Existing concussion education programs for preteen athletes typically do not result in sustained improvements in concussion symptom recognition or reporting behaviors. Virtual reality (VR) technology offers an innovative tool that may improve concussion symptom recognition and reporting behaviors among preteen athletes.We aimed to describe the design and development of a VR concussion education app, Make Play Safe (MPS), and present findings on the usability and preliminary efficacy of MPS in improving concussion recognition and reporting intentions among soccer athletes aged 9-12 years.A collaborative user-centered design process was implemented to develop and evaluate MPS, a semi-immersive VR concussion education app designed to address two behavioral outcomes in preteen athletes aged 9-12 years: (1) recognizing concussion and (2) reporting concussion. The development of MPS occurred in three phases: (1) design and development, (2) usability testing, and (3) preliminary efficacy testing. During phase 1, consultations were completed with 6 experts. Additionally, 5 interviews with children who had a history of concussion were conducted to collect feedback about the proof of concept of MPS. During phase 2, a participatory workshop with 11 preteen athletes and a small group discussion with 6 parents and 2 coaches were conducted to explore the usefulness and acceptability of MPS from the perspective of end users. Finally, phase 3 included preliminary efficacy testing with 33 soccer athletes aged 9-12 years to examine changes in concussion-related knowledge, attitudes, and reporting intentions from pre- to postintervention. The data generated from each phase of this study informed the development of the final version of the proof of concept of the VR concussion education app, MPS.P<.05), while changes in attitudes toward reporting concussions were not (P=.08).Experts positively rated the features of MPS and noted that the design and content were innovative and age-appropriate. Preteens with a history of concussion indicated the scenarios and symptoms portrayed in the app represented well what they experienced while concussed. Further, they stated that the app would be an engaging way for children to learn about concussions. The 11 healthy children in the workshop perceived the app positively, noting that the scenarios were informative and engaging. Results from preliminary efficacy testing revealed increases in many athletes’ knowledge and reporting intentions from pre- to postintervention. Others demonstrated no significant changes or a decrease in knowledge, attitudes, or reporting intentions from pre- to postintervention. Group-level changes in concussion knowledge and intention to report concussions were statistically significant (Results suggest VR technology may be an effective and efficient tool to equip preteen athletes with the requisite knowledge and skills to recognize and report future concussions. Further research is recommended to examine the use of VR as an effective strategy to improve concussion-reporting behaviors in preteen athletes. Organized sports are extremely popular among children, with approximately 36 million US school-aged children participating in at least one organized sport each year . Sports Concussion, a type of mild traumatic brain injury, is a public health concern among children and adolescent athletes . ApproxiMultiple strategies, including both primary and secondary prevention strategies, have been designed and implemented to prevent concussions in sports and change the culture of concussion reporting ,21. One While these traditional approaches can help children and adolescents learn about concussions -26, theyVirtual reality (VR) technology, which enables users to have a multisensory experience in a simulated and all-encompassing interactive 3D world, offers several advantages over traditional and existing approaches to concussion education. First, it engages preteens by allowing them to “experience” concussive events and symptoms without the risk of actual injury in a safe, computer-generated, immersive, and 3D environment that resembles the real world. Second, VR technology is underpinned by the principles of the Experiential Learning Model, which emphasizes the importance of learning by doing and posits that knowledge is constructed through physical interaction ,29. It aVR technology has been extensively used in other health domains, including child pedestrian safety training -35, the MPS is a semi-immersive VR concussion education app designed to address two primary behavioral outcomes of athletes aged 9-12 years: (1) recognizing concussion signs and symptoms and (2) reporting a suspected or actual concussion to an appropriate adult . The version of the MPS app developed and then evaluated in this report begins with a brief introduction to concussion, providing the definition of concussion, describing concussion signs and symptoms, and explaining why it is important to report concussion symptoms to an appropriate adult. After the introduction, app users are directed through a scenario in which they virtually experience a concussive event while playing soccer. Specifically, preteens are immersed in the virtual environment as a soccer player playing on an outdoor soccer field when a virtual soccer ball unexpectedly hits them in the head, causing them to start experiencing various simulated concussion symptoms  both on the field and after virtually returning home. Filmed video clips and scenarios of actual preteen soccer players reinforce the virtual-to-physical relationship by allowing users to see familiar, relatable projections of themselves and their environment in the virtual space.MPS was developed using a collaborative user-centered design process that involved VR experts, a clinician-researcher with expertise in concussion assessment and management, injury prevention scientists, psychologists with expertise in developmental and educational psychology, and a health education specialist. As detailed below, development occurred in three phases: (1) design and development, (2) usability testing, and (3) preliminary efficacy testing .Before the development of MPS, a literature review on existing concussion education programs was conducted. Shortcomings and lessons learned were noted to guide the development of the initial proof of concept. The MPS app was designed for athletes aged 9-12 years, with an initial focus on soccer athletes, as soccer is a sport with a high risk of concussion  and a spPhase 1 identified the key features of effective VR technology needed to ensure the MPS app simulated lifelike concussion symptoms and scenarios.Six consultations with experts in design, communication, developmental psychology, sports medicine, and technology were conducted. These interviews had two goals: (1) to collect feedback about the initial proof of concept MPS app, including the appropriateness of the information for the target age group and relevance to pediatric concussion, and (2) to elicit recommendations on how to improve the visual and sensory design, the content, and the scenarios presented in the proof of concept app. The consultations took place from December 2019 to January 2020. Experts were identified by the research team.Prior to the consultation, experts completed a preconsultation questionnaire that solicited recommendations for essential elements and features of VR apps as well as how to make VR apps available to and engaging for the target audience. During the consultations, experts viewed the proof of concept MPS app and then provided feedback on the app, including its features, the simulation of concussion symptoms, and the scenarios presented. Finally, after viewing the proof of concept of MPS, experts completed an app experience form to rate their experiences with MPS on a 5-point scale (1=positive to 5=negative), to describe their emotions while using the app, and to indicate the features they “liked most” and “liked least.” All consultations were conducted by a qualitatively trained member of our research team and were audio-recorded and transcribed.Five interviews with children and adolescents  who had a history of a physician-diagnosed concussion were conducted. The purpose of these interviews was threefold: (1) to collect feedback about the proof of concept MPS app , (2) to examine the accuracy of the concussion symptoms and scenarios presented in the proof of concept of MPS , and (3) elicit recommendations on how the visual design, content, and scenarios could be improved.Participants were identified through other hospital concussion studies and invited to participate. Inclusion criteria included a diagnosis of concussion within the past 12 months and full recovery from their concussion by the time of their interview. Prior to each interview, written and verbal assent or consent was obtained from participating children and a parent or legal guardian, respectively. Children and adolescents then viewed the MPS VR app session and provided feedback on MPS in four domains: (1) lessons about concussion, (2) their experience during the app, (3) the simulation of concussion symptoms, and (4) the design of the app. All interviews were conducted between November 2019 and January 2020 using an interview guide developed specifically for this purpose. Each interview lasted approximately 30 minutes and was audio-recorded and transcribed.Based on feedback received during this phase of development, a prioritized list of app updates and feature design modifications was created. The highest-ranked priorities for modification included (1) adding onboarding instructions with a tutorial to familiarize users with the virtual environment and how to get started with the app, (2) enhancing the delivery of the symptoms directly simulated by the MPS app  so that users can feel and experience them more, (3) adding age-appropriate educational interludes and a branching decision tree that leads to different outcomes depending on the decisions made within MPS , and (4) creating a conclusion or ending for the app that reiterates signs and symptoms of concussion and what to do if the user experiences a suspected concussion in the future.To evaluate the usability and acceptability of MPS by preteen athletes and their parents and coaches, a workshop with athletes and a small group discussion with parents and soccer coaches were conducted.Soccer athletes aged 9-12 years old participated in an activity-based workshop designed to elicit design ideas and preferences for a VR app that educated preteens aged 9 to 12 years about sport-related concussions in a relevant and engaging way. A total of 11 preteen soccer athletes participated. Following written informed consent (parents) and assent (children), participants were assigned into small groups of 3-4 participants and guided through a series of activities. These activities included “Scenarios and Storytelling,” where children engaged in role-play portraying a concussed athlete and walked through the decision to disclose or not disclose symptoms of a suspected concussion; and “Virtual Reality Watching,” where each athlete individually viewed the proof of concept MPS app. After completing these activities, in researcher-led small group discussions, the preteens shared their views toward and acceptance of MPS as well as provided