Chrna2, Chrna3, Chrna10, and Chrnd mRNA of each brain region correlated with withdrawal-induced changes in FOS expression. Finally, by mining the Allen Brain mRNA expression database, we were able to identify 1755 gene candidates and three pathways  that may contribute to nicotine withdrawal-induced FOS expression. These results identify the dual contribution of the basal forebrain and brainstem-thalamic cholinergic systems to whole-brain functional connectivity during withdrawal; and identify nicotinic receptors and novel cellular pathways that may be critical for the transition to nicotine dependence.Chronic nicotine results in dependence with withdrawal symptoms upon discontinuation of use, through desensitization of nicotinic acetylcholine receptors and altered cholinergic neurotransmission. Nicotine withdrawal is associated with increased whole-brain functional connectivity and decreased network modularity, however, the role of cholinergic neurons in those changes is unknown. To identify the contribution of nicotinic receptors and cholinergic regions to changes in the functional network, we analyzed the contribution of the main cholinergic regions to brain-wide activation of the immediate early-gene FOS during withdrawal in male mice and correlated these changes with the expression of nicotinic receptor mRNA throughout the brain. We show that the main functional connectivity modules included the main long-range cholinergic regions, which were highly synchronized with the rest of the brain. However, despite this hyperconnectivity they were organized into two anticorrelated networks that were separated into basal forebrain projecting and brainstem-thalamic projecting cholinergic regions, validating a long-standing hypothesis of the organization of the brain cholinergic systems. Moreover, baseline (without nicotine) expression of Discontinuation of nicotine use in dependent users is associated with increased whole-brain activation and functional connectivity and leads to withdrawal symptoms. Here we investigated the contribution of the nicotinic cholinergic receptors and main cholinergic projecting brain areas in the whole-brain changes associated with withdrawal. This not only allowed us to visualize and confirm the previously described duality of the cholinergic brain system using this novel methodology, but also identify nicotinic receptors together with 1751 other genes that contribute, and could thus be targets for treatments against, nicotine withdrawal and dependence. Chronic nicotine use causes adaptive changes throughout the brain that lead to drug dependence , the eme2–α10 and β2–β4. Alpha4 and beta2 are the most prevalent, but all subunits are expressed throughout the brain. A large number of brain regions (40+) have cholinergic neurons, characterized by the expression of choline acetyltransferase (ChAT), however, most of them are interneurons and only eight brain regions have long-range projecting cholinergic neurons ]. The matrix was then hierarchically clustered, based on the Euclidean distances calculated from the correlations [Heatmap.2(data)] Modules were obtained by cutting the clustering dendrogram at half height [cutree].Average R values were calculated for each treatment  within the basal forebrain cholinergic regions , withing the brainstem-thalamic cholinergic regions , and between both cholinergic subgroups (n=12). Average R values were also calculated per treatment for the interaction of all cholinergic regions with the major anatomical groups in the brain. Two-way ANOVA was then performed to examine the effect of treatment condition on the average R value for each comparison.https://github.com/aestrivex/bctpy), derived from the MATLAB implementation of Brain Connectivity Toolbox ] or [betweenness(graph)] with the R package iGraph [graph_from_data_frame]. Participation coefficient was obtained using a customized version of the bctpy Python package . In cases of multiple comparisons, p-values were corrected using Benjamini-Hochberg (BH) false discovery rate (FDR) with q = 0.05  as Bonferroni multiple comparison correction of p-values was considered too stringent  [p.adjust(method = ‘BH’)]. Significance of Pearson correlations [cor(method = ‘pearson’)] was obtained with [cor.test(method = ‘pearson’)].BioRender.com. Figures were combined and edited with Adobe Illustrator.Graphs were constructed using the R package ggplot2 included in the tidyverse package or the ggpubr package for barplots. Heatmaps were constructed using the R packages gplots and ComplexHeatmap. Networks were visualized by plotting [plot] in the R package igraph or using Gephi software. Pathway illustrations were created with c-Fos captured the neuronal reactivity over a period of 1-2 h during nicotine withdrawal. Automated registration onto an anatomical reference atlas using the ClearMap pipeline .To investigate the role of the cholinergic regions in the organization of the whole-brain network, we first tested whether the long-range cholinergic groups  were sigb. In line with the overall increase in correlation, the posthoc test showed an increase in the correlation of the cholinergic groups with the cortical plate (p<0.0001), striatum (p<0.0001), thalamus (p=0.0035), and hypothalamus (p<0.0001).We then looked at how these long-range cholinergic groups correlated with the different anatomical groups throughout the brain using hierarchical clustering within the main anatomical structures  -E. ThereFinally, we looked at the interaction of the long-range cholinergic regions with brain regions that have been shown to be critical to nicotine addiction and nicotine withdrawal including the anterior cingulate area (ACA) , infralic, d, To better understand the role of the cholinergic groups within the whole-brain network and validate their role as hub regions or identify others, we calculated the network centrality measures: degree (number of connections a region has) and betweenness (number of shortest paths between two regions that go through a region). Nicotine withdrawal significantly increased the average degree , and that the localization of each group within each module was consistent with known anatomical and functional connections for these groups . Cholinec-Fos transcription  were not incorporated in this minimal network under control conditions. The organization of a basal forebrain and a brainstem cholinergic system resembles the original anatomical descriptions of the basal forebrain and brainstem cholinergic systems by Woolf and Mesulam , which nThird, the central hubs of the network changed. In the saline control network, the cuneiform nucleus and parastrial area acted as hub regions with high degree and betweenness, measures of network centrality  Wheeler. FollowiChrna2, Chrna3, Chrna10, and Chrnd (Chrna2 has been identified in human GWAS studies in association with smoking related behaviors, like smoking status, smoking initiation, cigarettes smoked per day, and smoking cessation (Chrna3 is part of a locus on chromosome 15q25 with Chrna5 and Chrnb4, which has also been identified in human GWAS studies to be associated with smoking related behaviors and nicotine dependence. One SNP is specifically localized in Chrna3 (Chrna10 was identified through linkage analysis in sibling pairs for nicotine withdrawal (Chrnd to increase the risk for nicotine dependence in an African-American population subset (All nicotinic receptor genes, except the muscle-type CHRNB1, including eight genomic regions containing 11 neuronal CHRN genes and three genomic regions containing four muscle-type CHRN genes, have been significantly associated with nicotine dependence and/or alcohol dependence . Analysind Chrnd -D. Chrnaessation . Chrna3 n Chrna3 . Chrna10thdrawal  and founn subset . It is in subset  and wholn subset  studies Sox2, Oct4, and Nanog, which are highly expressed in proliferative adult neurogenesis precursor cells in discrete brain regions (We then extended the gene analysis to include the mRNA transcript levels of all 19,413 genes of the In Situ Hybridization gene expression Allen Brain Atlas database, which resulted in a list of 1755 genes that had significant correlations between their expression and the nicotine withdrawal-induced FOS changes. Through pathway analysis , we iden regions  that are regions  and have regions . The sec regions . The thi regions . MeCP2 k regions , dysfunc regions , and som regions . MeCP2 h regions . GABA si regions  . DespitChrna2, Chrna3, Chrna10, and Chrnd mRNA throughout the brain was correlated with the nicotine withdrawal-induced FOS changes. Finally, we’ve identified a list of over ~1700 genes for which the baseline expression correlates significantly with the altered brain reactivity in the nicotine withdrawal state and identified cellular pathways that may contribute to neuronal activation during nicotine withdrawal.In conclusion, these results demonstrate that cholinergic regions increased functional connectivity with the rest of the brain through two anticorrelated sub-networks separated into basal forebrain projecting and brainstem-thalamic projecting cholinergic regions. The expression level of Supplement 1


To systematically review the scientific evidence comparing the clinical effectiveness of bulk-fill versus incrementally layered conventional resin composites and to evaluate if one method offers clear merits with specific clinical outcomes.Using relevant mesh terms and pre-established eligibility criteria in PubMed, Embase, Scopus and Web of Science, a thorough scientific search was conducted with an end-date of 30.04.2023. Randomized controlled clinical trials that involved the direct comparison of Class I and Class II resin composite restorations applied using incremental layering techniques versus bulk-filled in permanent teeth with an observation period of at least six months were considered. To evaluate the bias risk of the finalized records, a revised version of the Cochrane risk-of-bias tool for randomized trials was implemented.Out of the 1445 records determined, 18 eligible reports were chosen for qualitative analysis. Data obtained was categorized as per, the cavity design, the intervention, the comparator(s), the methods of success/failure assessment, the outcomes, and follow-up. Two studies demonstrated an overall low-risk of bias, fourteen studies raised some concerns, and two studies exhibited high-risk.Bulk filled resin composite restorations demonstrated clinical outcomes similar to those of incrementally layered resin composite restorations within a review interval of 6 months to 10 years. Key pointsProvides a comparison of the clinical efficacy of incremental layering versus bulk-fill techniques for the application of direct resin composites.Indicates bulk-fill technique as a good alternative to incremental layering.Suggests that the outcome of any restorative technique is dependent on patient, operator, material, cavity, and oral condition factors.1. Resin composite materials offer the benefits of ease of handling, superior physical properties, ability to polymerize on demand, availability in wide range of colors and translucencies, biocompatibility, and satisfactory adhesion to the dental hard tissues2. Composites have reported excellent clinical performance with annual failure rates ranging from 1% to 5% and 1% to 3% for anterior and posterior teeth respectively3. Furthermore, composite resin materials may offer the scope to allow repair4, strengthen the residual tooth structure, and their prescription may facilitate conservative cavity preparation5.Resin composite has been the preferred direct restorative material by dental practitioners for the treatment of anterior and posterior teeth, with acceptable success rates and longer-term clinical performance reported in the literature6.The main challenges encountered when using direct composite resins are, polymerization shrinkage and shrinkage stress, the degree of polymerization conversion, and their limited depth of cure. These factors may influence clinical performance. Adequate polymerization and the use of proper placement techniques are critical for the optimal clinical performance of these restorations7. The three-site method