjective measures of vividness. All children imagined tomorrow’s breakfast, an event for which children typically have control , allowinbased on , but obt–5 years  and 8–12–5 years . This co–5 years .It is unclear when an ESI could be expected to produce sufficiently strong effects, if indeed ESI depends on a mature “episodic” system. The hippocampus, a critical structure for episodic constructive processes, continues to develop along with episodic memory into adolescence . ConsistThe cognitive load of our ESI could also perhaps be reduced through a shift to past-oriented ESI, given that future thinking in general is more demanding and less rich than episodic memory . Similarcf.As pointed out in the introduction, cognitive development creates particular challenges for future-thinking interventions. There are also several degrees of freedom when designing an ESI suitable for young children. For example, drawing could be leveraged as a tool to facilitate episodic elaboration and stimulate engagement . On a caFuture thinking permeates the daily lives of children and their parents: A parent prepares a glass of milk at the request of their child only to discover minutes later that their child has lost interest in the drink. A child does not put a pet back in its cage, failing to foresee the damage that could ensue. A child does not anticipate the boredom of a rainy day on a camping trip and fails to bring indoor games. Educational pursuits could inspire countless additional examples. The improvement of future thinking, even on a temporary basis, holds promise for immediate and long-term benefits. Our child-friendly ESI demonstrated potential immediate benefits on the recollection and imagination of events through an increase of details. In contrast, the minimally-verbal outcome tasks offered no hints of improvement, possibly due to an order effect or the cognitive functions involved in the tasks. Therefore, we found no indication that this version of ESI could translate to better choices for the future or remembering actions in children. Even though we conclude on the limited benefits of relying on the episodic memory system at young ages, our findings along with others e.g.,  support atang027@uottawa.ca.The datasets presented in this article are not readily available because of ethical restrictions. End-stage data will be made available. Requests to access the datasets should be directed to AT, The studies involving humans were approved by Health Sciences and Sciences Research Ethics Board of the University of Ottawa. The studies were conducted in accordance with the local legislation and institutional requirements. The Ethics Committee/Institutional Review Board waived the requirement of written informed consent for participation from the participants or the participants’ legal guardians/next of kin because of the online administration; verbal consent/assent were obtained.AT, OG, JA, GA, and CA: conceptualization and methodology. AT: formal analysis and visualization. OG and JA: investigation. CA: resources, supervision, and funding acquisition. AT, OG, JA, and GA: data curation. AT and OG: writing – original draft. GA, OG, JA, and CA: writing – review and edition. AT and CA: project administration. All authors contributed to the article and approved the submitted version.


Recently, researchers have found that the tumour microenvironment plays an important role in tumours. We aimed to investigate the effects of plasma lipids on the prognosis of patients with pancreatic cancer and the infiltration of CD8+ T lymphocytes in tumour tissue.We enrolled patients with pancreatic ductal adenocarcinoma (PDAC) who underwent pancreatic surgery between 2012 and 2021. Clinical pathological data were recorded; total cholesterol (TC) and triglyceride (TG) levels were measured; and tissue samples were collected. A tissue microarray was generated using collected tissue samples, and CD8 staining was performed to determine immunoreactive scores (IRSs). The correlations of TC and TG levels with clinicopathological characteristics and prognosis were analysed. Finally, the correlations of TC and TG levels with CD8+ T‐cell infiltration were analysed.A total of 90 eligible PDAC patients were included. TC levels were significantly correlated with tumour grade and lymph node metastasis, and TG levels were significantly correlated with