recommendations on how to improve the app. The 1-hour workshop was conducted at a local community center.Six parents and 2 soccer coaches participated in a small group discussion led by members of our research team. Consent for participation and permission to take notes were obtained from each participant prior to the 40-minute small group discussion. After viewing the MPS proof of concept, parents and coaches took part in a script-guided discussion and were provided an opportunity to give general feedback on MPS. Discussion questions centered on parents’ and coaches’ views and experiences regarding preteen concussion education, perceived disadvantages for their child or team to receive concussion education via a VR app, and feedback on the current proof of concept of MPS, including visual app navigations, features, and core functionalities, as well as suggestions for app improvement.To evaluate the preliminary efficacy of MPS in improving concussion symptom recognition and reporting, a pre- or posttest design was conducted with 33 preteen athletes.All aspects of the study were approved by the institutional review board at Nationwide Children’s Hospital (IRB17-00260). Written informed consent (parents) and assent (children) were obtained from study participants prior to the study procedures. All participant data were deidentified to ensure the privacy and anonymity of participants. Participants were compensated with a US $25 ClinCard (Greenphire Inc) upon completion of the study.Soccer leagues and clubs in central Ohio were contacted via email and asked to disseminate information about the study to families with a child aged 9-12 years participating in a soccer league. Participants were also recruited by word of mouth and snowball sampling. Interested families contacted the research team to learn more about the study. Following eligibility screening, written consent and assent were obtained from interested parents or guardians and preteens, respectively. Children aged 9-12 years were eligible if they played organized soccer in the past 12 months and spoke English.A 1-group pre- and posttest design was used with 33 children. Children completed a preintervention survey to examine concussion-related knowledge, attitudes toward concussion reporting, and reporting intentions. They then completed the 10-minute MPS VR educational session using a VR headset and app, with guidance provided by a researcher as needed. After completing the training, children completed the postintervention survey assessing concussion-related knowledge, attitudes toward concussion reporting, and reporting intentions. Data were collected from June to December 2021.Concussion knowledge was assessed pre- and postintervention using an adapted version of the Rosenbaum Concussion Knowledge and Attitudes Survey–Students (RoCKAS-ST) . The surAttitudes toward concussion reporting were assessed pre- and postintervention. Participants rated their views toward 8 statements  using a 5-point scale from 1=strongly disagree to 5=strongly agree. A total attitude score was calculated by summing the 8 responses (possible range 8-40), with higher scores indicating more favorable attitudes toward concussion reporting. These questions were developed by pediatric concussion researchers and pilot-tested prior to use in this study.Concussion reporting intentions were measured using the reporting behaviors and intentions section of the RoCKAS-ST . ChildreThe following demographic variables were also collected from participants: sex, age, medical history of concussion (yes or no), and prior concussion education received (yes or no).Demographic characteristics of the preteens were described, and descriptive data were considered. The methods of open coding , axial cChildren and adolescents with a history of concussion indicated that the scenarios and symptoms portrayed in MPS were very similar to those they experienced in real life. They reported the app would be a helpful and engaging way for preteen athletes to learn about concussions because it allowed them to experience various real-world simulated concussion symptoms both on the simulated soccer field  as well as after returning to their virtual home. Participants also provided some recommendations on how the app could be improved, such as by adding instructions on how to navigate the app .Preteen athletes in the workshop were receptive to a VR app that taught them concussion signs and symptoms and the importance of concussion reporting. They indicated that they liked the MPS VR session because the scenarios presented were realistic, making them feel like they were on an actual soccer field. They also reported that they enjoyed being able to experience concussion signs and symptoms in a virtual environment. Data collected from end users were incorporated into the design of the updated proof of concept, as described below.Parents and coaches in the small group discussion reported that they enjoyed the storyline portrayed in the MPS app and felt that the MPS educational session was informative, realistic, and age-appropriate. Parents and coaches reported that the MPS app would prepare children to successfully recognize and respond to future concussions. Parents and coaches also noted that the completion of MPS would help facilitate communication about concussion safety, which may enhance children’s understanding of the importance of concussion reporting.A total of 33 preteen soccer players participated, including 20 (61%) boys and 13 (39%) girls. Of the 33 participants, 14 (42%) were 9 years old, 10 (30%) were 10 years old, 5 (15%) were 11 years old, and 4 (12%) were 12 years old. The mean concussion knowledge score significantly increased from 12.2 (SD 1.9) to 12.9 (SD 1.9) from pre- to postintervention. An increase in concussion knowledge from pre- to postintervention was demonstrated by 18 (55%) participants, whereas 7 (21%) demonstrated no change in concussion knowledge and 8 (24%) showed a decrease in concussion knowledge during the same period .P=.08. More favorable attitudes toward concussion reporting from pre- to postintervention were demonstrated by 17 (52%) preteens, 7 (21%) showed no change in attitudes, and 9 (27%) reported less favorable attitudes toward concussion reporting following the intervention.There was no statistically significant increase in mean attitudes toward concussion reporting scores from pre-  to postintervention , Finally, there was a significant increase in mean concussion reporting intention scores from pre-  to postintervention . Increased intentions to report future concussions from pre- to postintervention were reported by 16 (49%) participants, while 7 (21%) showed no change in reporting intentions, and 10 (30%) reported decreased concussion reporting intentions following the intervention.This pilot study reports the 3-phase process to design, develop, and examine the feasibility and preliminary efficacy of MPS, a VR concussion education app. We used a systematic and iterative process to develop a proof of concept for the VR app intervention. Our findings showed that it was well accepted by preteen athletes as well as their parents and coaches. The findings also showed that the intervention had a positive effect on many preteen athletes’ concussion-related knowledge and reporting intentions immediately following the concussion education VR intervention. These results provide support for the continued development, refinement, and evaluation of MPS as a VR app to educate and train preteen athletes to recognize and report signs and symptoms of concussion.The use of VR technology to teach preteens about concussions offers many potential advantages over traditional approaches to concussion education. First, unlike traditional concussion education approaches, VR technology allows athletes to virtually feel and experience what it is like to have a concussion in a safe, computer-generated environment ,46. VR aOur findings highlight the value of using a systematic and collaborative user-centered approach to design and develop intervention programs like VR technology for children. Active engagement from experts and end users contributed valuable insights that guided the iterative development of our proof of concept app, including recommendations on how to make the app more engaging and to increase the realism of the concussion symptoms simulated within the app. The final proof of concept app was accepted by our target audience and offered an engaging strategy to equip preteens with the requisite knowledge, skills, attitudes, and intentions to recognize and respond to future concussions. The participatory approach used to design and develop MPS could be applied to the development of other interventions for children, including those incorporating VR technology.We found improvements in many preteens’ concussion-related knowledge, attitudes toward reporting, and reporting intentions following the VR session, although the effect of the intervention on changes in attitudes from pre- to postintervention was not statistically significant. Prior studies suggest that increased concussion-related knowledge, favorable attitudes toward concussion reporting, and reporting intentions are associated with a decreased likelihood of continuing to play in a game or practice while symptomatic from a concussion ,49. HoweSeveral limitations of this pilot study warrant attention. First, the sample size was small; the results may not generalize to other populations of athletes. Second, we did not include a comparison or control group. Third, we did not examine the effectiveness of the MPS app in improving actual concussion-reporting behaviors or monitor changes in concussion-related knowledge, attitudes toward reporting, or reporting intentions over time following the intervention. Prior to the broad-based implementation of concussion education via VR technology, future studies should determine the effectiveness and dosage of intervention required to improve preteens’ concussion symptom recognition and reporting behaviors using a more rigorous evaluation design with a larger sample. Fourth, the adapted survey we used was not validated in preteens aged 9-12 years. Psychometric research should be conducted to develop and validate instruments to examine concussion-related knowledge, attitudes, reporting intentions, and behaviors in this population of athletes.This study provides insights into how we can use VR technology to teach preteens about concussions as well as highlights the importance of using an iterative, user-centered approach when designing VR technology for preteens. Our findings suggest that VR technology may be an effective tool to teach preteens about concussions and thereby reduce the negative and potentially long-lasting health consequences of concussions by increasing concussion symptom recognition and reporting among preteen athletes. However, further research with a larger, more diverse sample is needed to determine the effectiveness of VR technology in improving concussion-reporting behaviors and ensure that such technology has a positive effect on preteens. This study provides a critical first step in understanding how VR technology can be used to improve concussion symptom recognition and reporting behaviors among preteen athletes and should be extended with further research.