followed by the use of an oblique layering technique has been considered to be a good approach for composite layering technique and aid with the reduction of polymerization shrinkage8. The split, simultaneous modeling of separated increments has also been suggested to reduce shrinkage issues9. Lower levels of microleakage have been reported with the use of a split horizontal incremental technique at the gingival margin of a cavity10. At the occlusal margin of a Class II restoration, the application of a split horizontal incremental technique followed by the application of a centripetal and oblique placement technique has demonstrated the lowest levels of microleakage11.Incremental layering of composite resin using increments of ≤2 mm has been suggested to decrease shrinkage stress, improve the degree of conversion, evade disintegration of the restoration margin, and provide adequate esthetics12. A rise in the elastic modulus and post-photopolymerization shrinkage has been observed with increasing number of increments13.The incremental application of resin composite is, however, time consuming. It can be challenging whilst restoring more conservative cavities and is associated with the increased risk of contamination. The incremental application technique also has the scope for unwanted air entrapment between successive layers, which may culminate in adhesive failure between layers12.The challenges with incremental layering have paved the development of bulk-fill composite materials which may be applied in layers of thickness of 4–5 mm, thereby offering the merit of reduced treatment time and the potential of reduced volumetric shrinkage stress as well as improved curing depth whilst maintaining the desired micromechanical properties14. Polymerization depth is enhanced by better light transmission to deeper areas because of lowered light dispersion at the filler-matrix meeting point by reducing filler load, and/or improving filler particle size15.Polymerization shrinkage of bulk fills is decreased by incorporation of stress modulators like addition-fragmentation monomer (AFM), aromatic urethane dimethacrylate (AUDMA); high molecular mass monomers such as BisEMA, UDMA, BisGMA, Procrylat; and highly reactive photoinitiators. Initiator system optimization and the inclusion of fillers like zirconium / silica, ytterbium trifluoride, proacrylate, mixed oxides, and barium aluminum silicate particles in bulk fill resins have also improved their radiopacity and curing depth16. Furthermore, some constituents and modifications have been reported by the manufacturers of bulk-fill composites, but still certain constituents are unrevealed which may affect the ultimate clinical performance14.Nevertheless, there is inconsistency in determining the curing depth in the literature and a concern among clinicians regarding the degree of conversionThere is a need for a new systematic review that helps clinicians understand the clinical effectiveness of the two composite placement techniques . This will also enable practitioners to decide if they can select bulk technique as a reliable alternative method to incremental technique.The aim of this review was to assess the efficacy of incremental layering versus bulk-fill techniques for the fabrication of direct composite restorations by evaluating their respective clinical outcomes for the restoration of permanent teeth. The objective was to systematically determine if one placement method offers clear benefits over the other by comparing their outcomes (success/failure) of clinical parameters like retention, recurrent/secondary caries, marginal discoloration/staining, marginal adaptation/integrity, fracture, postoperative sensitivity, anatomic form, color match, and surface texture/ roughness.18. This review was registered with the International Prospective Register of Systematic Reviews (PROSPERO) under the registration number CRD42021258095.The protocol was formulated considering the suggestions of the Cochrane Collaboration for systematic reviews and conforming with the Preferred Reporting Items for Systematic Reviews and Meta‐Analysis Protocols (PRISMA‐P) Statement recommendationsReview question was, “In permanent teeth restored with direct composites, does incremental layering or bulk-fill technique perform better clinically?”The PICO model for clinical questions was used as follows:Participants- Participants with permanent teeth restored using direct composite restorations.Intervention- Bulk technique.Comparison- Incremental layering technique.Outcomes- Bulk-fill versus incrementally layered techniques were compared based on their performance (success/ failure) with regards to specific clinical parameters such as; retention, recurrent/secondary caries, marginal discoloration/staining, marginal adaptation/integrity, fracture, postoperative sensitivity, surface texture/ roughness, color match, and anatomical form.Types of studies- Studies comparing Class I and Class II direct composite restorations restored by incremental layering technique versus bulk technique were involved. Randomized controlled trials (RCTs) were included in this review as bias is reduced by randomization and a meticulous instrument is provided to investigate the connection of an intervention and outcome by cause-effect association19. Non-randomized clinical studies, reviews, case reports, in vitro studies were excluded. Studies where a bulk fill composite was incrementally applied, or either bulk fill or incremental layering was individually assessed, were also excluded.Timing- Studies with a review period of at least six months were included.Language- Studies documented in English were selected.Publication status- Only full papers published in peer-reviewed journals were considered.PubMed / MEDLINE , EMBASE (OVID interface), Scopus (Elsevier B.V.), and Web of Science were the electronic databases that were applied. Scientific articles were selected from the electronic databases using different combinations of text words and medical subject headings (MeSH) related to ‘bulk-fill’, ‘incremental layering’, ‘conventional composite’, and ‘direct restorations.’ The electronic probing was accompanied by manually searching of the Journal of Esthetic and Restorative Dentistry, Operative Dentistry, and the Journal of Conservative Dentistry. Furthermore, reference lists of the studies involved were scanned to confirm information saturation of pertinent studies. There was no restriction with regards to the publication date of the literature search. Before the final analysis, the search was repeated and carried out until 30.04.2023.In RefWorks, “Close Duplicates” plus “Exact Duplicates” options were selected in the “View” tab and all associated citations were removed. The remaining studies were screened and evaluated as per their titles and abstracts. Trials that addressed the review question and met the eligibility benchmark, were shortlisted. Consequently, articles that met all the eligibility criteria were finalized. The whole process was provided by one researcher because the basis for this was a thesis project, and any concerns were discussed with the supervisors.A checklist of information was attained from the selected articles to provide relevant information. Data collected from the shortlisted studies were organized in the form of tables to enable the presentation and evaluation of the proof acquired.The Oxford CEBM (Centre of Evidence-Based Medicine) tool was utilized to ascertain the level and grade of evidence of the articles involved in the review ranging from the highest Level 1a to the lowest Level 5.20. If the Cochrane Handbook criteria was fulfilled by all the components, it was considered low bias risk; if the elements were questionable, it was viewed as high risk and, if inadequate attributes were found, it was marked as some concerns17.The bias risk of the randomized clinical studies involved in the present systematic review were determined with the help of revised Cochrane risk-of-bias tool for randomized trials (RoB 2)31, and their exclusion criteria are listed in Table As shown by Fig. All the included records displayed level 1b evidence [Individual randomized controlled trials ]. Since, consistent level 1 studies were observed, the Grade of Recommendation as per CEBM is A.Descriptive information obtained in this systematic review is shown in Tables 33 (11.1%). Two studies35 demonstrated high risk (11.1%); and fourteen studies48 showed some concerns (77.8%) for overall bias risk as shown in Figs. Overall, bias risk for all domains was low for two studiesDue to the variety of composite materials and bonding systems used, and the differences in the design of the involved studies, a meta-analysis that requires relatively homogenous studies could not be performed. Thus, the results were summarized in a qualitative way by listing data from the included studies as per the cavity design, the intervention, the comparator(s), the methods of success/failure assessment, the outcomes, and follow-up. The quality of the data obtained from the included studies was weighed via the risk of bias assessment, giving greater reliability to the higher quality study results.49. The evaluation of the systematic reviews by AMSTAR 2  from included studies on the outcome of meta-analysis was not assessed, and funding sources for involved studies was not reported. Two studies52 were graded critically low, as both had critical flaws such as review protocol was not registered before commencement and the publication bias was not assessed. Additionally, exclusion of individual studies was not justified in one study52.A 16 item-criterion appraisal tool known as AMSTAR-2 was applied to methodologically inspect the quality of systematic reviews with a relevant review question 2 Table  graded o13. Also, when incrementally layering composites, void formation between increments can take place, resulting in sensitivity, deterioration of the resin material, and cuspal deflection54. On the other hand, the bulk-fill application technique provides ease of placement, lower technical sensitivity, and is less time-consuming31. Bulk-filling also prevents disintegration of mechanical properties, with a reduced risk of void formation55. But, there is a significant variation between individual bulk-fill products regarding filler particle size and framework, plus method of clinical placement, which can impact their clinical performance14.Traditionally, the incremental application of resin composite is prescribed to allow efficient polymerization, to control polymerization shrinkage and stress, and to improve the C-factor. However, aggravated deformation of compromised cusps has been demonstrated with increasing number of incrementsAccording to the studies included in this review, bulk-fill technique demonstrated no notable distinction in clinical performance compared to the incremental layering technique for the specific clinical parameters considered.42. One report recorded retention failure with two Tetric EvoCeram bulk-fills and one incrementally placed Tetric EvoCeram restoration. The difference in retention was linked to the adhesive material or the method used35. Loss of retention was recorded with two Filtek Supreme XTE and none with Filtek Bulk Fill Posterior in a three-year study37. This was associated with the viscoelastic property of the bulk-fill material used due to which shrinkage stress was not a problem56. One Filtek bulk-fill was lost in a study due to technical error during restoration placement41.The success of a restoration in clinical studies is demonstrated by its endurance in the oral cavity, making retention the most significant evaluation criteria57. Even in this systematic review, the reasons for failures predominantly were tooth- and resin-fractures, followed by secondary caries in both bulk-fill restorations and incrementally layered conventional restorations respectively. Patients with temporomandibular disorders or parafunctional habits like bruxism can eventually impact the sound tooth, resulting in restoration- and tooth-fractures58. Two studies demonstrated a substantially notable number of failures caused by resin composite and/or tooth fractures, mostly