perineural invasion. The Kaplan–Meier survival analysis results indicated that the prognosis in the high TC group was significantly worse than that in the low TC group, and the prognosis in the high TG group was significantly worse than that in the low TG group. Cox multivariate analysis indicated that TC was an independent indicator of poor prognosis of pancreatic cancer patients after surgery. Spearman correlation analysis indicated that there were significant negative correlations between CD8 IRS and TC and between CD8 IRS and TG.TC and TG levels are significantly related to the prognosis of pancreatic cancer patients. They may be associated with tumour progression to higher grades, lymph node metastasis and/or nerve invasion. More importantly, TC and TG may reduce CD8+ T lymphocyte infiltration into pancreatic cancer tissue, affecting antitumour immune functions and immunotherapy efficacy. Tumour‐infiltrating immune cells, including T lymphocytes, B lymphocytes, macrophages, neutrophils and dendritic cells, are important for antitumour immune responses. Among them, CD8+ T lymphocytes play a major role in killing tumour cells. Researchers have proposed different categories of tumour immune phenotypes/immunophenotypes based on the infiltration of immune cells into tumour tissue.Previous studies have shown that lipids are closely related to tumours and are likely to affect tumour immunity. Studies have found that the inhibition of cholesterol esterification enhances the antitumour activity of CD8+ T cells.The results of the above studies indicate that lipids can modulate the antitumour effects of immune cells, potentially promoting the malignant progression of tumours. However, studies of lipids in pancreatic cancer are very rare, and in particular, it remains to be determined whether lipids can affect CD8+ T lymphocytes in tumour tissues. It is not known whether lipids can influence patient prognosis or immunotherapy efficacy by affecting the spatial infiltration of CD8+ T lymphocytes in tumour tissue. The aim of this study was to investigate the effect of plasma lipids on the prognosis of patients with pancreatic cancer and on the infiltration of CD8+ T lymphocytes in tumour tissues.22.1We retrospectively analysed the data of 90 pancreatic cancer patients who underwent surgery at the Second Affiliated Hospital of Jiaxing University between 2012 and 2021. All patients with pancreatic cancer were treated with the first‐line chemotherapy regimen recommended by the guidelines. The postoperative pathological diagnosis was pancreatic ductal adenocarcinoma (PDAC). All patients had complete clinicopathological information and prognostic follow‐up data, including sex, age, grade, tumour size, tumour site, local invasion, perineural invasion, vascular invasion, lymph node metastasis, T stage, N stage, M stage, AJCC stage, smoking history, drinking history, diabetes history, survival status and survival time. In addition, plasma total cholesterol (TC) and triglyceride (TG) levels were measured before surgery. Cancer and paired paracancerous tissues were collected from 90 patients. A tissue microarray (TMA) was generated using the procedure described by Jawhar et al.2.2TMA paraffin blocks were cut into 4‐μm sections, and the sections were deparaffinised in xylene and an ethanol gradient. Antigen retrieval was performed using citric acid, followed by staining with an anti‐CD8 alpha antibody . Preliminary observation was performed under a microscope, and undesirable spots in the image during the preparation and staining process were eliminated. TMA readings were analysed by two independent pathologists . The digitally scanned image files of the IHC chip were analysed using an Aperio ImageScope digital pathology system. IHC of CD8 was performed using the immunoreactive score (IRS) method. The IRS is the product of the staining intensity and percent of positive nuclei scores. In brief, the staining intensity score ranged from 0 to 2: 0 = no positivity, 1 = weak and 2 = moderate. The percent of positive nuclei score ranged from 0 to 4: 0 = 0% cells stained, 1 = 1%–10% cells stained, 2 = 11%–50% cells stained, 3 = 51%–80% cells stained (strong reaction) and 4 = 81%–100% cells stained. The IRS was calculated according to the following formula: IRS = staining intensity score×percentage of positive nuclei score. Hence, the IRS