The importance of the epidermal barrier as a multifaceted mechanistic unit supporting whole-body homeostasis continues to grow in prominence. The spotlight again points towards this vital barrier tissue with the recent realization of significant importance for novel aspects in preventing skin diseases and skin infections, that have been tightly associated with atopic dermatitis (AD) and psoriasis. Unchecked, percutaneous defects progress to atopic diathesis and linked systemic manifestations. To this end, the skin provides interlinked mechanical, immunological, and antimicrobial protection, formed through keratinocyte differentiation, generating a multi-layered tissue with a structural and protective function. In addition, multiple cell types including keratinocytes, melanocytes, and immune cells crosstalk within the skin to create the first line of protection against pathogens and environmental threats, through the production of immunological mediators, recruitment of humoral and cellular immunity as well as the hosting of a commensal microbiome. The goal of this Research Topic was to bring together the latest advances and insights of epidermal biology, emphasizing both its immunological and physical aspects, providing a platform to present novel findings and summarise current knowledge, from both basic science and the clinical perspective. To this end, ten publications were accepted covering a broad range of research areas for epidermal barrier function in the form of 4 original articles and 6 reviews.STS) gene resulting in a cutaneous phenotype but also additional syndromic consequences. The mechanisms related to X-linked ichthyosis are still unclear and therefore McGeoghan et al. investigated the transcriptomic and lipidomic profiles related to STS gene deficiency. Findings included major alterations in epidermal differentiation and lipid metabolism as well as differentially expressed genes (DEGs) related to corneal transparency and behavioural disorders that are syndromic with the cutaneous phenotype. This work provides novel pathomechanistic data that will be important for future studies on X-linked ichthyosis.Multiple functions related to effective epidermal homeostasis has been implicated in various skin diseases. Protein and lipid-related barrier functions are associated with common, complex disorders such as atopic dermatitis (AD) but also many rarer and monogenic disorders with gene defects that result in ichthyosis or scaling phenotypes of the epidermis. One such condition is recessive X-linked ichthyosis. This disorder is caused by genetic defects in steroid sulfatase  gene, a skin barrier protein expressed in the upper layers of the epidermis. There is still much work to be done to further investigate DEGs in inflammatory skin diseases such as AD and Li et al., have taken a bioinformatics approach to this challenge. To identify potential effective diagnostic biomarkers in AD the team investigated gene expression from the Gene Expression Omnibus (GEO) database using comprehensive bioinformatics analysis. They identified biomarkers that were subsequently validated with immunohistochemistry showing CCR7, CXCL10, IRF7, MMP1, and RRM2 as potential biomarkers with diagnostic value. Finally, CIBERSORT algorithm was used to evaluate immune cell infiltration with CCR7 correlating with high levels of CD4+ naïve T cells.Atopic dermatitis (AD) is a chronic inflammatory skin disease with an itchy erythematous rash. The pathomechanism of AD is complex with major contributions from the skin barrier, inflammation and the exposome. The complex genetics of AD has revealed over 50 potential loci that are associated with the phenotype of which the strongest genetic risk factor is Dainichi et al., and co-workers have identified an orphan receptor, G protein-coupled receptor 15 ligand (GPR15L), encoded by C10Orf99 gene as an important protein increased in frequently occurring skin diseases atopic dermatitis (eczema) and psoriasis. Over-expression of GPR15L reduces the expression of cornified envelope and keratinocyte differentiation markers keratin 10, filaggrin and loricrin, and increased the inflammatory response. Gene expression analyses revealed that GPR15L upregulated pathways and cognate transcription factors involved in signal transduction and stress inducible transcription, while reducing lipid metabolism, all responses that have previous been associated with skin barrier dysfunction.Continuing the theme on skin inflammation, Kobiela et al., has reported alteration of cellular communication originating from epidermal keratinocytes, as the effect of allergic milieu and AD-relevant skin pathogens. Specifically, the study determined that the combined presence of AD cytokines and a common AD pathogen, C. albicans, resulted in an increased propensity of small extracellular vesicles (sEVs), a vehicle of long-distance communication, secreted by keratinocytes to interact with immunosuppressive Siglec receptors. The study also implicated the upregulation of specific enzymes involved in cellular glycosylation, i.e., β-Galactoside α-2,6-Sialyltransferase 1 (ST6GAL1) and Core 1 β,3-Galactosyltransferase 1 (C1GALT1) in remodelling of sEV surface glycans to allow for this keratinocyte related signalling effect to take place.The study by Baquero et al., who discussed the dynamic interaction between the epidermis and microbes and involving mechanisms of innate cell control. The authors explained how the threshold control is executed over overgrown pathogenic populations while allowing commensals to re-establish the original population density. The review further discussed the role of the epidermis in pathogen transmission and the factors behind interindividual variability in transmission efficiency.The theme of the microbiome is further addressed in the review by Visscher et al., discusses the formation of the epidermal barrier from the foetal stage through birth and into adolescence, encompassing detailed information on the process of barrier maturation. The authors highlight the remodelling of the barrier proteome and lipidome as well as the importance of the vernix at the various life stages, and also touch on the role of the skin microbiome evolution past birth. The review by Çetinarslan et al. and co-workers provide an update on the contribution of microbiome imbalance, or “dysbiosis”, in atopic dermatitis (AD). This covers the complex interplay between innate immunity, lipid metabolism and dysbiosis, discussing the microbiome beyond the role of Staphylococcus aureus.The manuscript by Szondi et al. and co-workers, the multiple roles of Arginase 1 are discussed, including the well-characterised role in macrophage polarity, and also pro- and anti-inflammatory responses via innate immunity modulation. The review is concluded by describing how defects in Arginase 1 or Arginase 1 function can alter the microbiome of wounds contributing to chronic and non-healing wounds.Arginase 1 is a key modulator of the skin microbiome but also regulates various mechanisms related to wound healing. In the review by MacLeod et al., provides a comprehensive description of the multiple functions of the IL-1 family in detecting external threats, controlling physical barrier function and inflammation. The authors also describe the deleterious effects on skin immunity when the IL-1 signalling pathways are defective. Finally, the potential role the IL-1 family has for translational therapeutics is reviewed with perspectives for future developments.Another key component of innate immunity is the IL-1 family of cytokines. The review by In conclusion, the articles in this Research Topic present original findings and aggregate prior knowledge on the novel aspects of the epidermal barrier. Appreciation of the importance of the epidermal barrier in physiology and pathology provides an important basis for development of new therapeutic strategies to address the diverse patient groups suffering from epidermal-associated skin diseases and their linked co-morbidities.


Oxidative stress participates at the baseline of different non-communicable pathologies such as cardiovascular diseases. Excessive formation of reactive oxygen species (ROS), above the signaling levels necessary for the correct function of organelles and cells, may contribute to the non-desired effects of oxidative stress. Platelets play a relevant role in arterial thrombosis, by aggregation triggered by different agonists, where excessive ROS formation induces mitochondrial dysfunction and stimulate platelet activation and aggregation. Platelet is both a source and a target of ROS, thus we aim to analyze both the platelet enzymes responsible for ROS generation and their involvement in intracellular signal transduction pathways. Among the proteins involved in these processes are Protein Disulphide Isomerase (PDI) and NADPH oxidase (NOX) isoforms. By using bioinformatic tools and information from available databases, a complete bioinformatic analysis of the role and interactions of PDI and NOX in platelets, as well as the signal transduction pathways involved in their effects was performed. We focused the study on analyzing whether these proteins collaborate to control platelet function. The data presented in the current manuscript support the role that PDI and NOX play on activation pathways necessary for platelet activation and aggregation, as well as on the platelet signaling imbalance produced by ROS production. Our data could be used to design specific enzyme inhibitors or a dual inhibition for these enzymes with an antiplatelet effect to design promising treatments for diseases involving platelet dysfunction. Oxidative stress has been associated with many pathologies such as cancer, diabetes mellitus, and cardiovascular diseases due to an increase in the production of reactive oxygen species (ROS) ,2. It isPDI is expressed in almost all mammalian tissues and is detected in the endoplasmic reticulum, nucleus, cytosol, cell surface, and extracellular space , playingPlatelets contain four members of the PDI family . These isoforms have been identified on the surface of resting platelets, or secreted and recruited to the platelet surface in response to platelet activation ,12. P4HB2.−) or its dismutation product hydrogen peroxide (H2O2) can act as second messengers when produced in excess can cause oxidative stress and cellular dysfunction [.NO) and serve as a precursor for other species such as H2O2 and peroxynitrite [NOX is the major contributor of ROS in cells; either superoxide  significantly contribute to ROS production and are considered responsible for platelet activation . Both isThe current manuscript aims to perform a complete bioinformatic analysis of the role and interactions of PDI and NOX in platelets, as well as the signal transduction pathways involved in their effects. In addition, we sought to examine whether these proteins collaborate to control platelet function. These data could be of great relevance for designing specific enzyme inhibitors or dual inhibition of both enzymes with an antiplatelet effect to design promising treatments for diseases involving platelet dysfunction.For the bioinformatic analysis, and considering the data reported in the literature ,19,20,21To detect the possible candidate targets, we entered each of the enzymes into four databases  analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis enrichment, we used the SRplot  and Monap-value, and the top ten biological processes and more relevant KEGG pathways (p < 0.05) were plotted.All genes were ranked by their We started our work by determining the fundamental importance of the platelet redox signaling of P4HB, PDIA3, and PDIA6 and how they influence platelet function. First, we constructed a network of the interactions for each PDI isoform at either all protein interaction levels A,C,E or From the data in We then wanted to determine the importance of each protein regulated by PDI. As shown in the intersection of the Venn diagram, we identified 38 and 10 common protein targets for all platelet proteins A,B. SomeUsing GO, we constructed a Chord Plot  and idenNow using the graphics GO Three Ontologies, Cnetplot, and Biological Processes Enrichment Score dot-plot, we analyzed the ten most essential processes regulated by these PDI isoforms and their respective interactions , and molThe Cnetplot  shows th2.