amongst bruxist patients47. One report recorded teeth fractures with Quixfil bulk restorations (n = 2) and incremental Tetric Ceram resin (n = 1)48. Fracture risk is higher when premature fatigue of the bonding agent occurs at the restoration-tooth interface59, which was considered by the study as the probable cause of failure with Quixfil bulk-fills (n = 3)48. High fracture rate of resin composites, both conventional and bulk composites has been reported by many included studies48, which could be a material-specific constrain of composites3.Fracture and recurrent caries formation are the primary causes for the failure of directly placed composite restorations60. High caries-risk individuals or low socio-economic status patients with restricted access to health services are susceptible to recurrent caries formation61. In this review, patients with substandard dental hygiene and high caries risk were excluded in several records48. However, two records included high-risk caries participants which was linked to SDR flowable bulk +Ceram X mono (n = 2) and incremental Ceram X mono (n = 1) restoration failures due to secondary caries47. Despite selecting patients with good dental hygiene, in one of the included trials, secondary caries was detected with incrementally placed Tetric N-Ceram resins (n = 2) and QuiXfil bulk-fills (n = 2). It was assumed to be connected to restorative material’s physical framework or the adhesive system efficacy39.Biological factors may be the cause of secondary caries instead of the material being used to restoren = 5) in another study62. According to an earlier systematic review, a lower failure rate of direct restorations was observed to occur with the use of rubber dam isolation than those performed using saliva ejectors and cotton rolls as a means of attaining the required moisture control63. Amongst the four studies in this review where rubber dam isolation was used, the absence of any restorative failures were observed45.During restoration placement, contamination with saliva and marginal adaptation faults were associated with secondary caries development  and Tertic N-Ceram Bulk Fill (n = 2). In comparison, no failures were seen with Sonic fill composite resin whose viscosity is reduced due to sonic vibration, resulting in better adaptation to the cavity walls and hence, improved marginal properties46.Marginal staining may be the first clinical sign of restoration failure79. According to a study, low bravo scores for marginal discoloration of both nano-hybrid (Tetric EvoCeram) and Tetric EvoCeram bulk-fill restorations may be because etching with phosphoric acid was not done42. Significantly lower marginal discolouration was observed with the restoration of cavities with bulk-fill composite in a single layer, compared to conventional composites in two included studies. But this was disregarded by both the studies as no additional treatment was required for minor surface discoloration45. Conversely, higher marginal discolouration was presented by bulk-fill composite systems than the conventional one in a study of this systematic review34.Marginal discolouration has been documented to occur more frequently in cases using the self-etch technique47. Depth and dimensions of the cavity prepared, marginal seal and liner application in deep cavities, can also influence postoperative sensitivity83. Most studies assessing postoperative sensitivity did not describe the cavity depth or involved more shallow cavities. Only one study proved all the cavities being restored were 4–5 mm deep by radiographic assessment40 and most cavities were described to be deep in two studies47. Application of liners in deep cavities shields the pulpo-dentin complex, reducing heat/electric stimuli, dentin sensitivity, and helps in reparative dentin formation84. No post-operative sensitivity was noted in studies with liner applied in deep cavities42. Following 12-months, sensitivity was recorded in only one tooth with a deep cavity among the ones filled with bulk-fill via sonic activation. This was regarded to be caused by the absence of a calcium hydroxide based liner40. Use of flowable composite linings reported no restorative failures in another study43. Contradictory evidence was found concerning the application of liners and post-operative sensitivity in a Cochrane review85. According to one report, higher sensitivity was recorded with the use of incrementally applied Filtek Z250XT compared to Filtek bulk-fill posterior restorative, which may be linked to adhesive failure or cusp deflection6.Majority of the included studies recorded no post-operative sensitivity in the teeth restored86. In one study, surface texture was rougher after finishing and polishing of some nanohybrid restorations, which was attributed to large fillers exfoliating from the matrix while polishing87. Slightly rougher surface was reported in a study with Filtek Ultimate group which was associated with void entrapment in the incremental layering method45. The differences in surface roughness / texture may be related to the fact that there is no specific finishing and polishing system for bulk-fill materials. For full-body bulk-fill resin composites, multistep finishing/polishing systems have been suggested to give greater polishability88.Surface texture modifications can be associated with the composite’s filler load, size, or hardness35 was contemplated to be influenced by intrinsic factors like resin’s organic matrix89. One trial reported better performance of incremental microhybrid Amelogen Plus compared to Filtek Bulk Fill Flow + Filtek Z350XT with regards to superficial staining33. This may be associated to better sorption ability of the nanoparticles in Filtek bulk fill90. Best results were observed with SDR + TPH3 as it was less prone to liquid absorption which was linked to the absence of triethylene-glycol dimethacrylate in TPH391. Good color stability was seen because of compact filler particles present in bulk- and conventional-resins used in a study46, and because of resistance to color modification provided by the presence of Urethane Dimethacrylate (UDMA) polymer matrix like that found in an earlier study92.Color stability in a study41.In one study, sonic fill was found to provide anatomically superior results compared to incremental Filtek bulk fill, Tetric EvoCeram bulk fill, and Clearfil photo posteriorThe comparative assessment of both the techniques was difficult, as the included studies involved in the review had a number of variables such as, different etching and bonding techniques for different restorative materials  along with varied patient, operator, cavity, and oral condition factors. Evaluation criteria methods were non-standardized and analysis for the clinical parameters were not always explicitly provided.The comparison between the two composite placement techniques will be more adequate with studies involving similar materials with fewer variable factors. Clinical trials with an extended observation period are required to attain stronger evidence and information regarding the performance of layering techniques clinically.Direct resin composite restorations fabricated using incrementally layered techniques performed clinically just as well as those formed using bulk-fill technique in the permanent dentition.The placement techniques demonstrated no significant differences with regards to, retention, recurrent/secondary caries, marginal discoloration/staining, marginal adaptation/integrity, fracture, postoperative sensitivity, surface texture/ roughness, color match, and anatomical form.The bulk-fill technique is a good alternative treatment option to incremental filling, offering reduced restoration time and the scope for a reduction in feasible operator errors.This systematic review disclosed that:


Entrustable Professional Activities (EPAs) are defined units of professional practice entrusted to professionals once they have attained the specific competencies required to complete the end-to-end task. They provide a contemporary framework for capturing real-world clinical skillsets and integrating clinical education with practice. Our scoping review question was: how are post-licensure EPAs reported in peer reviewed literature, in different clinical professions?We followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Extension for Scoping Reviews (PRISMA-ScR) checklist, Arksey and O’Malley and Joanna Briggs Institute (JBI) methodology. Searching ten electronic databases returned 1622 articles, with 173 articles included. Data extracted included demographics, EPA discipline, titles and further specifications.n = 162, 73%) describing medical sub-specialty EPAs . There were comparably few EPA frameworks reported in clinical professions other than medicine . Many articles reported only EPA titles without further explanation and limited content validation. The majority did not include information about the EPA design process. Few EPAs and frameworks were reported according to all the recommended EPA attributes. There was unclear distinction between specialty-specific EPAs and those that could be useful across disciplines.All articles were published between 2007–2021 across sixteen country contexts. The majority were from North America (Our review highlights the large volume of EPAs reported in post-licensure medicine, including the volume disparity compared to other clinical professions. Basing our enquiry upon existing guidelines for EPA attributes and features, our experience in conducting the review and our primary finding demonstrated heterogeneity of EPA reporting according to these specifications. To promote EPA fidelity, and quality appraisal, and to reduce interpretation subjectivity, we advocate: diligently reporting EPA attributes and features; including reference or citation to EPA design and content validity information; and considering distinguishing EPAs as specialty-specific or transdisciplinary.A large volume of post-licensure EPAs were identified in medicine relative to other clinical professions. EPA specifications were absent or variously reported in the literature, risking ambiguous interpretation. The authors recommend that future EPAs are reported with reference to established and evolving construct recommendations, which is integral to concept fidelity and translation to practice and education.The online version contains supplementary material available at 10.1186/s12909-023-04364-4. Clinical skills can be characterised as the practical and interpersonal skills required by health professionals to engage in and deliver a clinical healthcare service. Clinical skills may contain examination, practical procedures, communication and management skills with components of "how to", why, and the reasoning of what the outcomes might mean . IndividEntrustable Professional Activities (EPAs) are well-defined units of professional practice that can be entrusted to a professional once they have attained and can contextually link the competencies required to complete the end-to-end task . They ofSeveral recent investigations and scoping reviews have described the use of EPAs in entry-to-practice health professional education –13. Thesand independent critical analysis, with complex information synthesis aligned to originality of circumstances and challenges [Differentially, post-licensure clinical practice as a qualified clinician requires development and assessment of increasingly complex and specialist clinical skills. These skills are typically developed in post-licensure professional education. This includes managing unexpected challenges, higher order thinking, navigating uncertainty and making decisions in challenging situations at the “trusted” apex of Miller’s pyramid –16. Postallenges .A systematic review by O’Dowd and colleagues  and an uWe took a broad view to answer our research question: how are post-licensure EPAs reported in peer reviewed literature, in different clinical professions? Understanding the current perspective and landscape of EPAs representing post-licensure clinical practice is necessary to advance the future direction of post-licensure professional clinical education and practice frameworks. This scoping review extends on the current perspective of how EPAs are used in (i) pre-licensure healthcare and (ii) medicine only.EPA: A well-defined and conceptualized work unit of professional practice which requires contextualized integration of multiple competencies to proficiently complete .Clinical competency: A defined granular ability or specific learnable skill required for safe and proficient clinical practice matched to the context , 8, 15.after completion of the initial entry-to-practice qualification.  