ranged from 0 to 12.2.3t‐tests, and comparisons of qualitative data were performed using χ2 tests, the Fisher exact probability method or an R × C contingency table. Survival was analysed using the Kaplan–Meier method to generate survival curves, and the log‐rank method was used to compare differences in survival rates between groups. Multivariate analysis was performed using a Cox multivariate regression analysis model. The correlation between the blood lipid level and the level of CD8+ T‐cell infiltration was analysed using Spearman correlation analysis. In this study, p < 0.05 was considered statistically significant. The following graphing software packages were used in this study: SPSS 22.0, GraphPad Prism 6.02, Adobe Photoshop CS5 and Adobe Illustrator CS6.In this study, comparisons of measurement data were performed using 33.1A total of 90 PDAC patients were included in this study. The clinicopathological data of 90 patients are provided in Table 3.2p = 0.039) and lymph node metastasis (p = 0.033). Patients with high TC levels tended to have high tumour grades and were more likely to have lymph node metastasis , and the 45 patients with high TC levels were distributed into the high TC group 5.34 ± 1.20 mmol/L). Plasma TC levels were significantly correlated with tumour grade (0 mmol/L.3.3p = 0.01), and patients with high TG levels tended to develop neurological invasion , and the 45 patients with high TG levels were distributed into the high TG group (1.94 ± 0.72 mmol/L). Plasma TG levels were significantly correlated with perineural invasion  was used as a prognostic indicator. The Kaplan–Meier survival analysis results indicated that the prognosis of patients in the high TC group was significantly worse than that of patients in the low TC group . The clinicopathological characteristics perineural invasion (p = 0.026), vascular invasion (p = 0.003), lymph node metastasis/N stage (p = 0.000), and T stage (p = 0.014) were significantly correlated with OS (p ≤ 0.05) and were associated with poor prognosis , that is, tumour size, perineural invasion, vascular invasion, lymph node metastasis, T stage, TC and TG, were included in the Cox multivariate regression analysis. Cox multivariate regression indicated that lymph node metastasis (p = 0.004) and TC (p = 0.036) were independent prognostic factors for pancreatic cancer patients after surgery. Lymph node metastasis and TC were both unfavourable factors for the prognosis of pancreatic cancer patients, and the hazard ratios (HRs) were 2.485 (1.332–4.638) and 1.991 (1.045–3.790), respectively. TG may be affected by some indicators and was not ultimately identified as an independent prognostic factor for patients with pancreatic cancer, but the univariate analysis indicated that it was a significant factor.The relevant clinicopathological factors in the above univariate analysis . Lymph node metastasis may be related to the decrease in CD8+ T‐cell infiltration caused by high TC. These results suggest that there may be a close relationship between lymph node positivity and CD8+ T cells. This idea is supported by a study by Silvio Daster et al., who showed that a high frequency of CD8+ lymphocyte infiltration is significantly predictive of lymph node negativity in early rectal cancers.In addition, we found that patients with high TC had significantly more likely to have lymph node metastasis . Weiai Shen: Data curation (supporting). Xiaoguang Wang: Writing – original draft (lead). Jing Wang: Formal analysis (lead). Zhengxiang Zhong: Writing – review and editing (lead). Zhongcheng Zhou: Writing – review and editing (lead). Xujian Chen: Methodology (lead).The present study was supported by the Medical and Health Science and Technology Project from the Health Committee of Provincial Zhejiang (grant no. 2020KY951), the Medical and Health Science and Technology Project from the Health Committee of Provincial Zhejiang (grant no. 2023RC284) and project of the Bureau of Science and Technology of Jiaxing City (2020AY30017).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All patients or their guardians were informed of and agreed to the usage of the tissue/blood samples in our research.This article does not contain any studies with animals.This research was approved by the Ethical Committee of the Second Affiliated Hospital of Jiaxing University, Jiaxing, China .