− in several cell types, platelets, and macrophages. Superoxide then dismutates to H2O2 which can decrease the reduction potential of the cell, i.e., decreasing reduced glutathione levels, or activating cell signaling pathways [2.−; while collagen mainly induces ROS formation in platelets through NOX1, thrombin leads to NOX2 activation and platelet ROS generation. As previously performed for the PDI family, we analyzed the interactions between proteins for the two NOX isoforms  . All anaWe aimed to evaluate the interplay of NOX and PDI in platelet function by analyzing the main processes where the two enzymes participate during platelet activation and aggregation. The interaction network plots show a large number of protein interactions, where those proteins highly interacting with others were located in the middle of the plot while those with lower interactions were present in the borders. These results mean that if a protein is affected, depending on its interacting capacity, could have minor or major repercussions on many other proteins which can be associated with the development of dysfunctional processes involved in many diseases. PDI members’ interactions shown in 2.− and H2O2. Indeed, NOX led to an increase in oxidative stress affecting platelet mitochondrial function, and cell activation capacity [Several important processes for the correct function of platelets are related to the NOX function. NOX2 affects lamellipodium formation as well as cytoskeleton rearrangement and cell adhesion function . Platelecapacity . The latcapacity , in whic2.− formation by NOX [NOX active complex requires the formation of heterodimers with cytosolic and membrane proteins, some of them acting as active complex regulatory subunits such as p22phox. Both NOX1 and NOX2 are further dependent on association with a cytosolic ‘activator’ subunit, p67phox. The interaction of p67phox with protein domains on the intracellular loops of the NOX induces a conformational change necessary for the electron flow; in addition, the cytosolic subunit p47phox has an organizer role aiding in the correct migration to the membrane. Finally, a GTPase (Rac1 or Rac2) interacting with p67phox induces a conformational change in p67phox required for NOX activation. Several groups report an important role of PDI in the migration of p47phox and the cytosolic subunits’ correct interaction with gp91phox and p22phox at the membrane for On by NOX ,33,53. In by NOX ,55. In gn by NOX . Regardin by NOX , the plan by NOX . On the Our study reveals the direct correlation of PDI and NOX in platelets starting from the complexity of cell proteins through to the analysis of how each protein impacts platelet function. The data follow several experimental reports from our labs ,59,60,61


TNNI3 gene encodes for the cardiac isoform of troponin I, a pivotal component of the sarcomeric structure of the myocardium. While heterozygous TNNI3 missense mutations have long been associated with autosomal dominant hypertrophic and restrictive cardiomyopathies, the role of TNNI3 null mutations has been more debated due to the paucity and weak characterization of reported cases and the low penetrance of heterozygous genotypes. In recent years, however, an increasing amount of evidence has validated the hypothesis that biallelic TNNI3 null mutations cause a severe form of neonatal dilated cardiomyopathy. Here, we expand the case series reporting two unrelated patients afflicted with early onset dilated cardiomyopathy, due to homozygosity for the p.Arg98* TNNI3 variant, which had thus far been documented only in heterozygous patients and apparently healthy carriers, and the recurrent p.Arg69Alafs*8 variant, respectively. A review of previously reported biallelic TNNI3 loss-of-function variants and their associated cardiac phenotypes was also performed.The  Inherited cardiomyopathies (ICMs) are genetic disorders caused by defects hampering the contractile function of the cardiac muscle. Even though they represent a minority of the overall cases of cardiomyopathy ,2, ICMs On the molecular level, ICMs can occur whenever a genetic mutation disrupts any of the components of the functional unit of the myocardium, i.e., the sarcomere , but alsTTN), which accounts for up to 25% of DCM cases, although most often in older patients [MYH6, MYH7 and MYBPC3) actin proteins (ACTC1 and ACTN2), tropomyosin protein (TPM1), and troponin complex proteins  are instead involved in the more precocious forms of the disease [The multimeric structure of the sarcomere results from the orderly assembly and the precise interaction of thick filaments, made of myosin, with thin filaments, while the giant protein titin contributes to cardiac passive tension . Thin fipatients . Genes e disease . The tro disease . Convers disease .TNNI3 gene, which is located on chromosome 19q13.4. De novo or inherited TNNI3 variants have thus far been associated with a variety of pathological cardiac phenotypes. However, they demonstrate a rather consistent genotype–phenotype correlation [TNNI3, have consistently been recognized as benign, to the point where heterozygous loss of function mutations in genes associated with ICM have been explicitly made a primary example of why the PVS1 criterion of the American College of Medical Genetics (ACMG) variant classification system should not be automatically attributed to null variants without a proved correlation with the underlying disease mechanism [cTnI is a 24 kD protein encoded by the relation ,17,18,19relation ,21. Healrelation . As suchechanism . On the TNNI3 null mutations with, respectively, the highest and second-highest carrier frequencies in the general population. We also perform a review of previously reported cases of autosomal recessive TNNI3-related ICMs.In this work, we describe the clinical manifestations of two young, unrelated patients diagnosed with DCM and presenting with a homozygous genotype for the two TNNI3 gene are reported according to the RefSeq transcript NM_000363.5; the predicted change in the encoded protein is reported according to the RefSeq protein NP_001605.1. The clinical assessments and the genetic tests were part of the routine diagnostic and follow-up procedures. Written informed consent for molecular genetic studies was collected from the parents of the probands.Genomic DNA was extracted from peripheral leukocytes of probands and their parents using standard methods. Two different next-generation sequencing (NGS) technologies were used in the two probands according to the availability in our Laboratory at the time of admission: for the first patient, clinical exome sequencing was performed using the Illumina TruSight ONE Expanded kit on an Illumina NextSeq 550 system . Variants’ calling, annotation, selection and interpretation were performed as previously described . For theTNNI3 variants and DCM2A phenotype was performed. Pertinent English language articles were searched in PubMed  and Web of Science  using any combination of the key words ‘TNNI3’, ‘CMD2A’, ‘congenital inherited dilated cardiomyopathy’, ‘autosomal recessive’, and ‘homozygous’. Demographic and genetic data, characteristics of cardiomyopathy, and accessory phenotypic features were extracted from previously reported patients. Additional pathogenic or likely pathogenic variants associated with CMD2A were searched in mutation databases, including LOVD—Leiden Open Variation Database , ClinVar , and HGMD—Human Gene Mutation Database .A retrospective review of the literature on The proband was the first child of nonconsanguineous Italian parents; her family history was unremarkable in terms of cardiac or genetic diseases. She was born at full term without any prenatal or perinatal complications. Early developmental milestones and growth were reported as normal. At the age of 7 months, the child was admitted to the local emergency ward and subsequently transferred to the pediatric intensive care unit of our clinic following the onset of progressive dyspnea. Her electrocardiogram (ECG) showed sinus tachycardia and signs of biventricular overload, while transthoracic echocardiography (TTE) revealed a dilated left ventricle with thin walls and impaired systolic function with LVEF equal to 25% according to Simpson’s disk summation method; left atrial dilatation and mild mitral regurgitation were also reported, whereas the right chambers appeared normal. Virological testing documented a concomitant adenoviral infection, which initially raised the suspicion of viral myocarditis. Subsequent cardiac magnetic resonance (MRI) confirmed a dilated and markedly hypokinetic left ventricle, whose walls were particularly thinned in the inferior–lateral and middle–apical parts with discontinuous late gadolinium enhancement, in the absence of edema or myocardial fibrosis; despite the normal volume, the right ventricle was itself hypokinetic . A myocaThree weeks after the first admission, the patient developed acute-on-chronic heart failure and required veno-arterial extracorporeal membrane oxygenation and the insertion of a ventricular assistance device to support her cardiac function. During hospitalization, her general condition continued to worsen and major neurological complications occurred; in particular, she suffered from an ischemic stroke secondary to the occlusion of the insular segment of the right middle cerebral artery, causing her a right-sided hemiparesis. After multidisciplinary evaluation, she was listed for a heart transplant, which she received after 4 months. A significant clinical improvement was observed following the procedure. The pre-discharge echocardiography was normal, with no signs of rejection and a LVEF of 61%.Patient B was the fourth child of consanguineous (first-degree cousins) Moroccan parents. Two of his sisters were reported as healthy, while the third died of heart failure at 8 months. The pregnancy and perinatal history were unremarkable. In consideration of family history, both fetal and neonatal echocardiography were performed, without any abnormal findings. Symptomatology started insidiously with feeding difficulties, followed by sudden onset of dyspnea at the age of 6 months. A chest X-ray highlighted an enlarged cardiac silhouette, prompting further cardiologic examinations. ECG found sinus tachycardia and signs of overload of the left ventricle. TTE showed a severe dilatation of the left chambers with remarkable reduction of LVEF (25%) and mitral and tricuspid insufficiency, while the right ventricle appeared undilated with sufficient systolic function. Because of the rapid deterioration of his clinical conditions, the patient was listed for a heart transplant, which he received at the age of 8 months with favorable clinical outcome. Post-transplant pathological examination assessed dilatation of both ventricles, and microscopic evaluation found evidence of normally oriented cardiomyocytes with dysmetric proportions, extensive subendocardial cytoplasmic vacuolization from likely ischemic damage, interstitial edema, and mild fibrous thickening of the endocardium. These results were deemed compatible with DCM.To investigate the etiology of their disease and to provide a recurrence risk to their families, both children were subjected to the molecular analysis of a selection of genes associated with cardiomyopathies.TNNI3 gene: NM_000363.5:c.292C>T, which is predicted to cause a premature stop codon p.(Arg98*). Segregation analysis revealed that both the parents were heterozygous carriers of the variant, confirming the homozygous genotype of the proband  stop codon affects exon 6 of the nset DCM , and it nset DCM . The maiTNNI3 truncating variant NM_000363.5:c.204del p.