checklist , Arksey Detailed inclusion and exclusion criteria for our review are contained in Table Our research team and author panel were constructed to contribute scholarship and experience across interprofessional research, clinical education and clinical practice to generate varied relevant perspectives  to our eAll articles were imported and managed in Covidence . Two of A data charting form was prospectively developed and piloted to capture study design, country, discipline context, EPAs attributes, any further specifications and alignment to professional competency standards  in this review.After duplicates were removed, our search identified 1622 articles and 173 articles were included in our final review. Figure n = 173). All included article characteristics and demographics and reference list are reported with detailed tabulation in Supplementary Table Adopting the approach of Eva , herein n = 82, 53%). Post-licensure EPAs are well reported in the North American context, and in the medical discipline. They are also reported by a range of countries and different disciplines world-wide. In total, 16 countries and 4 different multi-national collaborates had published EPAs, with the majority in North America , specifically United States  and Canada . Article characteristics are summarised in Table n = 162, 94%), further differentiated into 40 medical sub-specialties. Non-medical professions represented were nursing , pharmacy , dentistry , physiotherapy  and respiratory therapy . Article distribution according to clinical profession and medical specialty context is shown in Table There is a growing trend to report post-licensure clinical practice expectations using the EPA concept. Publications were between 2007–2021 and proportionally in most recent years, including more than half in 2019–2021 n = 82, 5%. Post-lTable n = 130, 75%). Half of the articles further reported further specifications, for example discrete clinical skills required to perform the EPA or milestones to be acquired within EPAs . The most infrequently reported attribute was any form of EPA construct evaluation or fit-for-purpose testing .All EPAs were reported to represent essential work confined to trained and qualified health professionals. Most reported content validation with specific alignment to discipline-recognized professional association standards ((i)Many articles reported only EPA titles without further explanation.Our critical narrative synthesis of reported attributes arrived at four themes relating to how EPAs are reported:n = 87, 50%). This precludes any validity judgement of EPA concept based on reporting of title alone, which was nevertheless beyond the scope of this review. We interpreted that half of reported EPAs had title wording where an end-to-end task could be envisaged .(ii)Many articles did not include information about the EPA design process.Half the articles reported no further detail beyond EPA title(s)  reported on the EPA design process, specifically involving educators , clinicians  or a collaborative team of educators and clinicians . Half  of the articles reported further EPA specifications, such as discrete clinical skills required to perform the EPA. Our review was unable to differentiate between EPAs which were briefly reported versus insufficiently constructed according to the ten Cate attributes [(iii)Few EPAs and frameworks were reported according to all or most ten Cate recommenJust over half the articles n = 92, 3% reportn = 10, 6%).(iv)Limited attribute reporting precludes distinction between profession-specific EPAs and EPAs which could have transdisciplinary relevanceWhile all EPAs were reported according to several essential attributes through the inclusion criteria of this review, the proportion decreased when reported attributes were aggregated. Very few articles reported all the following details: alignment to professional competencies and standards, sub-competencies or milestones and evaluation or assessment . The majority of EPAs also represented work-units requiring performance of a combination of procedural and interpersonal skills . Our review did not discover any articles reporting EPAs as transdisciplinary. Most articles reported EPAs requiring discipline-specific qualifications  healthcare or medicine only , 9. FurtOur scoping review offers a pragmatic perspective including problems evident in the literature and reporting of current post-licensure EPAs. Our review encountered and navigated challenges, inconsistencies and non-specific EPA terminology, which is consistent with other contemporary perspectives around EPA concept conflation and viability , 40, 41.It was beyond the scope of this review to make validity judgments of EPAs based on only reported attributes and details, moreover when often the title alone was reported. It is not feasible to judge EPA construct and validity from the reporting if EPAs are reported without the full specifications. Nevertheless, it is pertinent to differentiate whether any critical insufficiency lies within the EPA construct or in reporting, also recognizing that either will impact fidelity and translation to practice.Our review demonstrates that many post-licensure EPAs are not reported according to contemporary construct recommendations, and demonstrates the problematic concerns and inconsistencies highlighted in existing literature.This is the first review focused on contextualizing the volume and reporting of post-licensure EPAs including both medical and non-medicine clinical professions. In a rapidly evolving field, our review captured a large volume of EPAs reported in medicine, proportionally well established in North America but also utilized by a range of nations worldwide. Specifically, these work-units were to be entrusted during and/or at end-of-training medical specialization fellowships, aligned with and beyond the CanMEDS transition to discipline stage towards advanced expertise . Other hDiscovering the volume of post-licensure medical EPAs alongside the relative few available in other healthcare professions extends on the perspective offered by previous reviews by O’Dowd et al.  and Lui Another important point of difference to these previous reviews was our aim to identify any potentially transdisciplinary EPAs. Although EPAs are conventionally discipline-specific rather than interprofessional , the conIn the context of other evidence our findings support the widely reported perspective of rapidly expanding EPA reporting for the primary purpose of aligning clinical skill education and practice , 9, 39. Articles in our review often reported EPAs by title only. Established recommendations for EPA construct include unambiguous titles , 16 and Placing the importance of all EPA specifications and attributes in perspective, EPAs are intended to represent task-integrated performance of essential clinical skills defined in health professionals' competency standards matrices . This puDespite incomplete reporting of EPA attributes, most EPAs were reported as aligned to relevant benchmarked clinical tasks endorsed by discipline-specific professional associations and/or content validated with alignment to professional competency matrices . This inOur review determined that most EPAs were reported as designed according to professional competency matrices, but without reporting any fit-for-purpose evaluation. Alongside ambiguous task definitions, this risks fidelity in the formation of ad-hoc expectations, and inequitable learning experience and assessment of essential skills. Further, the extent of EPA implementation and evaluation is integral to determining fit-for-purpose . Since sResolving inconsistency of EPA reporting would facilitate transparent real-world implementation for consumers in clinical education and clinical practice. Advancing popularity and EPA concept dilution led ten Cate & Taylor  to furthDiligently reporting EPAs with their attributes and features, or directly citing towards the full specifications.Including reference to EPA design and content-validity information.Distinguishing the EPA as profession-specific or transdisciplinary relevantAdvocating, synthesizing and reporting future EPAs according to the current existing recommendations underpins our further three recommendations based on our experience of interpreting how post-licensure EPAs are reported in peer reviewed literature. We discovered a vast number of EPAs representing post-licensure clinical practice in different healthcare disciplines and acknowledge the variety of EPA reporting contexts in our review. To promote EPA fidelity, quality appraisal and reduce subjectivity in consumer interpretation, we assert the proportional value of the following recommendations towards reporting EPA features and attributes:Previously published recommendations for EPA construct and reporting in the context of our further reporting recommendations are represented in Supplementary file n value represents reporting volume, with some EPAs in duplicate. We grounded a feasible-sized synthesis using the first reported EPA title in each article, so further examples of high-integrity EPAs may have been omitted. It was beyond our resource to consider the large volume of heterogeneous evidence using a consistent Quality Appraisal Tool, which would have been valuable for commenting on the overall rigour of available evidence. Our review included all post-licensure EPAs, without discerning the organization of EPAs along the staged continuum of post-licensure professional training. This review can inform identification of focus points and approach for future work.Our review included EPAs referred to as EPAs by article authors in peer-reviewed literature. Some EPAs were reported in several different articles, so our Instead of optional additional stakeholder consultation, we drew upon the varied expertise within our research team. We prospectively acknowledged the subjectivity of our scoping review methodology, and further extend this to our critical narrative perspective and interpretation of EPAs. We acknowledge any influence of the wholly physiotherapy-discipline authorship, including any prior assumptions, beliefs and experience as practicing physiotherapists, active researchers and interprofessional education scholars. Reciprocally, we attest our combined and different extensive experiences in these fields qualify us to perform this review, leveraging our combined and different scholarly perspectives to formulate a valid perspective. We attest to the rigorous design and transparent reporting of our work. We developed our conclusions and recommendations with these limitations in mind, advocating the review size enabled us to demonstrate the large heterogenous body of literature and a robust subjective overview of the current evidence base.EPAs are an established approach to capturing post-licensure clinical skill requirements in different healthcare professions and extensively in medical specialist training. EPAs are typically designed to align clinical skill education and practice according to professional competency standards matrices. In answer to our research question, a large volume of post-licensure EPAs is reported in medicine and very few in other clinical professions. EPA specifications beyond the title were absent or variously reported in the literature, risking ambiguous interpretation of the education and practice expectations. We recommend that future EPAs are reported with reference to established and evolving construct recommendations, which is integral to concept fidelity and translation to practice and education.Additional  file 1. Additional  file 2. Additional  file 3. Additional  file 4.