Subjects with SFL, had significant lower TLC (p = 0.029). The MFL‐group had more air trapping (p = 0.036). Throughout the sample, air trapping correlated significantly with residual volume (RV) in L (p < 0.001). An increase in air trapping was related to an increase in BV5 (p < 0.001). Mean BV5 was 65% (IQR 5%). BV5% in patients with PASC was higher than in patients with acute COVID‐19 infection. This increase in BV5% in patients with PASC is thought to be driven by the air trapping in the lobes. This study reveals that symptoms are more driven by occlusion of the small airways. Patients with more physical complaints have significantly lower TLC. All subjects encounter physical limitations as indicated by suboptimal VO2max. Treatment should focus on opening or re‐opening of small airways by recruiting alveoli.Patients with post‐acute sequelae of COVID‐19 (PASC) present with a decrease in physical fitness. The aim of this paper is to reveal the relations between the remaining symptoms, blood volume distribution, exercise tolerance, static and dynamic lung volumes, and overall functioning. Patients with PASC were retrospectively studied. Pulmonary function tests (PFT), 6‐minute walk test (6MWT), and cardiopulmonary exercise test were performed. Chest CT was taken and quantified. Patients were divided into two groups: minor functional limitations (MFL) and severe functional limitations (SFL) based on the completed Post‐COVID‐19 Functional Status scale (PCFS). Twenty one patients , mean age 44 (IQR 21) were studied. Eighteen completed the PCFS . VO Common signs and symptoms were fever, cough, and tiredness. While many patients were asymptomatic or experienced moderate symptoms, 15% of the patients evolved towards a more severe disease and requiring hospitalization , commonly referred to as long COVID, affect COVID‐19 survivors at all levels of disease severity, even younger adults, children, and those not hospitalized Yong, . The defThe burden of these and other long‐lasting symptoms lead to a decrease in physical fitness.2 (BV5%) in cross‐sectional area, was associated with higher need for intubation and mortality in patients with acute COVID‐19 .2.22.2.1Data of patients at MedImprove with persisting respiratory symptoms at least 4 weeks after the initial infection with COVID‐19 were collected between August 2021 and October 2022. All male and female patients >18 years were enrolled (convenience sample). Only subjects with symptoms of comorbidities before COVID‐19 infection, such as myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) or previous heart or lung diseases, that might interfere with the symptoms of long covid were excluded.2.3Demographic data were collected including age, gender, height, and weight. After a general clinical examination, assessment of current symptoms and concomitant medication, baseline measurements were executed.2 (20.9%), CO (0.3%), CH4 (0.3%), and N (78.5%). To evaluate the carbon monoxide diffusion constant, the DLCO was divided by the alveolar volume (VA).All patients performed a pulmonary function test (PFT) . Dynamic lung volumes as expiratory capacity in 1 s (FEV1) and forced vital capacity (FVC) were measured by spirometry. Static lung volumes as total lung capacity (TLC) and functional residual capacity (FRC) were measured by body plethysmography. The diffusing capacity of the lung for carbon monoxide (DLCO) was measured by a 10 s breath hold maneuver at TLC, after inhalation of the diffusion gas O2 cross sectional area (down to a cutoff of 1.25 mm2) was termed “BV5”. We additionally defined BV5‐10 as the volume of blood contained in vessels with cross‐sectional area between 5 and 10 mm2, and BV10 as the volume contained in vessels with cross‐sectional area above 10 mm2. We refer collectively to these quantities as BVX. To account for variation in lung volume, we chose to normalize BVX by total pulmonary blood volume. This permits for the computation of a “BV spectrum”, a curve representing the percent of total pulmonary blood volume contained within vessels of a given caliber as a function of cross‐sectional area. It had previously been observed in analysis of scans from healthy volunteers that this yielded values with very low variance over all scales.Non‐contrast, thin‐slice, volumetric chest CT was acquired at TLC and functional residual capacity (FRC). 