(Arg69Alafs*8) in exon 5 of the TNNI3 gene. Allelic contribution from both parents was confirmed by segregation analysis  and died shortly after the disease onset. In 2018 Streff et al. [TNNI3. Another biallelic variant, although in relation to a remarkably different cardiac phenotype, was published in 2019, when Pantou et al. [TNNI3 apparently synonymous variant, c.150G>A p.(Lys50=), which was demonstrated to lead to exon 4 skipping, resulting in a protein lacking 14 amino acids. The variant was identified in two siblings from remotely consanguineous healthy parents, presenting with slightly different phenotypes: the proband developed a low LVEF acute myocarditis triggered by parvovirus B19 infection, while her sister was diagnosed with severe DCM at the age of two years. Both of them required a heart transplant. The latest contribution to the genotypic spectrum of loss-of-function TNNI3 variants has been made by Yu and colleagues [While performing a whole-exome sequencing analysis on a cohort of 42 pediatric patients, who were selected on the basis of acute myocarditis symptoms and histological evidence of inflammatory infiltrates within the myocardium, Belkaya et al.  found a f et al.  reportedu et al.  identifiu et al.  further u et al. , who repu et al.  and Pezzu et al.  further u et al.  reportedlleagues , who repTNNI3 carriers in the literature (16 out of 20 documented cases), sometimes in conjunction with LVNC, while RCMs and HCMs accounted for a minority of cases. They also confirm the early onset and the severe progression of these forms, and the almost constant requirement for heart transplantation within a short time after diagnosis. Notable exceptions in this regard are raised by the families reported by Murphy et al. and Pantou et al., which distinguished themselves both for a slower clinical course and, in the Pantou family, in remarkably different pathological findings, which are more reminiscent of the autosomal forms of TNNI3-related ICMs rather than the autosomal recessive ones. However, it should be noted that these two families shared a similar mutational landscape as well, both harboring homozygous TNNI3 missense mutations instead of null mutations. As was already hinted by Carballo et al. [The patients diagnosed in our clinic shared many of the most prevalent characteristics described in previous reports. They both presented with DCM phenotype, which is the most represented in biallelic loss-of-function o et al.  regardinTNNI3 biallelic carriers. In this regard, a plausible association between acute myocarditis and ICMs in general has already been established by several observations in the literature [Interestingly, the clinical presentation of our Patient A was associated with histopathological features of myocarditis in conjunction with viral infection, as described in the case studies of Belkaya and Seidel. However, these studies suffered from a selection bias in this respect, having both being identified from two distinct cohorts of myocarditis patients. It is nonetheless possible to hypothesize that viral infections in these patients may have played a role in triggering or anticipating the onset of dilated cardiomyopathy in terature ,40,41,42TNNI3-related dilated cardiomyopathy, despite the fact that this variant exhibits the highest relative carrier frequency in population databases . The frameshift variant p.Arg69Alafs* is the most common loss-of-function mutation reported in TNNI3 homozygous DCM patients (9 out of 16 families), possibly because of the combination of a relatively high carrier frequency  and its occurrence in populations characterized by a higher degree of consanguinity.The variant p.(Arg98*) is described here for the first time in association with an autosomal recessive form of TNNI3 biallelic loss-of-function mutations, consolidating the severe cardiac outcome shared by affected individuals with little to no variability, and the limited or even absent clinical impact of heterozygous variants on carriers. The non-negligible frequency of healthy carriers of TNNI3 null mutations in the general population  makes it desirable to improve general awareness regarding their reproductive risk.The patients reported in this work serve as a further confirmation of the association between DCM2A phenotype and


However, understanding its tension stiffening and crack spacing and width is of critical importance in fulfilling serviceability requirements. Therefore, this research aims to evaluate the tension stiffening and cracking performance of alkali-activated (AA) concrete. The variables considered in this study were compressive strength (fc) and concrete cover-to-bar diameter (Cc/db) ratios. After casting the specimen, they were cured before testing at ambient curing conditions for 180 days to reduce the effects of concrete shrinkage and obtain more realistic cracking results. The results showed that both AA and OPC concrete prisms develop slightly similar axial cracking force and corresponding cracking strain, but OPC concrete prisms exhibited a brittle behavior, resulting in a sudden drop in the load–strain curves at the crack location. In contrast, AA concrete prisms developed more than one crack simultaneously, suggesting a more uniform tensile strength compared to OPC specimens. The tension-stiffening factor (β) of AA concrete exhibited better ductile behavior than OPC concrete due to the strain compatibility between concrete and steel even after crack ignition. It was also observed that increasing the confinement (Cc/db ratio) around the steel bar delays internal crack formation and enhances tension stiffening in AAC. Comparing the experimental crack spacing and width with the values predicted using OPC codes of practice, such as EC2 and ACI 224R, revealed that EC2 tends to underestimate the maximum crack width, while ACI 224R provided better predictions. Thus, models to predict crack spacing and width have been proposed accordingly.Alkali-activated concrete is an eco-friendly construction material that is used to preserve natural resources and promote sustainability in the construction industry. This emerging concrete consists of fine and coarse aggregates and fly ash that constitute the binder when mixed with alkaline activators, such as sodium hydroxide (NaOH) and sodium silicate (Na Concrete made with Ordinary Portland Cement (OPC) has been commonly used as a construction material for various types of structures for almost 200 years ,2. Nowad2SiO3), in a process called geopolymerization [Recently, there has been an increased motivation among concrete practitioners and researchers to explore the potential of using AA concrete as an eco-friendly alternative to OPC concrete. In addition to the fine and coarse aggregates, FA is a primary component in AA concrete that is produced through mixing all previous components with an alkaline activator, such as sodium hydroxide (NaOH) and sodium silicate  ratio on tension stiffening, crack spacing, and crack width and propose models for crack spacing and width based on the experimental results.The outcomes of the present study on tension stiffening and cracking performance are expected to help better understand the serviceability limit state behavior of AA concrete. This is because the tension-stiffening behavior plays a significant role in the width and spacing of cracks, stiffness, and deformation of the structural member; thus, it is often the governing design criterion for longer-span reinforced concrete members. Understanding tension stiffening also enables engineers to perform accurate deflection calculations for serviceability limit state analysis. This is also significant when dealing with deformability analysis, particularly when cracking is involved, as it is a major source of nonlinearity. Furthermore, relying on the existing models of OPC concrete to estimate the crack spacing and crack width of AA concrete could lead to an unsafe or suboptimal design of structural members; the developed crack spacing and crack width models in this study could provide a safer design basis for structural engineers. Therefore, this study aims to investigate the effects of compressive strength and concrete cover-to-bar diameter  and are presented in STM C618 . The OPC2SiO3) and 14M sodium hydroxide (NaOH). The percentage of Na2SiO3 solution to the 14M NaOH was maintained at 1.5 by weight for all mixes, as suggested by Abdulrahman et al. [2SiO3 and 12.72% NaOH by weight. It is worth mentioning that the three optimum concrete admixtures developed by Abdulrahman et al. [The alkaline activator solution employed in this study was a combination of premixed sodium silicate  was applied to the protruded part of the steel bar until its yielding limit. Although a standard test setup for concrete elements under tension does not exist, the direct tensile test is the most widely used experimental layout . The loaThe results of the scanning electron microscopic (SEM) analysis of the FA and OPC powder are given in 2), mullite (Al6Si2O13), magnetite (Fe3O4), and hematite (Fe2O3). The figure also shows that increasing the intensity occurs at angles between 27 and 44, where the components are high.The results of the X-ray diffraction (XRD) analysis of FA are given in cr) and the corresponding cracking strain (ɛcr) of all prisms, together with the tension force  and strain  of reinforcement, as well as the cracking force (Pcr) and stress (Fcr) of concrete. The characteristics compressive strengths (fc) of AA concrete vary between 24 and 43 MPa, while the characteristics compressive strengths of OPC concrete were 28 MPa and 35 MPa, respectively. Despite the slight difference in concrete strength for both AA and OPC specimens, the obtained results were used to compare the mechanisms of tension stiffening and crack response for both concretes.The experimental results of tension stiffening for AA prisms along with the OPC control prisms are summarized in cr), the corresponding cracking strain (ɛcr) of OPC prisms was slightly smaller, as shown in cr). This enabled the OPC concrete to exhibit higher tensile stresses (Pcr) before cracking, which suddenly deteriorated after the initial crack form. However, the tensile stress (Pcr) in AA concrete was slightly lower at the initial crack, but it continued to carry more tensile stresses after cracking, resulting in a better tension stiffening than OPC concrete. These premature cracks observed in the current study are similar to those reported in the literature for AA concrete [Although both AA and OPC prisms developed slightly similar axial cracking force . This stage occurred when crack patterns in at least two consecutive load stages remained constant, and only the crack width increased. Thus, the response of the cracked prisms started to approach that of the bare bar, and the maximum applied load was limited by the yield strength of the reinforcement. This pattern agrees well with that in the literature for OPC and AA concrete ,49,51. TOne unusual observation can be made from Meanwhile, most of the AA prisms, observed in cr), as shown in Equation (4). This normalized tensile stress is known as the tension-stiffening factor (β), where the post-cracking phase of this factor represents the tension-stiffening contribution [m is the average member strain, Pcr is the concrete cracking stress, and β is the tension-stiffening bond factor.Tension stiffening is commonly analyzed using the normalized tensile stress in concrete that was obtained using the average strain–stress approach suggested in the literature ,53,54,55ribution . This faribution ,49.