Wnt signalling has been implicated as a driver of tumour cell metastasis, but less is known about which branches of Wnt signalling are involved and when they act in the metastatic cascade. Here, using a unique intravital imaging platform and fluorescent reporters, we visualised β-catenin/TCF-dependent and ATF2-dependent signalling activities during human cancer cell invasion, intravasation and metastatic lesion formation in the chick embryo host. We found that cancer cells readily shifted between states of low and high canonical Wnt activity. Cancer cells that displayed low Wnt canonical activity showed higher invasion and intravasation potential in primary tumours and in metastatic lesions. In contrast, cancer cells showing low ATF2-dependent activity were significantly less invasive both at the front of primary tumours and in metastatic lesions. Simultaneous visualisation of both these reporters using a double-reporter cell line confirmed their complementary activities in primary tumours and metastatic lesions. These findings might inform the development of therapies that target different branches of Wnt signalling at specific stages of metastasis. Summary: Using intravital imaging and an avian embryo xenograft model, we show that canonical Wnt and ATF2 signalling pathways have complementary activity patterns during human cancer cell invasion and metastasis. Human cancers often exploit signalling pathways that are involved in normal tissue growth and organ development. Wnt signalling is one of the best-known examples of a developmental pathway that is commonly hijacked by cancer cells. During normal development, Wnt signals control multiple processes, including stem cell maintenance, cell polarity and individual and collective cell migration . Similarhigh) in vitro show increased capacity for tumour initiation in vivo  model -dependent gene transcription and is mainly implicated in stem cell maintenance and cell proliferation . The can in vivo . Throughfunction . In earlgnalling . In brea readily . Metastainvasion . Upon arinvasion . Moreoveinvasion . Finallyinvasion . In glioxpansion . MoreoveC) model .high cells were shown to have only a marginal increase in their tumour-initiating properties. Genetic interference with Wnt/β-catenin signalling was found to increase colon cancer cell invasive potential, contrary to what was expected. In a patient setting, the activation of the Wnt/β-catenin–TCF axis was found to be significantly lower in the CD133+ colon CSC-like population and its inhibition led to a boost in tumour growth in mouse xenografts  Wnt signalling during cancer cell metastasis, yet the consensus is that it promotes cancer cell invasion in a broad range of human cancers when activated ,b, inclureporter . The usereporter . Neverthreporter  and in creporter , to Wnt-reporter  and to creporter  in primareporter . Adding reporter .Together, these studies suggest that the spatio-temporal map of β-catenin/TCF-dependent and ATF2-dependent signalling activation during cancer progression is likely to be complex. During the metastatic cascade, Wnt signals might be activated at distinct locations and at different times within the same tumour, metastatic lesion, and even in individual cancer cells. Until recently, continuous intravital visualisation of Wnt pathway activation during cancer progression has not been possible. Here, we use fluorescent β-catenin/TCF-dependent and ATF2-dependent reporters as tools to visualise their activities during cancer cell invasion, intravasation and metastatic lesion formation. We show that canonical Wnt signalling is mostly downregulated during all these steps and is restricted to a limited population of cells with low motility. In contrast ATF2-dependent signalling is active in most invasive cells, both in primary tumours and in metastatic lesions. Throughout the metastatic cascade β-catenin/TCF-dependent and ATF2-dependent signalling pathways appear to complement one another, being active in distinct zones within the primary tumour and in metastatic lesions.in vivo systems to study Wnt signalling activity during development  with cWntlow cells constituting the majority (>80%) of the cells at the invasive tumour zone  was located within the main tumour mass . In accordance with these results, exogenous activation of Wnt/β-catenin with recombinant Wnt-3a protein and CHIR99021 reduced migration of HT1080 cells in in vitro t-scratch assays  population in the primary tumour, they would still be responsible for most of the invasion and metastatic dissemination, as suggested previously  as previously described . In thisour zone A–C. In cour mass A–C. HT10. These switches were not due to changes in tdTomato fluorescence intensity, but to the changes in Venus fluorescence (driven by the TCF/LEF-binding sites in pBARV) and could occur within a relatively short (∼12 h) period of observation (Movie 1).Surprisingly, during long term time-lapse analysis of cancer cell invasion at the primary tumour front, we found that invasive HT1080 cells readily switched between cWnthigh cells were responsible for the majority of the intravasation and early stages of metastatic lesion dissemination of tumour cells. Although relatively rare, intravasation events can be routinely observed in this model when cancer cells protrude into the vessel lumen and are carried away by the flow of the blood. First, detailed visualisation of the vasculature at the primary tumour front was surveyed in order to detect intravasation events. This found that in all the cases observed (>30), all the cells that were completely intravasated or rolling along the vascular endothelium or carried by the blood flow were cWntlow . High-resolution visualisation of cancer cell interaction with the tumour vasculature showed that, even though in some cases cWnthigh cells were observed to the contact vascular wall, their ability to protrude into the vascular lumen was significantly reduced when compared to that of cWntlow cells (Movie 2). To further study this behaviour, we employed an in ovo spontaneous metastasis assay, where primary tumours are grown on the upper CAM within the uncracked egg and cancer cells are allowed to metastasise spontaneously to the lower CAM and to other organs via the vasculature  of cWnthigh cells were located in the contact with the vasculature within the primary tumours grown in in ovo or ex ovo  with the majority of the colonies comprising a mixed population (cWnthigh and cWntlow), although there were also colonies consisting entirely of cWnthigh and cWntlow cells. Interestingly, similar to what was observed in primary tumours, we noticed a striking difference in the behaviour of cWnthigh and cWntlow HT1080 cells within metastatic lesions (Movie 3). HT1080 cWntlow cells were significantly more invasive, displaying increased vasculotropism, cell track velocity and displacement rates (Movie 3). In contrast, cWnthigh cells were less invasive, forming fewer contacts with the vasculature and often displaying a more central position within the metastatic lesions (Movie 3). Long-term time-lapse analysis showed that, like primary tumours, invasive HT1080 cells could switch between cWntlow and cWnthigh states within similar periods of time (Movie 3). Again, these switches resulted from changes in Venus fluorescence, as tdTomato fluorescence did not change significantly during the observation time  and imaged metastatic colony formation within the CAM tissue daily for 5 consecutive days after tumour cell injection  of HT1080 cells in primary tumours displayed moderate ATF2-dependent GFP reporter activity . There was a small proportion of cells with a low GFP/tdTomato fluorescence ratio  that was significantly less invasive and was predominantly located within the main tumour mass  was engineered to expressed both ATF2-GFP and 7TCF-mCherry reporters, as well as constitutively expressing the cyan fluorescent protein mCerulean to normalise the fluorescence intensities of GFP and mCherry. FACS and immunofluorescence analysis confirmed HTDR cells responded to stimulation by recombinant Wnt-3a protein and Wnt-3a conditioned medium by activating 7TCF-mCherry, and to exogenous expression of Wnt-11 by activating ATF2-GFP, confirming the dual functionality of these reporter cells . Notablyhigh HTDR cells  were more rounded and located within the central regions of metastatic lesions, where cells are normally closely packed . Indeed, cWnthigh HTDR cells did not actively participate in invasion, in clear contrast to ATF2pos HTDR cells, which rapidly and persistently invaded out of metastatic lesions, as confirmed by tracking analysis . Even though cWnthigh HTDR cells constituted a significant part of the main tumour mass , they were significantly underrepresented within the invasive tumour front, displaying slower and less directional invasion. In contrast, ATF2pos HTDR cells rapidly invaded, both at the primary tumour front and within the main tumour mass, confirming the data obtained using cells expressing single-pathway reporters (Movie 5).Live imaging of primary tumours formed by HTDR cells further confirmed the activation patterns observed in the metastatic lesions during cancer cell invasion, with ATF2in vivo in real-time within the same invading cancer cells. Canonical Wnt reporter activity was observed to be significantly downregulated during cancer cell invasion, both in primary tumours and in metastatic lesions. Cells displaying increased canonical Wnt reporter activity (cWnthigh) tended to remain within the primary tumour or in non-invasive zones of metastatic lesions. This was found to be the case in multiple cancer types – fibrosarcoma (HT1080), head and neck cancer (HEp3) and glioblastoma  – and was observed using both Venus and mCherry as reporting fluorophores for β-catenin and TCF-dependent activation. Even though cWnthigh cells were often found to be in close association with the primary tumour vasculature, they formed less extensive contacts and did not participate in intravasation. Time-series analysis found that the vast majority of metastatic lesions consisted of cWntlow cells during the initial stage of metastatic lesion formation, further suggesting that cWntlow cells are responsible for the initial steps of the metastatic cascade . Importantly, we showed that invading cancer cells rapidly (within less than 24 h) undergo shifts between cWnthigh and cWntlow states, both in primary tumours and metastatic lesions. In clear contrast, invading cancer cells maintained relatively high levels of ATF2-dependent reporter activity (ATF2pos), both within the metastatic lesions and in primary tumours. Moreover, time-series analysis of metastatic lesions found that the majority of metastatic cells were ATF2pos and that the ATF2low cells were located in the central areas of metastatic lesions and displayed non-invasive behaviour. Simultaneous visualisation of both reporters confirmed that they display distinct and highly coordinated activation patterns during metastatic cancer progression.We have visualised the activation patterns of β-catenin/TCF-dependent and ATF2-dependent reporters during human cancer cell invasion and metastasis. To our knowledge, this work is the first to simultaneously image these reporter activities Based on these results, we hypothesise that, during the initial steps of the metastatic cascade , low Wnt canonical activity is a pre-requisite for invasion and intravasation. In contrast, high ATF2-dependent signalling promotes cancer cell invasion out of the primary tumour and dissemination to secondary sites of metastasis. Shortly after extravasation at a secondary site, canonical Wnt activity is upregulated, which could facilitate the proliferation of the metastatic lesion, whereas ATF2-dependent activity remains high in cells invading out of the metastatic lesions, leading to the invasive expansion of the distant metastatic lesion. These observations are summarised in the model shown in high tumour cells within the main tumour mass, as quantified for single HTTOP or dual HTDR cancer cell lines, which can be explained by the cells being selected for optimal reporter responses to different signals during FACS sorting for cell line generation. Although crosstalk between canonical and noncanonical Wnt signalling has been reported previously . The dual reporter cell line gave us striking confirmation of the findings observed using single reporters, yet these reporters are different. For example, these reporters showed differences in the percentages of the cWnteviously , this isat brain . Many caat brain . These tat brain . These cf Wnt-11 . Althougf Wnt-11  their usf Wnt-11  and furtf Wnt-11 . Conversf Wnt-11 . The mecR stress . Activatactivity F, and inactivity .high cells truly responsible for driving cancer cell proliferation within the primary tumour and metastatic lesions? Can any cancer cell switch into the cWnthigh state, or is this ability limited to a CSC-like population? How specific are the β-catenin and TCF- and ATF2-dependent reporters in vivo, which Wnt ligands are responsible for their activation and what is their source? Finally, despite a long history of use in the field of developmental and cancer biology, questions remain on how closely the results obtained in the chick CAM model, as a xenograft host, can be translated into human cancer settings. For instance, the embryonic environment could be providing a rich source of chick Wnt ligands that share high sequence identity with human Wnts and are therefore likely to activate the reporter cells monitored here. This might also be the case in mouse xenograft studies, although Wnt-dependent activity in adults may be more niche-restricted than in embryos  and pLVE-eGFP (Addgene #52581) using HEK293 FT Thermo Fisher ViraPowerTM lentiviral packaging mix transfection protocol, as described by the manufacturer. HT1080-ATF2-GFP cells were generated as described above transducing HT1080-tdTomato with ATF2-GFP vector  was used for multicolour imaging of cancer cells and chicken embryo CAM vasculature. Image drift was corrected using the ImageJ StackReg plugin .All experimental data were plotted  and analysed for statistical significance using GraphPad Prism analysis module (unpaired two-tailed 10.1242/joces.260285_sup1Supplementary informationClick here for additional data file.