3D reconstructions of the lungs and pulmonary vasculature were created. An automated blood vessel segmentation algorithm performs an eigenvalue analysis of the Hessian matrix to enhance and identify tubular structures, by returning the probability of each voxel belonging to tubular structure based on shape analysis ‐based air trapping is defined as all the intrapulmonary voxels with HUs between −1024 and −850 using the expiratory scans at FRC. A Binominal Blur filter is applied and a mask within these thresholds is created, being intersected with each lobe mask to make the regional air trapping masks. The 3D models are then created from these masks.By identifying and grouping the voxels that represent the air in the lungs, the lung volume (L) can be determined from the scans. During segmentation, lung lobes are separated by identifying the fissure planes on the CT images and using these surfaces as cutting objects. This means that not only the total lung volume is determined, but also the volume of each lobe individually.2 peak in mL/kg/min) was measured by a maximal incremental cardiopulmonary exercise test using Vyntus CPX device  and an ergocycle (GE Vivid S60 N NOR v204) with monitoring of ECG (Cardiosoft GE). After a 3 min warm up, the protocol started with 5, 10, or 20 W, followed by an increasing of the workload with 5–15 Watt per minute, depending on the patient's baseline condition. The patient was asked to cycle with a cadence of 60 rotations per minute. This protocol was followed until exhaustion.Cardiorespiratory fitness , Borg Dyspnea Scale (Borg) and the Post‐COVID‐19 Functional Status scale (PCFS)  was used for analysis. All analyses were evaluated using a significance cutoff of U was used to analyze the difference between groups and Spearman's rho for correlations.Demographic data were examined descriptively to gain an understanding of the participating population. Discontinuous variables were described using frequency distributions. Variables were described using mean and SD (parametric), or median and interquartile range (nonparametric). Mann–Whitney 33.1Between August 2021 and October 2022, a total of 21 subjects , mean age 44 (SD 13) years, presented at the clinical practice of MedImprove with persisting respiratory symptoms at least 4 weeks after the initial COVID‐19 infection. Previous to the latter, these 21 patients did not have any comorbidities, respiratory disease, nor issues (ME/CFS), and were all enrolled in the retrospective study. None of the patients were hospitalized during their acute COVID‐19 infection. All subjects were initially treated symptomatically with paracetamol and rest. Only one subject was administered oral prednisolone. None of the cohort was on inhaled therapy previously to their first consultation. Descriptive data of the basic characteristics are given in Table The results of the PFTs, 6 MWT, and ergospirometry are given in Table The median of the baseline Borg Dyspnea score of 18 subjects was 2, with a range of 7 (IQR 2).p = 0.657), BMI (p = 0.23) or Borg Dyspnea Score (p = 0.693) between these groups.Eighteen subjects completed the PCFS. With eight subjects experiencing mild functional limitations (MFL) and 10 subjects with SFL with no differences in age  and ergospirometry peak load (160 W).p = 0.043) between the MFL and SFL group was found reflecting the severity of the functional impairments in the two groups  and the SFL‐group reached a mean VO2max predicted of 74.40% (SD 19.43%). Between the two groups, although numerically different, no significant difference was found in the maximal exercise tolerance (p = 0.13). Even no significant differences were found between the two groups in O2pulse (p = 0.72), EqO2 at peak (p = 0.42), nor EqCO2 at peak (p = 0.66).The overall functioning of all patients was low when considering the ergospirometry results. The group with MFL reached a mean VO3.3p = 0.018; Figure p = 0.029) , FEV1 predicted , and DLCO .Lung function parameters uncovered large differences between patients with wide ranges in FEV1, RV, TLC, and DLCO/VA. When comparing the MFL and SFL group, significant differences were found in RV Liter  Figure , and DLC3.4Scans were evaluated by trained radiologists. Visually, all subjects had a normal CT‐thorax scan. There were no signs of ongoing parenchymal infiltrates or fibrotic tissue.Table p = 0.036; Figure p = 0.016; Figure p = 0.016; Figure The quantified CT scans revealed significant differences between the two groups for several parameters, including air trapping (p < 0.001), RV predicted (p = 0.012) and TLC %  and BV5 . The mixed model considering BV5 and air trapping on a lobar level, showed that an increase in air trapping was related to an increase in BV5 (p < 0.001).Throughout the sample, air trapping correlated significantly with RV in L . In the SFL group, a decreased TLC was observed what might lead to a higher proportion in DLCO/VA and might explain the significant difference between both groups. The BV5 correlated significantly with air trapping. The more air trapping, the higher the BV5. The expansion of the lobes due to the air trapping can cause compression on the (not quantified) intra‐alveolar blood vessels with a shift of blood flow to the blood vessels with a diameter of 5 mm2. It is also known that