(1)NThe diagrams reported in cr). This trend agrees well with that reported for LCFA prisms in the literature [An important observation from terature ,57. Anotterature . HoweverThe tension-stiffening factor (β) starts to decay at the end of the stabilized cracking stage, as shown in By referring to In contrast, the tension-stiffening factor of OPC  ratio on the tension stiffening was evaluated by employing the reinforcement of either 10 mm, 12 mm, or 16 mm in diameter. This corresponds to a Cc/db ratio of 3.25, 2.63, and 1.84, respectively. Referring to c/db ratio slightly improves the elongation resistance of the prisms by adding more stiffness to the bare bar response. This can be explained by the fact that increasing the confinement (Cc/db ratio) around the steel bar improves the tensile capacity of the surrounding concrete, which delays the formation of internal cracks [c/db ratio enhances the tension stiffening as increasing the confinement means increasing the effective concrete area that carries tensile stresses [In addition to the compressive strength, the effect of concrete cover to bar diameter  of the prism. Since many building codes formulas evaluate the cracking in terms of average crack spacing , the evolution of cracks in the current study followed a similar approach. Thus, the average crack spacing was plotted as a function of the average axial strain, as shown in A significant aspect of the investigation herein concerns crack spacing and its evolution to provide a clear understanding of concrete cracking. Thus, crack spacing results that include the maximum, average, and minimum crack spacing for all specimens were monitored during the test and reported in −6) and 1750 (10−6). However, Comparing the response of AA specimens with OPC counterparts in To better capture the influence of reinforcement ratio (ρ%) on the average crack spacing, the average crack spacing was plotted with respect to the reinforcement ratio (p%) in By referring to Many building codes included a 𝜙/p parameter in estimating the crack spacing of OPC concrete. Thus, it is worth mentioning that The crack patterns of all tested specimens are given in The crack width monitoring began upon crack initiation and continued whenever a new crack appeared on the concrete surface. After the crack pattern stabilized, the monitoring process was carried out at different loading intervals during the test with an increment that varied between 10 and 15 kN. An example of crack width monitoring is given in By referring to As given in The crack width growth results shown in This section thoroughly discussed the verification of the experimental crack spacing and width through comparisons with that computed using the OPC codes formula. Regressed models were also proposed, and their predictions were statically compared with OPC codes.One of the main goals of the current work is to check the applicability of OPC concrete approaches in various codes’ provisions by comparing the estimated values of the crack spacing with the experimental results of the present study. The design codes considered were EC2  and MC901 = 0.8). In contrast, the predicted values of the average and maximum crack spacing by CEB-FIP [By referring to  CEB-FIP  provisioc, bond factors, k1, and loading factor, k2, in the EC2 [The discrepancy in the predicted values between both design codes can be attributed to considering the concrete cover, C the EC2  formulat the EC2  formula max) based on the maximum crack spacing. In contrast, the ACI 224R [The expressions of EC2  and CEB-ACI 224R  approachAn overall comparison between the experimental and predicted maximum crack widths is summarized in max,exp) rather than using the theoretical maximum crack spacing . An improvement in code predictions has been obtained, and therefore, the crack width code formulas of EC2 [s of EC2  have beeComparing the experimental and the prediction values of the maximum crack width by the ACI 224R  provisioc/db ratios. Following this, the applicability of OPC concrete approaches in various codes of practice to predict the crack spacing and width of this concrete has been examined. Based on the results of the present work, these conclusions were drawn.Alkali-activated concrete is a sustainable construction material, and understanding its tension stiffening and crack spacing and width is of critical importance. These properties are essential indicators of the concrete suitability for structural applications, particularly at service limit states. Therefore, the current work aimed to investigate the tension stiffening and cracking performance of AA concrete by considering a wide range of compressive strengths and Ccr). Both concrete types also have a global response consisting of three stages: elastic, cracking, and stabilized. However, the OPC prisms experienced a brittle cracking mechanism, resulting in a sudden drop in the load–strain curves at the crack location.Both AA and OPC concrete prisms developed slightly similar axial cracking force  of AA concrete exhibited better ductile behavior than OPC concrete due to the strain compatibility between concrete and steel even after the crack ignition. In contrast, OPC concrete experienced a gradual reduction in the tension-stiffening factor (β) after the crack formation.c/db ratio) around the steel bar improves the tensile capacity of the surrounding concrete, which delays the formation of internal cracks and, consequently, enhances the tension stiffening of AA concrete.Compressive strength was generally found to be an influencing parameter in the global response of tested prisms because it improves the cracking resistance and bond between concrete and reinforcement. In addition, increasing the confinement  and the high bond behavior considered. In contrast, CEB-FIP predictions of crack spacing are mostly on the top side of the equality line. In addition, code provisions tend to underestimate the maximum crack width, especially EC2 , and thi


A comprehensive characterization of cell subpopulations involved in the immune response against bladder cancer has not been performed so far. In addition, due to the high prevalence, recurrence and progression capacity of non-muscle-invasive bladder cancer (NMIBC), the identification of novel biomarkers of tumor progression and response to therapy is of utmost importance. The detailed analysis of the immune landscape in these patients is highly relevant to anticipating tumor behavior and optimizing diagnosis methods and tumor management. We present here the results of the first detailed characterization of immune cell populations in the normal bladder, tumor samples and peripheral blood from patients with NMIBC. We have found specific immune cell subsets differentially expressed in these samples and identified potential markers of tumor progression and patient outcome in peripheral blood. These findings provide relevant information about the host immune response against bladder cancer and set the basis for novel non-invasive procedures for patient stratification and monitoring.Background. Bladder carcinoma has elevated morbimortality due to its high recurrence and progression in localized disease. A better understanding of the role of the tumor microenvironment in carcinogenesis and response to treatment is needed. Methods. Peripheral blood and samples of urothelial bladder cancer and adjacent healthy urothelial tissue were collected from 41 patients and stratified in low- and high-grade urothelial bladder cancer, excluding muscular infiltration or carcinoma in situ. Mononuclear cells were isolated and labeled for flow cytometry analysis with antibodies aimed at identifying specific subpopulations within T lymphocytes, myeloid cells and NK cells. Results. In peripheral blood and tumor samples, we detected different percentages of CD4+ and CD8+ lymphocytes, monocyte and myeloid-derived suppressor cells, as well as differential expression of activation- and exhaustion-related markers. Conversely, only a significant increase in bladder total monocytes was found when comparing bladder and tumor samples. Interestingly, we identified specific markers differentially expressed in the peripheral blood of patients with different outcomes. Conclusion. The analysis of host immune response in patients with NMIBC may help to identify specific markers that allow optimizing therapy and patient follow-up. Further investigation is needed to establish a strong predictive model. Bladder cancer has high morbidity and mortality , and it Approximately 75% of bladder cancers are confined to the urothelial mucosa , which hTransurethral Resection of Bladder Tumor (TURB) is the main approach to diagnosing and treating non-muscle-invasive bladder cancer (NMIBC). The risk of recurrence and progression of NMIBC after the first TURB varies from 48–70% and 7–40%, respectively [www.nmibc.net; accessed on 15 November 2022). This system includes different variables such as age; if the tumor is de novo or recurrent; its size and numbers; associated carcinoma in situ; the pathological stage according to the TNM classification; and the differentiation degree (low-grade or high-grade) [It is crucial to determine the risk of recurrence and progression to muscle-invasive disease in the short and medium term. This stratification is determined at the diagnosis, and tumors may be classified into four risk groups: low, intermediate, high, and very high, following the system developed by the European Association of Urology (EAU) (h-grade) ,10.The treatment of low-risk NMIBC varies from different agents, and nowadays, Mitomycin C is widely used in low-grade tumors. On the other hand, Bacillus Calmette-Guérin (BCG) is the treatment of choice for high-grade NMIBC; therefore, both treatments are standard of care.The interaction of cancer with the immune system promotes the recruitment and activation of different myeloid and lymphoid cell populations, as shown by infiltrates of macrophages, dendritic cells, mast cells, granulocytes, and lymphoid cells found in immunopathological studies . The immThere is an increasing interest in studying the precise mechanisms by which immune cells promote cancer cell death and response to different drugs. Previous data suggest that, in patients with bladder cancer, the content of immune cells in urine is similar to that in the tumor microenvironment, but this issue needs further clarification . On the We present here a descriptive study conducted in a population of 41 patients with NMIBC, in which immune cell subpopulations from tumor tissue, adjacent healthy bladder, and peripheral blood have been analyzed. We show the differential distribution of immune cell subsets among the different tissues and provide preliminary evidence for biomarkers predictive of patient outcomes in peripheral blood.This descriptive study was performed in the period between February 2019 and January 2022. Samples from peripheral blood, urothelial bladder cancer and adjacent healthy urothelial tissue (confirmed by a Pathologist) were collected from 41 patients treated at the Department of Urology of La Princesa University Hospital, Madrid, Spain. The epidemiological and clinical characteristics of these individuals are detailed in Blood and bladder samples , PBMC-containing interphase was collected with a pipette, transferred to a new tube and washed twice with PBS. Finally, isolated cells were cryopreserved in media containing 90% fetal bovine serum  and 10% DMSO (Inilab) until analysis.After disaggregation of fresh samples from tumors and normal bladder urothelium with a razor blade and subsequent filtration through a 40 μm-pore cell strainer , mononuclear cells were isolated using a density gradient centrifugation . Peripheral blood mononuclear cells (PBMC) were isolated as follows: peripheral blood collected in EDTA-treated tubes was diluted 1:1 with PBS and carefully added to Ficoll-Paque  in order to avoid mixing. After centrifugation (400× Panel 1 (T lymphocytes 1): CD49b, CD127, CD3, LAG3, CD4, CD25 and CD8;Panel 2 (T lymphocytes 2): TIM3, PD1, CD3, ICOS, CD27, CD4 and CD8;Panel 3 (myeloid cells): MHCII, CD 127a/b (SIRP), IDO1, CD14, CD11c and CD123;Panel 4 (NK cells): KIR, NKG2a, NKG2d and CD56.Mononuclear cells from blood and tissues were incubated with Human FcR blocking reagent for 10 min at room temperature and then labeled with the different antibodies for 30 min at 4 °C. Finally, the cells were washed and resuspended in 200 μL PBS 1×. The antibodies were distributed in the following panels:Fluorochromes and providing