Resistance and tolerance are two important strategies employed by the host immune response to defend against pathogens. Multidrug-resistant bacteria affect the resistance mechanisms involved in pathogen clearance. Disease tolerance, defined as the ability to reduce the negative impact of infection on the host, might be a new research direction for the treatment of infections. The lungs are highly susceptible to infections and thus are important for understanding host tolerance and its precise mechanisms. This review focuses on the factors that induce lung disease tolerance, cell and molecular mechanisms involved in tissue damage control, and the relationship between disease tolerance and sepsis immunoparalysis. Understanding the exact mechanism of lung disease tolerance could allow better assessment of the immune status of patients and provide new ideas for the treatment of infections. COVID-19 has been prevailing for nearly two years and has infected more than 500 million people across 218 countries, leading to more than 6 million deaths. In addition to viruses, a variety of bacteria and fungi can infect the respiratory tract and cause significant morbidity and mortality . Many stAs the primary site of gas exchange, the lungs are the most common target of infections. The understanding of disease tolerance in the lung holds promise because, unlike antibiotics and vaccines, it points to methods of treating infections that exert evolutionary pressures on pathogens . In this22.12+ signaling to AECs and macrophages, and reduce endotoxin-induced lung inflammation  that can control and contain the spread of TB without directly reducing the pathogen burden. As a disease tolerance medium, granulomas maintain proinflammatory signaling in the core and anti-inflammatory signaling in the periphery , inhibitrocesses .2.3Several classes of antibiotics are beneficial for infections, which cannot be explained by their direct antibacterial activities alone. A group of anthracyclines at low doses is very effective in conferring protection against severe sepsis in mice. The mode of protection manifests at the level of DNA damage response and autophagy in the lung, which confers disease tolerance to severe infection occurring irrespectively of the pathogen burden . Colaco 2.4A typical endotoxin induces a proinflammatory response to counteract the growth and dissemination of gram-negative bacteria. Initial exposure to LPS can decrease responsiveness to LPS challenge, a condition known as LPS tolerance . Low-dos3The specific mechanisms underlying disease tolerance remain unclear. The tolerance effect caused by different triggers varies according to the location or form of injury. However, most studies focused on the regulation of the host inflammatory response to control tissue damage . The rel3.1Legionella pneumophila infection, AREG treatment decreased albumin levels in the bronchoalveolar lavage fluid (BALF) and pulmonary infiltrate and significantly decreased mortality without affecting viral and bacterial burdens (Amphiregulin (AREG), a member of the epidermal growth factor (EGF) family, was originally described as an epithelial- and mesenchymal-derived factor. Recent studies have shown that AREG can be expressed by multiple populations of activated immune cells, such as mast cells, basophils, group 2 innate lymphoid cells (ILC2s), and macrophages . The pro burdens . In addi burdens . These sToll-Like Receptor 4 (TLR4) is a member of the TLR family, which comprises germline-encoded receptors expressed by cells of the innate immune system . As an iIndolamine-2,3 dioxygenase (IDO) is a crucial intracellular enzyme in tryptophan (Trp) catabolism and related immune regulation . It can + T regulatory cells, further mediating immunosuppression and attenuating lung fibrosis (Aryl hydrocarbon receptor (AHR) is a ligand-operated transcription factor that is mainly expressed in barrier organs such as the skin, intestine, lung, and associated immune cells . AHR canfibrosis . Tryptopfibrosis . AHR canfibrosis . Loss offibrosis . Anotherfibrosis .IL-22 is a member of the IL-10 cytokine family and is mainly produced by T helper (Th) 17 cells, γδT cells, NKT cells, and innate lymphoid cells (ILCs). It can promote tissue regeneration and repair after lung injury by inducing proliferation and inhibiting apoptosis of epithelial cells . The proin situ monocyte-derived macrophage proliferation by promoting the production of IFN-α by pulmonary interstitial macrophages and controlling tissue damage in influenza A virus (IAV) infection  were induced. The absence of Treg cells leads to deterioration of chronic lung inflammation and aggravation of fibrosis (Regulatory T (Treg) cells are a special CD4ammation . Treg ceammation . Similarammation . Treg cefibrosis . In addifibrosis . The tisfibrosis . In a mofibrosis .Innate lymphoid cells (ILCs) are a group of tissue-resident innate immune cells that share characteristics with T lymphocytes . ILCs pavia connexin 43 (Cx43). Calcium ions transmit immunosuppressive signals between these two types of cells and inhibit the production of proinflammatory factors and neutrophil recruitment in LPS-induced inflammation  infections. However, enhanced Mtb antigen-specific T cell responses cannot effectively eliminate tuberculosis but cause death in mice. Tzelepis et al. found that mitochondrial cyclophilin D (CypD) controls the expansion of activated T cells and limits disease severity, contributing to disease tolerance , was found to induce Treg cells in the lung at a steady state and prevent lung inflammation (+ DCs in COPD patients were insensitive to LPS and favor the generation of IL-10-secreting CD4+ T cells which play an immunosuppressive role hinder T-cell effector functions (Dendritic cells (DCs) in the lungs comprise functionally distinct subsets. The aggregation and maturation of classical DCs in the lungs lead to an increase in neutrophil infiltration and Th1 cell activation after LPS stimulation, which aggravates pulmonary edema, acute pulmonary inflammation, and injury . DCs notammation . IDO, asammation . GITRL iammation . Dendritammation . CD1c+ Dunctions , this toKlebsiella pneumoniae-infected mouse model, monocyte MDSCs played a key role in reducing bacterial load and controlling lung injury by continuously producing IL-10  have a strong immunosuppressive ability and play an important role in immune regulation in cancer and chronic inflammatory diseases . In a Klng IL-10 . In the ng IL-10 . The rolng IL-10 .2+ signaling and inhibit the recruitment of neutrophils. Alveolar macrophages expressing TGF-β can induce naïve T cells to differentiate into Tregs. IDO secreted by DCs promotes tryptophan metabolism, and the metabolite (kynurenine) acts as a ligand of AHR to promote naïve T cells differentiation to Treg cells. Low-dose LPS induces the downregulation of GITRL in dendritic cells, which can also promote the differentiation of Treg cells. MDSC has a strong immunosuppressive ability and can also induce Treg cell differentiation and infiltration. In addition to inhibiting the lung tissue damage caused by inflammation, Treg cells can also promote tissue repair by producing AREG. AREG can also be generated by activated ILCs. IDO: indolamine-2,3 dioxygenase; DCs: dendritic cells; GITRL: glucocorticoid-induced tumor necrosis factor receptor ligand; MDSC: myeloid-derived suppressor cell; AREG: amphiregulin; ILCs: Innate lymphoid cells.Alveolar macrophages receive immunosuppressive signals from epithelial cells through Ca3.3E. coli, mice cultured at 22°C have better body temperature management and immunological consumption to ensure lower mortality. Simultaneously, the mice fasted the next day, recovered their body temperature and weight faster, and had reduced mortality without affecting the bacterial burden. The results show that energy-saving and low-metabolic states could promote tissue tolerance during bacterial infection , a member of the TGF-β superfamily, is associated with many biological processes such as cancer, obesity, energy homeostasis, and body weight regulation . GDF15 pMtb infection was observed in FTH-knockout mice. A decrease in FTH levels leads to an excessive inflammatory response, which aggravates the disease. Abnormal iron metabolism was also observed in the lung tissues of patients with tuberculosis  through oxidative stress, thereby preventing liver gluconeogenesis and resulting in hypoglycemia. The ferritin H chain (FTH) can resist this inhibition and maintain blood glucose in the range of host survival . This inrculosis . These sKlebsiella pneumoniae (Kp) infections may be achieved by promoting the metabolic pathway of disease tolerance in the host. Kp induces glutamine decomposition and fatty acid oxidation, creating an oxidant-rich microenvironment that promotes the accumulation of MDSCs and M2 macrophages. This process induces disease tolerance by inhibiting inflammatory responses. However, Kp survives by upregulating the type VI secretion system to adapt to airway oxidants -associated phagocytosis (LAP) is an important means of macrophage-mediated pathogen clearance and inhibits proinflammatory cytokine production during the innate immune response. Compared with young mice, elderly murine bone marrow-derived macrophages lack LAP, resulting in impaired bacterial killing and enhanced proinflammatory response after infection with umoniae . In a moumoniae . Reducedumoniae . In eldeumoniae .The renewal and differentiation of type II alveolar epithelial cells (AEC2s) are important for maintaining alveolar integrity and lung barrier function. Researchers have found that the rates of bronchiolar club progenitor cell self-renewal and differentiation decrease with aging. In addition, the overall density of type I cells was decreased, and changes in genes related to proliferation and differentiation were found in the gene expression profile of epithelial cells, which is associated with age . TelomerIn conclusion, an uncontrolled inflammatory response and decreased alveolar regeneration may be important reasons for impaired disease tolerance in the elderly.3.5Obesity has a significant influence on respiratory diseases. Obesity has marked effects on lung function, which is attributed to metabolic dysfunction and leads to increased susceptibility to infectious pathogens . Obesity3.6Staphylococcus aureus showed significantly reduced lung injury following influenza virus infection. Staphylococcus aureus recruits peripheral monocytes into the lungs. Monocytes differentiate into M2 alveolar macrophages and secrete anti-inflammatory factors that inhibit inflammatory responses and enhance tissue tolerance  leads to persistent immune paralysis, which affects the clearance of pathogens and secondary infections. These two phenomena have a fatal impact on patients through diverse mechanisms. They cannot be distinguished as they involve