endothelitis .However, a total occlusion of the small airways resulting in micro‐atelectasis and shunting, will cause more SFL and contributes to the decrease in total lung capacity. The DLCO/VA was significantly higher in patients with SFL, which might indicate that they have no impairment in their diffusing capacity. These patients with SFL have less lobe volume resulting in significantly less blood volume in the lower lobes compared to patients with MFL  and therefore not reduced as it was seen in patients with acute Covid infection where a BV5% of 35% was measured  is needed.This study has several limitations. All data was retrospectively collected in one center. All included subjects had chosen to present to a clinic with post‐COVID symptoms which might introduce unmeasured confounders. The sample size used in this proof of concept study is discrete and needs to be expanded for further validation and to phenotype these patients more in detail. Most patients were suspected of being infected with the Omicron variant, however this was not always confirmed and therefore not included in the manuscript. In this study no blood parameters were included. Blood analysis could be used in additional research to verify the findings. The quantified CT has no reference values but can be used for intra patient comparisons and for this population, the baseline measurement will be used to evaluate the impact of pulmonary rehabilitation in future research.Additional research needs to be focused on the impact of pulmonary rehabilitation and the evolution of the clinical outcomes of those patients.52max. The treatment should focus on the opening or re‐opening of the small airways by recruiting alveoli. This is work for a multidisciplinary team of pulmonologists, physiotherapists and nurses, to provide pulmonary rehabilitation including exercise training, medical treatment if needed, and a follow‐up of the pulmonary function parameters, especially TLC.The BV5% in patients with PASC was higher than measured in patients with acute COVID‐19 infection. This increase in BV5% in patients with PASC is thought to be driven by the air trapping in the lobes. However, this study reveals that symptoms are more driven by occlusion of the small airways. Patients with more physical complaints have a significantly lower TLC compared to patients with less physical complaints, but also patients with higher TLC encounter physical limitations as indicated by suboptimal VOWendel Dierckx—conceptualization, data curation, formal analysis, investigation, methodology, project administration, software, supervision, validation, visualisation, writing original draft, writing review and editing. Wilfried De Backer—conceptualization, data curation, formal analysis, investigation, methodology, software, supervision, validation, visualisation, writing original draft, writing review and editing. Kris Ides—conceptualization, data curation, formal analysis, methodology, project administration, supervision, validation, visualisation, writing original draft, writing review and editing. Yinka De Meyer—data curation, project administration, writing review and editing. Eline Lauwers—formal analysis, writing review and editing. Erik Franck—conceptualization, writing review and editing. Jan De Backer—software, visualisation, writing review and editing.All subjects in this practice signed an informed consent approving that their medical data can be pseudonymized utilized for research purposes. This monocenter study was reviewed and ethically approved by the Ethical Committee of the University Hospital of Antwerp on December 12, 2022 (Project ID 5050).


Multiple myeloma (MM) ranks second among the most prevalent hematological malignancies. Recent studies have unearthed the promise of cuproptosis as a novel therapeutic intervention for cancer. However, no research has unveiled the particular roles of cuproptosis‐related genes (CRGs) in the prediction of MM diagnosis.Microarray data and clinical characteristics of MM patients were obtained from the Gene Expression Omnibus (GEO) database. Differentially expressed gene analysis, least absolute shrinkage and selection operator (LASSO) and support vector machine‐recursive feature elimination (SVM‐RFE) algorithms were applied to identify potential signature genes for MM diagnosis. Predictive performance was further assessed by receiver operating characteristic (ROC) curves, nomogram analysis, and external data sets. Functional enrichment analysis was performed to elucidate the involved mechanisms. Finally, the expression of the identified genes was validated by quantitative real‐time polymerase chain reaction (qRT‐PCR) in MM cell samples.ATP7A, FDX1, PDHA1, PDHB, MTF1, CDKN2A, and DLST. Our gene signature‐based nomogram revealed a high degree of accuracy in predicting MM diagnosis. ROC