Companies are listed in t-test. We used a multivariable logistic regression model to assess the association of the expression of molecules in the different leukocyte populations with TNM independently of known risk factors . Variables included in the model were those found to be statistically significant, with p < 0.1 in the univariable analysis. Data analyses were performed using Graph Pad Prism 8 Software  and Graph Stata .Quantitative variables obtained by flow cytometry were represented in bar graphs as mean ± standard deviation (SD) or in box-and-whiskers plots. Analysis of variance was performed to determine differences in cell subsets between blood and tissues , and Sidak’s multiple comparison post-test was used. For correlation analyses, Spearman’s coefficient r was calculated. Differences between the 2 groups  were determined using an unpaired The study has been approved by the institutional review board and ethics committee at La Princesa University Hospital . It meets international standards of data protection and is in line with practice guidelines established in the Declaration of Helsinki. Informed consent was signed by all the patients before surgery.In order to carry out a detailed characterization of immune cell populations involved in anti-tumor response to urothelial bladder cancer, we analyzed by flow cytometry the percentage of different immune cell subsets in peripheral blood, healthy bladder tissue and tumor samples from a cohort of 41 patients .When comparing peripheral blood and tumor infiltrate A, we fouComparative analysis of samples from peripheral blood and healthy bladder tissue B showed p < 0.0001) and in LAG3 MFI  of CD8+ lymphocytes between tumor and peripheral blood. Likewise, a significant correlation was found in monocyte SIRP MFI  between both samples.We next determined the expression levels of different surface markers present in immune subpopulations by quantifying their mean fluorescence intensity (MFI) in flow cytometry analyses. When comparing peripheral blood and tumor samples, significantly higher expression of CD127 and LAG3 within CD8+ lymphocytes, and increased IDO within MDSCs, were found in the tumors. On the other hand, higher levels of SIRP in monocytes were identified in peripheral blood A. FurtheFurthermore, when considering healthy bladder and peripheral blood, we detected significantly increased expression of LAG3 in CD4+, CD127, and LAG3 in CD8+ bladder lymphocytes. Similarly, the expression of IDO in both monocytes and MDSCs was higher in bladder tissue. Only SIRP expression in monocytes was significantly lower in bladder samples than in blood B.Interestingly, when comparing bladder tissue and tumor samples, increased levels of LAG3 were seen in CD4+ lymphocytes, CD8 and CD127 in CD8+ lymphocytes, as well as SIRP within the monocyte population was found in the bladder C.In view of the differences observed in the immune profile among samples, we sought to investigate the differential expression of immune markers in relation to parameters of tumor evolution and patient outcome . As shown in In order to determine whether previously analyzed immune markers could be used as independent markers for tumor progression or patient outcome, a multivariable logistic regression analysis was performed considering relevant variables such as histological grade and tumor size. This analysis showed that the percentage of CD4+ CD27+ cells and IDO+ monocytes remained independent predictors of tumor progression A. This mMany studies characterize different aspects of anti-tumor immune response in various types of cancer. Thus, in non-small cell lung cancer, a higher density of CD8+ tumor infiltrate lymphocytes (TILs) may promote immune response at the level of the tumor microenvironment . LikewisChronic stimulation of T cells by tumor antigens leads to a process of lymphocyte exhaustion, accompanied by increased surface LAG3 and PD1 levels . LAG3 reMost of the current evidence on the immune response to bladder cancer focuses on murine tumor models, on determining global lymphoid populations through immunohistochemistry in tissue, or on studying immune profiles in urine in patient samples. Some studies performed by immunohistochemistry techniques have demonstrated that high levels of dendritic cell infiltrates were associated with a worse response to BCG . SimilarOur study presents for the first time a detailed analysis by flow cytometry of the immune infiltration profile in tumor tissue and its correlation with healthy bladder tissue and peripheral blood in patients with NMIBC. Due to the high prevalence and recurrence rate of NMIBC, we have focused primarily on this entity independently of muscle-invasive cancers. Likewise, we have not included CIS-containing NMIBC samples because the coexistence of CIS radically modifies the prognosis of the disease. However, in view of the results obtained and the statistically significant differences achieved in NMIBC, it will be of great interest to extend the study to T2 and CIS.We have found differences in specific immune subsets and differentially expressed markers between peripheral blood and tumor, while similar changes can be observed between peripheral blood and normal bladder urothelium. Interestingly, CD127 and LAG3 expression in CD8+ lymphocytes, and SIRP expression in monocytes, show a correlation between tumor samples and blood, indicating that peripheral expression of these parameters reliably reflects the composition of tumor infiltrate. Of note, few significant differences are found when comparing tumor and healthy bladder . ParticuIn our univariable analysis, we observed a significant increase in the percentage of CD4+ TIM3+ in the peripheral blood of patients who have not relapsed. Similarly, we have observed increased CD8+ lymphocytes expressing TIM3 and PD1 exhaustion-related markers in normal bladder urothelium from non-relapsed patients . The expWe have obtained a predictive model for TNM which combines peripheral blood parameters  with histological grade and tumor size and discriminates pTa and pT1 tumors with a sensitivity of 75% and specificity of 96.65%. This model may help to reliably stratify patients with NMIBC in uncertain cases as an extra tool for patient follow-up and management.As a limitation of our study, we have not assessed the presence of lymphovascular invasion that may affect tumor-peripheral blood recirculation of immune cells since Pathologists in our hospital only describe the presence or absence of lymphovascular invasion in radical cystectomy but not in transurethral resection samples. Another limitation is that we have conducted a descriptive study, not focused on monitoring the immune response throughout the natural progression of cancer. Therefore, we have not detected any patients with progression. This is an essential aspect that will be addressed in future research.Determining the immune marker profile in peripheral blood and tissue may provide relevant information on tumor pathogenesis and have diagnostic and prognostic implications in NMIBC. In this regard, we have detected an increase in populations with immunosuppressive characteristics in the healthy bladder of patients with high-grade tumors. These findings highlight the relevance of bladder immune response in the pathogenesis of NMIBC and support the hypothesis that bladder cancer is a pan-urothelial disease. In addition, in a highly prevalent disease like NMIBC, this type of study is of paramount importance since it can provide early information about the immune response in the tumor microenvironment. The use of a panel of antibodies to assess immune populations in peripheral blood by flow cytometry is feasible, minimally invasive and reproducible in clinical practice. Therefore, it may facilitate better follow-up and decision-making in the diagnosis and treatment of these patients. Despite the heterogeneity in the study population, we still observe significant differences among diverse immune parameters and provide preliminary predictive models that may help to stratify patients. Thus, our findings set the basis for further analyses of immune cell subsets in specific scenarios of NMIBC, which allow us to reliably identify biomarkers and establish predictive models of disease progression.


Chaperone-mediated autophagy (CMA) is a process that rapidly degrades proteins labeled with KFERQ-like motifs within cells via lysosomes to terminate their cellular functioning. Meanwhile, CMA plays an essential role in various biological processes correlated with cell proliferation and apoptosis. Previous studies have shown that CMA was initially found to be procancer in cancer cells, while some theories suggest that it may have an inhibitory effect on the progression of cancer in untransformed cells. Therefore, the complex relationship between CMA and cancer has aroused great interest in the application of CMA activity regulation in cancer therapy. Here, we describe the basic information related to CMA and introduce the physiological functions of CMA, the dual role of CMA in different cancer contexts, and its related research progress. Further study on the mechanism of CMA in tumor development may provide novel insights for tumor therapy targeting CMA. This review aims to summarize and discuss the complex mechanisms of CMA in cancer and related potential strategies for cancer therapy. The synthesis and degradation of proteins determine cell growth and differentiation, playing important roles in the growth and development of organisms. The two pathways work in a coordinated manner to maintain cellular homeostasis . As earlAutophagy, which occurs in lysosomes containing multiple hydrolases, is a major mechanism for protein degradation in the intracellular lysosomal pathway, and its function is to degrade unnecessary components both inside and outside the cell. Autophagy contributes to the maintenance of cellular homeostasis, cellular quality control, the ability to defend against damage from inside and outside the cell, and the maintenance of cellular energy homeostasis, which plays a positive role in cell survival . There amice [The CMA degradation pathway starts with the selective recognition of substrate proteins containing pentapeptide motifs (KFERQ) by heat shock protein family A member 8 (HSPA8), which subsequently binds to lysosome-associated membrane protein type 2a (LAMP-2A) molecules on the lysosomal membrane and initiates multimerization to rapidly degrade substrate proteins after they enter the lysosome . Within mice . Basal Cmice ,8.In this paper, we will first outline the main features of the CMA pathway and its physiological functions, with a focus on its dual role in various types of cancer. We will then elaborate on the mechanisms of CMA action in cancer and finally discuss relevant strategies for cancer therapy. These findings may provide meaningful references for targeted therapy of cancer by studying the role of CMA.The substrate protein is recognized and binds specifically to the CMA receptor. Early studies identified RNase A as the first CMA substrate; it was confirmed to contain the pentapeptide motif of KFERQ. These motifs were recognized by the chaperone molecule HSPA8, which targets them for the lysosome. Subsequently, researchers identified KFERQ motifs similar to the pentapeptide motif in other proteins. Therefore, identifying proteins containing KFERQ-like motifs is a natural way to mark CMA substrates. The consensus sequence usually contains one or two positively charged amino acids , one or two hydrophobic residues , and a negatively charged Glu or Asp, flanked by glutamine residues . About 3HSPA8, which is constitutively expressed and distributed throughout the cytoplasm and lysosomes, plays a primary role in the CMA pathway as a core component required for targeting substrates for lysosomes . HSPA8, LAMP2 gene. The other two variants, LAMP-2B and LAMP-2C share the lumenal structural domain that is common among the three, but they differ from the LAMP-2A type in the transmembrane and cytoplasmic