a variety of positive and negative feedback mechanisms, as well as processes related to disease tolerance .Endotoxin tolerance is an important mechanism that protects hosts against excessive inflammatory damage after exposure to endotoxins, such as LPS. Endotoxin tolerance is also considered an important mechanism that leads to immunosuppression in sepsis because of its negative regulation of the inflammatory response . ResearcLung LPS tolerance significantly reduces TNF-α expression in lung lavage fluid, but it does not affect the number of neutrophils, myeloperoxidase activity, or expression of chemoattractants such as CXCL1 and CXCL2, indicating that it not only inhibits the inflammatory response but also preserves the antibacterial ability of the lung . HoweverImmunoparalysis and disease tolerance are both states of immune hyporesponsiveness, and their mechanisms are complex. Many studies consider immunoparalysis to be a consequence of excessive tolerance in many studies. Whether there is an obvious boundary between the two definitions or to find a balance point, the host can not only maintain an efficient immune response but also be reasonably regulated to limit tissue damage.5Disease tolerance is one of the most important strategies of the host against infection, as it ameliorates both the pathogen-induced and inflammatory response-induced effects and limits damage to the host tissues. It occurs mainly by inhibiting the inflammatory response, promoting tissue repair, and regulating metabolism to maintain the integrity of the respiratory barrier and preserve lung function. Although many studies have provided a preliminary understanding of the mechanism of disease tolerance, it is still not known how to use the tolerance mechanism to effectively regulate the immune response. Disease tolerance is like a double-edged sword. It inhibits excessive inflammatory responses and protects the function of tissues and organs, but it also has the potential to affect pathogen clearance and induce immune paralysis. Therefore, accurately identifying the immune status of patients, limiting inflammatory injury by inducing tolerance, and changing the tolerance state to restore immune activity will be the focus of future research.The first draft of the manuscript was written by JX and NX, and the manuscript was revised by DZ and LX. All authors contributed to the article and approved the submitted version.


Exercise confers numerous salutary effects that extend beyond individual organ systems to provide systemic health benefits. Here, we discuss the role of exercise in cardiovascular health. We summarize major findings from human exercise studies in cardiometabolic disease. We next describe our current understanding of cardiac-specific substrate metabolism that occurs with acute exercise and in response to exercise training. We subsequently focus on exercise-stimulated circulating biochemicals (“exerkines”) as a paradigm for understanding the global health circuitry of exercise, and discuss important concepts in this emerging field before highlighting exerkines relevant in cardiovascular health and disease. Finally, this Review identifies gaps that remain in the field of exercise science and opportunities that exist to translate biologic insights into human health improvement. Regular exercise remains one of the bedrocks of human health promotion, benefiting cognition, immune and neurohormonal networks, and the musculoskeletal and cardiovascular systems. The multiorgan effects of regular exercise — including but not limited to improved cardiorespiratory fitness (CRF) — prevent and combat a broad spectrum of human diseases, including cancer, cardiometabolic disease, and frailty, and promote longevity –5. TheseHere we summarize major insights from human studies demonstrating the benefits of regular exercise and improved CRF for cardiometabolic health. We describe the regulation of substrate metabolism required across the spectrum of exercise, detailing the cardiac-specific substrate metabolism that underlies acute exercise and adaptations to exercise training. With this as background, we focus in particular on the emerging field of “exerkines,” and highlight exerkines relevant in cardiovascular disease. We subsequently discuss the challenges and knowledge gaps that remain in the field, as well as opportunities to translate the increasingly vast molecular information into knowledge that can improve human health.The growing epidemics of obesity, T2D, nonalcoholic fatty liver disease, and their downstream sequelae have highlighted the urgent need to decrease sedentary behavior and increase physical activity. Regular exercise — a broad term reflecting structured, purposeful physical activity  — has pr2max) and glucose homeostasis were seen in those undergoing high-intensity interventions , is less clear. Individuals with higher levels of CRF have lower risk of both incident AF and AF burden , 33, andOur understanding of the relationship between exercise and aortic disease is more limited, with cross-sectional data suggesting that high volumes of endurance exercise are associated with thoracic aortic dilatation . WhetherPerhaps no concept so clearly conveys the value of physical activity and regular exercise to human health as Charles Darwin’s “survival of the fittest” . While eThe ability to perform locomotor activity is wholly dependent on the skeletal muscle receiving a constant supply of adenosine triphosphate (ATP) for fuel. The body’s ability to make ATP available under conditions ranging from maximal-power-generating bursts to prolonged endurance exercise highlights its remarkable metabolic plasticity. Numerous dedicated reviews have described the processes that govern exercise metabolism and its adaptations to training –54. NotaO2) may increase up to 10-fold (2max) and decreasing during high-intensity exercise (75% VO2max)  , 64. Whi VO2max) , it may  VO2max) , 67. The VO2max) ; however VO2max) , 69. In  VO2max) .Since Randle et al.  first deIn addition to the pathways that govern the Randle cycle, the serine-threonine kinase 5′-AMP–activated protein kinase (AMPK) has emerged as an important metabolic regulator in the heart. AMPK has been described as a cellular energy gauge — stimulating fatty acid and glucose oxidation and glycolysis and inhibiting energy-consuming biosynthetic processes under conditions of high AMP/ATP ratios . Rat andBeginning with the landmark Dallas Bed Rest and Training Study, performed in 1966, numerous human studies have helped characterize the cardiac structural and functional adaptations that occur with exercise training –85. A ceImportantly, several changes in cardiac metabolism occur in response to regular exercise, and studies contrasting physiologic versus pathologic cardiac hypertrophy have greatly informed our understanding of the biochemical pathways that underlie them , 90. WhiHi and GlycoLo, respectively). As PFK2 is a central regulator of glycolysis through allosteric control of the enzyme PFK1, the authors showed that low PFK1 activity through expression of a kinase-dead PFK2 transgene (GlycoLo hearts) recapitulated physiologic cardiac hypertrophy — including a similar gene expression profile  signaling mediates physiologic hypertrophy but also metabolic adaptations after endurance training . Through profile . More re profile . Other p profile .Reductionist approaches to studying exercise metabolism have generated a wealth of knowledge regarding individual organs but, by their nature, have yielded fewer insights into how health benefits are communicated at the global level of an organism. The increased recognition of exerkines  — signalAn important consideration in the field is the temporal relationship between exercise bouts, training, and exerkine effects. While the acute and chronic effects of exercise-induced transcription at the skeletal muscle level have been well established , less isAs described above, exerkines represent a diverse group of compounds that enter the circulation through different means. It has long been established that proteins with a secreted signal peptide sequence  can be stimulated by exercise and released into the circulation and impart health effects . SimilarExercise and increased physical activity provide protection against the development of CVD that extends well beyond that expected from their effects on traditional risk factors . Indeed,The diverse effects of exerkines on metabolic and vascular health coupled with increasing data supporting interorgan crosstalk with the heart  support 12,13-Dihydroxy-9Z-octadecenoic acid  is an oxidized linoleic acid metabolite secreted by brown adipose tissue (BAT) that was recently shown to increase significantly in male and female human subjects after cold (14°C) exposure as well as in response to an acute bout of moderate-intensity exercise , 144. 12Perhaps the most well-recognized myokine, IL-6 is an important metabolic regulator in skeletal muscle whose exercise effects were briefly described above and have been extensively reviewed elsewhere , 121. Mo–/–Apoe mouse models  is an additional immunologic protein and unique chemokine that is both membrane-bound and secreted into the circulation. Human studies have shown that acute bouts of endurance exercise lead to significant increases in both skeletal muscle mRNA and protein levels — mainly in endothelial cells — as well as circulating protein levels , 148. Fre models , 150. Hoe models . Thus, aMurine studies of myonectin, a member of the C1q/TNF-related protein (CTRP) family, have shown that its skeletal muscle expression and circulating levels increase in response to regular aerobic exercise , 153. Us+2/Akt/FOXO1 signaling pathway. Using a murine musclin (Ostn) knockout model, the authors then showed that Ostn-knockout mice had reduced exercise tolerance despite exhibiting an otherwise normal resting metabolic phenotype, and that musclin administration “rescued” their exercise capacity. Ostn-knockout mice demonstrated reduced VO2max after 5 days of training, an effect mediated by musclin’s potentiation of the effects of atrial natriuretic peptide (ANP) on cyclic guanosine monophosphate (cGMP)/PGC-1α regulation and subsequent mitochondrial biogenesis (Ostn-knockout mice demonstrate exaggerated cardiac dysfunction in a murine pressure overload (transverse aortic constriction) model, and that AAV6-mediated skeletal muscle musclin overexpression attenuates cardiac dysfunction and myocardial fibrosis in the same model  cohort of young, sedentary male human participants after acute resistance exercise (Angiopoietin-1 (ANG1) is a secreted member of the angiopoietin/TIE growth factor receptor pathway that mediates vascular protection through inhibition of vascular inflammation and plasma leakage and attenuates fibrosis . Its thetraining ; howevertraining . Similarexercise .Despite the many advances in our