curves showed the signature had dependable predictive ability across all data sets, with area under the curve values exceeding 0.80. Additionally, functional enrichment analysis suggested significant associations between the signature genes and immune‐related pathways. The expression of the genes was validated in MM cells, indicating the robustness of these findings.The optimal gene signature was identified using LASSO and SVM‐RFE algorithms based on the differentially expressed CRGs: We discovered and validated a novel CRG signature with strong predictive capability for diagnosing MM, potentially implicated in MM pathogenesis and progression through immune‐related pathways. We identified a novel signature associated with cuproptosis‐related genes, which exhibited a strong diagnostic value for multiple myeloma (MM). Our study highlights the involvement of cuproptosis‐related genes in the diagnosis of MM and investigates the underlying molecular mechanisms, offering novel insights into the pathogenesis and progression of MM. We identified a CRG signature for MM diagnosis by analysis of differentially expressed genes, least absolute shrinkage and selection operator (LASSO), and support vector machine‐recursive feature elimination (SVM‐RFE) algorithms. Then, we validated our findings using nine external data sets. Furthermore, we conducted functional enrichment analysis to investigate the potential mechanisms. Additionally, the expression of the identified genes in MM cell samples was validated using quantitative real‐time polymerase chain reaction (qRT‐PCR). Our study discovered a novel role of CRGs in MM diagnosis and explored the underlying molecular mechanisms. Our findings are expected to contribute to more effective decision‐making processes in establishing and implementing strategies for MM diagnosis and treatment.22.1https://www.ncbi.nlm.nih.gov/geo/). The raw data of all analyzed data sets were normalized to remove batch effects. The GSE118985 data set served as the training cohort for subsequent analyses, whereas the GSE5900, GSE6477, GSE24870, GSE27838, GSE46053, GSE113295, GSE113736, GSE133346, and GSE146649 data sets were selected to serve as diverse external validation cohorts for result verification. Based on our literature review,Ten data sets . The “rms” and “rmda” packages were employed to perform these analyses and visualize their results.2.5The ability of the gene signature to differentiate between MM and controls was assessed in nine validation cohorts: GSE5900, GSE6477, GSE24870, GSE27838, GSE46053, GSE113295, GSE113736, GSE133346, and GSE146649, using ROC curve analysis. Visualization of the results was performed using the “glmnet” and “pROC” packages.2.6Gene set enrichment analysis (GSEA) software was employed to explore the potential mechanisms by identifying significantly enriched pathways for each signature gene using the gene set (Kegg.v7.4.symbols.gmt). Moreover, the CIBERSORT algorithm was applied to compare the levels of 22 distinct infiltrating immune cell types between the MM and control individuals. We performed Spearman correlation analysis to assess the correlation between the signature genes and the infiltrating immune cells. The results of these analyses were visualized by the “clusterProfiler,” “enrichplot,” “DOSE,” “org.Hs.eg.db,” “preprocessCore”, “ggpubr,” “tidyverse,” “ggplot2,” and “reshape2” packages.2.7https://www.mirdb.org/), TargetScan (https://www.targetscan.org/vert_80/), and miRanda (www.microrna.org) databases based on the signature genes. Subsequently, we searched for potential lncRNAs interactions with miRNAs on the spongeScan database (http://spongescan.rc.ufl.edu/). Visualization of the mRNA–miRNA–lncRNA ceRNA network was performed with Cytoscape software (version 3.7.0).A ceRNA network was built by exploring the interaction relationships among messenger RNAs (mRNAs), microRNAs (miRNAs), and long noncoding RNAs (lncRNAs). The mRNA and miRNA interaction pairs were predicted by the intersection of miRDB . These cells were cultured in RPMI‐1640 medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin–streptomycin (Gibco). Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors using Ficoll‐Hypaque density gradient centrifugation after obtaining informed consent. This study was approved by the Ethics Committee of Shanghai Yangpu Hospital (LL‐009). All cells were incubated at 37°C in a humidified atmosphere with 5% carbon dioxide. Total RNA was extracted using TRIzol reagent (Invitrogen), following the manufacturer's protocol. Subsequently, complementary DNA was reverse‐transcribed from 1 μg of total RNA using the PrimeScript™ RT Reagent Kit (Takara). qRT‐PCR was performed in 96‐well plates using the SYBR Green PCR Master Mix (KAPA) and the Applied Biosystems 7900HT Fast Real‐Time PCR System (Thermo Fisher Scientific). Gene expression levels were determined using the 2.9t test, whereas Wilcoxon test was employed for skewed data. A two‐sided p < .05 was considered to indicate a statistically significant difference. The significance levels are denoted as follows: *p < .05, **p < .01, and ***p < .001.Data analysis was conducted using R software (version 4.3.0) and GraphPad Prism software (version 8.0.1). For quantitative variables, differences between groups with normally distributed variables were analyzed using Student's 33.1FDX1, LIPT1, PDHB, GLS, and CDKN2A were upregulated, whereas the expression levels of NLRP3, ATP7B, ATP7A, DLD, PDHA1, MTF1, and DLST were downregulated in the MM samples. Besides, the identified genes had significant synergistic effects, which are visualized in Figure Figure 3.2ATP7A, FDX1, PDHA1, PDHB, MTF1, CDKN2A, and DLST) were identified as the optimal CRG signature for diagnostic prediction in MM patients  was identified as the optimal cuproptosis‐related signature for predicting MM diagnosis, with an AUC value of 0.887 in the training cohort. Additionally, the nomogram based on this gene model had high accuracy in predicting MM diagnosis, suggesting that the developed model may serve as a powerful diagnostic tool for MM. Validating the developed gene signature was performed on nine external data sets, confirming its robustness with consistently exceeding AUC values of 0.80. The identified signature genes have various roles. For instance, ATP7A functions as an ATP‐driven copper ion pump, thus playing a crucial role in maintaining intracellular copper ion homeostasis.FDX1 is essential for the synthesis of various steroid hormones.PDHA1 and PDHB catalyze the conversion of pyruvate to acetyl‐coenzyme A (CoA) and carbon dioxide, linking the glycolytic pathway to the tricarboxylic cycle.MTF1 acts as a transcription factor that induces the expression of metallothioneins and other genes involved in metal homeostasis in response to heavy metals.CDKN2A was found to be capable of inducing cell cycle arrest in G1 and G2 phases and to be involved in the regulation of autophagy and caspase‐independent cell death.DLST, a mitochondrial protein belonging to the 2‐oxoacid dehydrogenase family, catalyzes the conversion of 2‐oxoglutarate to succinyl‐CoA and carbon dioxide.CDKN2A has previously been reported in MM patients.In this study, 12 of the 19 CRGs were identified as differentially expressed genes between the MM and control samples, underscoring the critical role of CRGs in the development and progression of MM. Leveraging these differentially expressed CRGs, we employed LASSO and SVM‐RFE algorithms to screen the feature variables and construct an optimal diagnostic model. A total number of 11 and 7 genes were extracted using the LASSO and SVM‐RFE algorithms, respectively. Ultimately, a gene signature consisting of seven overlapping genes  and mast cells. Tregs are a diverse population of CD4In this study, we successfully identified and validated a novel signature consisting of CRGs, showcasing its potential as a promising biomarker for MM diagnosis. Importantly, the gene signature revealed AUC values exceeding 0.80 in all the data sets, solidifying the robust predictive capacity of our model. Moreover, the expression patterns of the identified genes remained consistent in the MM cell samples, further bolstering the potential for effective clinical application. Of note, the results of functional enrichment analysis indicated notable associations between the signature genes and pathways related to the immune system. However, some limitations of our study need to be clarified. Although the differential expression of the identified genes was validated by qRT‐PCR analysis in our study, more prospective investigations are needed to validate the predictive power. In addition, the underlying molecular mechanisms of the genes were not fully understood, which will be the focus of our future research efforts.5We identified a novel signature associated with CRGs, which showed a strong diagnostic value for MM with AUC values exceeding 0.80 across 10 data sets. Functional enrichment analysis indicated that the signature genes might enhance the survival and progression of myeloma cells by influencing immune‐related signaling pathways. Our study highlights the involvement of CRGs in the diagnosis of MM and investigates the underlying molecular mechanisms, offering novel insights into the pathogenesis and progression of MM.Yidong Zhu: Conceptualization (lead); writing—original draft ; formal analysis (lead). Shuaikang Chang: Experimental validation ; writing—original draft . Jun Liu: Writing—review and editing . Bo Wang: Writing—review and editing ; experimental validation .The authors declare no conflict of interest.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.