tail regions [EF1 α). GFAP interacts with EF1 α, whereas guanosine triphosphate (GTP) induces GFAP to release EF1 α, inhibits CMA, and promotes the disassembly of the multimeric LAMP-2A [LAMP-2A is a receptor protein on the lysosomal membrane for the CMA pathway and is one of the splicing variants of the  regions . LAMP-2A LAMP-2A . The exp LAMP-2A . TherefoCMA plays a vital role in physiological processes, including protein balance, cell energy, the cell cycle, cell survival, immune response, and cell metabolism .Under conditions of nutrient deficiency in organs and cells, the CMA pathway is activated in the body. However, under starvation conditions, the human body first activates the macroautophagy pathway. When macroautophagy reaches its maximum activity, CMA is activated . When huCMA has antioxidant effects. The latest research shows that under oxidative conditions, the activated CMA pathway is directly involved in the degradation of the oxidized protein Kelch-like ECH-associated protein 1 (Keap1) . The lonCheckpoint kinase 1 (CHK1) is one of the substrates of CMA. CMA plays an important role in regulating the cell cycle in response to DNA damage by timely degrading target proteins associated with the regulation of the cell cycle . For exaFirst of all, due to the specificity of CMA, there are many regulatory mechanisms, and studying CMA activity regulation is conducive to finding focuses for disease treatment. The most studied and critical restriction point is the number and assembly of the substrate protein LAMP-2A on the lysosomal membrane . The chaCMA activity can be regulated by several signaling pathways, including the calcium-regulated phosphatase–NFAT pathway, which is essential for CMA activation in T-cells. This pathway also contributes to the CMA response to oxidative stress and was the first signaling mechanism identified in CMA activation. During T-cell activation, the generation of reactive oxygen species (ROS) promotes the nuclear translocation of NFAT1 and upregulates LAMP-2A expression, thus further promoting CMA activity . EndoplaeEF1α), which mediate the negative regulation of CMA via guanosine triphosphate (GTP) [Bandyopadhyay et al. identified two regulatory proteins, glial fibrillary acidic protein (GFAP) and eukaryotic elongation factor 1 alpha (te (GTP) . The prete (GTP) . RARα inChava et al. detected increased expression of LAMP-2A in hepatocellular carcinoma (HCC) while identifying a compensatory mechanism between macroautophagy and CMA: the activation of CMA can replace the impaired macroautophagy pathway, leading to increased survival in cirrhotic hepatocellular carcinoma . InteresThe tumor microenvironment refers to the cell environment where tumor cells are located, including various stromal cells , immune cells, and extracellular components  . The tumResearch on the role of CMA in the tumor microenvironment shows that the CMA pathway in the tumor microenvironment can promote tumor growth. Glioblastoma cells interact with pericytes to increase the activity of CMA in pericytes through high levels of ROS produced by pericytes, thereby promoting the proliferation of tumor cells and eliminating the immune anticancer response . The tumRecent studies have together suggested a dual role of CMA in cancer. In short, the anticancer functioning of CMA in healthy cells is changed in cancer cells . The reaAlthough the decline in CMA activity and its functional impediment was first revealed in aging, later studies have gradually indicated an intricate link between CMA and cancer pathogenesis because of the close association between aging and cancer . The antThe CMA pathway regulates cancer cell growth by remodeling the proteome: downregulating CMA activity prevents the degradation of proteins required for cancer development, whereas upregulating its activity selectively degrades proteins that inhibit cancer cell growth. Analysis of the proteomics and transcriptomics of glioblastoma revealed that CMA engages in various processes affecting glioblastoma stem cell (GSC) activity. It clarified that low activity of LAMP-2A decreases the mitochondrial activity of GSC, which leads to reduced ATP production, thus leaving cancer cells lacking the energy required for survival . In normMYC by promoting its ubiquitination and degradation in a degradative manner involving protein phosphatase 2A (CIP2A), thereby eliminating its oncogenic activity [MYC, exacerbating its phosphorylation in cells with impaired CMA functioning, resulting in significant changes in tumorigenesis metabolic capacity. Therefore, reducing and preventing the accumulation of MYC is crucial in preventing malignant transformation in healthy cells.Existing studies have shown that this triggering mechanism of CMA can also exert anticancer effects by degrading the corresponding promoter of tumor occurrence . As mentactivity . BlockinTp53 encodes the p53 tumor suppressor, yet mutations in Tp53 contribute to the growth of cancer cells. Activated CMA targets mutant p53 for lysosomal degradation to inhibit cancer progression. CMA-mediated degradation of mutant p53 in cancer cells is a new way to treat Tp53-type cancer [Many other tumor promoters , hexokinase 2 (HK2), galactose lectin-3, etc.) are degraded via the CMA pathway to deactivate them and inhibit tumor growth . In 2010e cancer . In addie cancer ,50. CombWhen cancer cells form new tumors, they leave the primary tumor and metastasize to other organs or tissues. CMA is thought to be possibly linked to tumor metastasis, as it has been shown that blocking LAMP-2A in the CMA pathway can significantly reduce the tumor metastatic ability of lung cancer cells . As XuanAlthough CMA can timely prevent the accumulation of intracellular and extracellular damage in healthy cells, its protumor role in malignantly transformed cells is dominant. Several studies have shown that CMA activity may be upregulated after tumor formation, mainly through energy regulation, cell cycle regulation, and oxidative stress, increasing tumor survival and contributing to tumor metastasis . The conYAP1 and IL6ST , by CMA promotes tumor growth, and it demonstrated that YAP1 and IL6ST are novel substrates for CMA [CMA exerts a procancer effect by preventing the degradation of tumor-associated proteins. The pro-survival protein MCL1 is one of the critical components of lung cancer cell survival, and studies have demonstrated the presence of a CMA-mediated MCL1 protein stabilization system in lung cancer cells, which may contribute to cancer progression . The lat for CMA . CMA regmice was able to reduce the metastatic ability of lung cancer cells [Although high levels of CMA activity have a procancer function in cancer cells, they can also promote cancer cell development by deactivating tumor suppressors, pro-apoptotic factors, and anti-proliferative factors through CMA . An increr cells . Recent er cells . Similarer cells . Furtherer cells . TherefoPED is an anti-apoptotic molecule that occurs in two forms in cells: phosphorylated PED (as a tumor promoter) and unphosphorylated PED (as a tumor suppressor) [PED is not only a bona fide CMA substrate but also that it interacts with HSPA8. Unphosphorylated PED in lung cancer is degraded by targeting lysosomes through the CMA pathway, which may promote cancer progression [p73 through the CMA pathway. P73 is one of the tumor suppressors of the p53 family [p73 on cancer cell growth through NGFR, we found that the new mechanism of CMA positively affects cancer cell survival. CMA degrades the antiproliferative protein RND3 (Rho family GTPase 3) to maintain the rapid proliferation of gastric cancer cells [p21 [pressor) . Quintavgression . In addi3 family . Therefoer cells . The CMAlls [p21 . SNX10 dATG5)-dependent macroautophagy pathway activity [Compared with normal cells, tumor cells still produce ATP through aerobic glycolysis to meet their energy consumption needs, even when the oxygen supply is sufficient. This phenomenon is known as the Warburg effect . Pyruvatactivity ,71. Enhaactivity . This alThe current views on CMA in existing cancers are mostly biased toward its cancer-promoting role. However, for cancer treatment, there are still breakthroughs and challenges in studying the CMA pathway and finding ways to reduce the growth and metastasis of cancer cells by blocking the cancer-promoting mechanism .Despite the complex links between CMA and cancer biology, we can still find breakthroughs in cancer treatment when the roles and limitations of the various influencing factors are individually analyzed. LAMP-2A is a breakthrough in current therapeutic drug development and is critical to the CMA pathway . This sumouse liver attenuates hepatic protein water toxicity and reduces the accumulation of cellular damage. The positive effect of this intervention on liver homeostasis may prevent the malignant transition of this organ [In addition to treating cancers that have already occurred, several studies have suggested that preventive measures can be taken before they occur. Zhang et al. has found that genetic prophylaxis of age-dependent reduction of CMA in is organ . Given tmouse liver, Gomes et al. found disrupted protein homeostasis and reduced stress capacity, increasing the probability of liver tumorigenesis in CMA-deficient aged mice [Because the odds of tumorigenesis are associated with a decrease in CMA activity with age, it is not difficult to explain the greater incidence of cancer in older age groups. For example, in an earlier study of CMA-deficient ged mice . When reged mice . It is hged mice . This paIndeed, in various cancer cell lines, downregulation of LAMP-2A resulted in a significant reduction in proliferation, which not only sensitized the cells to stressors but also reduced the ability of xenografts to form cancer cells. Thus, the antitumor effect of CMA downregulation could be used as a cancer treatment strategy ,53. In aMost recent studies suggest inducing CMA during cancer treatment may be a key mechanism for drug resistance in different types of cancer. For example, Cao et al. suggested that the downregulation of CMA activity in patients with esophageal squamous cell carcinoma (ESCC) inhibited ESCC cell proliferation and colony formation, with increased sensitivity to therapeutic agents . ResistaMoreover, the study revealed that the involvement of CMA drives the balance between stem cell proliferation and cell differentiation, which illustrates the close relationship between cancer and the activities performed by these two types of cells . It can The activity of CMA is directly related to the function of LAMP-2A, which is on the lysosome membrane and is an essential part of the CMA pathway. In addition, CMA activity can also be regulated by mediating the levels of molecular chaperones and substrate proteins. However, it is yet unknown exactly how molecular chaperones other than HSPA8 contribute to the control of CMA. As CMA is predominantly upregulated in cancer, most of the research on CMA and cancer has focused on its procancer mechanisms. However, it is imperative to direct greater attention toward CMA that assumes an oncogenic role in cancer. As such, promoting the oncogenic mechanism in cancer therapy and considering the dual role of CMA are essential.Although the regulation of CMA activity in various tumors has been preliminarily explored, there is still a lack of effective potential drug targets in tumor therapy. It becomes difficult to develop effective CMA modulators due to the crosstalk between CMA and other forms of autophagy and its complex physiological functions. We need to develop selective inhibitors of CMA, not just ones that disrupt all autophagy pathways by hydrolyzing proteins. It is posited that an investigation into the function of CMA in different stages of tumor progression may yield targeted therapeutic interventions. Additionally, we believe investigating the transition of CMA from an oncogenic effect in non-transformed cells to a tumor-promoting function in cancer cells is a promising avenue in cancer treatment.