understanding of the substrate metabolism involved in exercise performance and adaptation, as well as emerging knowledge of the whole organ circuitry involved, numerous gaps in the field remain .2max? Many questions stem, in part, from intra- and interindividual differences in exercise training response according to clinical outcome , sex differences, and evolutionary biology. These concepts have been previously reviewed . Second,Similarly, enthusiasm for findings regarding the role of exerkines in human health and disease is tempered by many questions. What are the effective concentrations of candidate exerkines needed to promote physiologic effects in humans? What are the temporal relationships of exerkines across acute exercise bouts compared with regular exercise? What are the specific tissue sources of exerkines, and how do they differ from the resting  state?These challenges highlight the rich opportunities to advance the field of exercise biology.A convergence of factors, including increased appreciation for exercise’s pleiotropic health benefits, renewed commitment to its study, and the dissemination of high-throughput molecular profiling technologies, has led to a surge of new information in exercise biology. How to harness this information and translate findings into therapeutic advances remains an important challenge. Several approaches may help bridge this gap and inform future directions in the field .First, while much of the molecular biology work performed thus far has focused on single organ systems and/or used one, or possibly two, omics-based approaches, it is crucial to develop a molecular “road map” of exercise’s biochemical effects given the maturity of these technologies. Efforts to do so are ongoing, most notably in the NIH’s Molecular Transducers of Physical Activity Consortium (MoTrPAC) study, a multicenter, clinical trial of resistance and endurance exercise training that aims to generate such a road map across the plasma, skeletal muscle, and adipose tissue in sedentary and highly active human subjects, and across animal studies using genomics and epigenomics, proteomics and phosphoproteomics, metabolomics, and transcriptomics among other molecular profiling methods , 167. ThSecond, connecting observations made in exercise intervention studies with longitudinal studies of human health to assess long-term outcomes can help triage findings. The example of exercise response biomarkers helps illustrate this concept. While far from ready for clinical adoption, several studies highlight the potential of using molecular biomarkers to predict an individual’s responsiveness to a given exercise intervention  , 169. MoIn the same vein, large human genetic studies may also provide opportunities to assess causality for an exercise-relevant biochemical or molecular pathway. As technological advances have enabled researchers to perform molecular profiling techniques at very large scale, Mendelian randomization experiments have been increasingly used to assess putative causal relationships between genetic variation, intermediate molecular phenotypes , and health outcomes –177. TheFinally, retro-translating human exercise findings to animal and model systems is important not only to generate mechanistic insights, but also to establish the tissue source of exerkines. Indeed, as discussed above, many of the known exerkines  and undoubtedly many yet-to-be-identified exerkines are expressed in multiple tissue sources. While the study of extracellular vesicles in the transport of exercise-secreted proteins and microRNAs has provided important insights into their relevance , little Our understanding of the organ- and even cell-specific activity involved in the cardiometabolic adaptations to exercise has evolved considerably over the past several decades, fueled by advances in molecular and cellular biology. These gains have helped reveal the extraordinary metabolic plasticity that enables such a broad spectrum of human locomotor activity — from short, maximal-intensity bursts of activity to ultra-endurance athletic events. While the skeletal muscle, liver, and adipose tissue remain central actors, the heart remains a crucial engine whose study may lead to novel therapeutic advances in cardiometabolic health and disease. Integrative multi-omics and systems biology approaches to studying exercise have helped unmask the complex whole-organism circuitry that is marshalled in response to physical activity, and offer promise to understand the systemic health benefits that this circuitry provides at the molecular level. Much work remains to be done in the field of exercise science, and the gaps that remain should be viewed as an enormous opportunity for the medical community. And finally, it remains important to emphasize the public health message that any physical activity is better than none while working toward a more complete understanding of the molecular and biochemical pathways that underlie exercise’s rich health benefits.


Dupilumab is a monoclonal antibody inhibiting key drivers in the inflammatory signaling cascade. It has been considered a saving grace for many patients suffering from severe atopic dermatitis and has quickly become a widespread treatment option for many inflammatory pathologies. However, recent reports have linked dupilumab with exacerbating symptoms and accelerated disease progression of cutaneous T-cell lymphoma (CTCL). This report presents the case of a 60-year-old African American female who was diagnosed with CTCL three months after her initial treatment with dupilumab for presumed atopic dermatitis. An appreciation of this unique relationship, as well as the morphological differences seen in patient presentation, is important for physicians to better guide timely diagnoses while working to uncover the true relationship between dupilumab and CTCL. Dupilumab is a monoclonal antibody that antagonizes the alpha subunit of interleukin-4 (IL-4) and interleukin-13, thereby inhibiting key drivers in the inflammatory signaling cascade . It has A 60-year-old female with a past medical history of eczema, gastroesophageal reflux disease (GERD), fibromyalgia, type 2 diabetes mellitus, and osteoarthritis presented to the emergency department due to a worsening rash that had spread all over her body. The patient had a three-year history of mild-to-moderate eczema without any identifying triggers. She started dupilumab for her eczema three months prior to the current admission due to a persistent rash of the same intensity. However, since starting dupilumab therapy, the patient experienced a worsening rash with fluctuating periods of intensity. She previously visited the emergency department (ED) one month after starting dupilumab treatment due to worsening itch with spreading erythematous rash and was given a course of systemic steroids and antibiotics that showed partial resolution of symptoms. Her last injection of dupilumab was administered three weeks prior to her current presentation. Since the last dose, she had experienced a diffuse, desquamating rash without pustules, sloughing, or blistering. It initially appeared on her arms and thighs and gradually spread, now covering her entire body from scalp to toes, including areas on the lips and vulva Figure . Her lowShe was hemodynamically stable and afebrile at presentation but exhibited significant atypical lymphocytosis. Additionally, new palpable lymphadenopathy was observed in the axillary and inguinal regions. Skin biopsies were performed, and the leukemia and lymphoma panel indicated 26.8% CD4+/CD7- cells. The patient was diagnosed with cutaneous T-cell lymphoma (CTCL) and started on a daily dose of 25mg of acitretin. She was also referred for extracorporeal photopheresis (ECP).Dupilumab is a monoclonal antibody that antagonizes the interleukin-4 (IL-4) alpha subunit and interleukin-13, thereby inhibiting key drivers in the inflammatory signaling cascade . It has It is important to note that other documented cases have also reported a paradoxical worsening of skin scaling, lymphadenopathy, severe pruritis, and extension of rash borders one to two months after the initial administration of dupilumab ,5. The pAdult-onset atopic dermatitis affects approximately 1%-3% of the general population. Its diverse symptoms and stubborn nature make it challenging to diagnose, often necessitating additional diagnostic studies, such as a biopsy of the affected skin . Since tThe approval of dupilumab for the treatment of moderate-to-severe atopic dermatitis has led to a significant expansion of its usage and exploration in various dermatological disorders. Consequently, it is crucial to provide medical professionals with an understanding of a potentially detrimental pitfall of dupilumab. This becomes increasingly important as dupilumab becomes a widely adopted treatment option for numerous inflammatory dermatological conditions, including atopic dermatitis. Furthermore, we aim to shed light on the variances in patient presentation in the skin of color patients with CTCL. The heightened morbidity and delayed treatment observed in such patients should prompt clinicians to recognize and educate themselves about morphological differences. Further studies are needed to evaluate this possible relationship and uncover the cellular mechanisms behind CTCL and dupilumab.


Cat scratch disease (CSD) is a benign condition caused by the inoculation of Bartonella henselae. The imaging findings are non-specific, and it is difficult to diagnose the disease via imaging. However, imaging studies help exclude other differential diagnoses in diagnostic dilemmas. We encountered a case of a 17-year-old adolescent who presented with painful neck swelling. CT showed multiple bilateral cervical lymphadenopathies with triangular soft tissue mass at the anterior mediastinum likely to be thymic hyperplasia, which is unusual in CSD and was mistaken for a lymphoproliferative disorder. Tissue diagnosis with a thorough clinical history yielded the diagnosis of cat scratch disease, and follow-up imaging showed resolution of the cervical lymphadenopathy and thymic hyperplasia. A 17-year-old adolescent presented with painful right-sided neck swelling for two weeks; this gradually increased in size and was associated with right otalgia. The patient had no history of ear discharge or skin redness and denied any history of fever or contact with tuberculosis patients. However, he had an episode of upper respiratory tract infection before he developed otalgia. Examination of the patient revealed multiple tender, enlarged bilateral cervical lymph nodes measuring up to 1 cm in size. Otoscopy examination revealed an intact tympanic membrane with no evidence of inflammation. No enlarged lymph nodes were palpable elsewhere, and there was no hepatosplenomegaly. Patient then proceeded with contrast-enhanced computed tomography (CECT) neck .