This study aimed to systematically review and summarize the available data regarding the influence of overweight and obesity across the lifespan on obstacle crossing during walking. Four databases were systematically searched with no limitation on publication date following the Cochrane Handbook for Systematic Reviews and PRISMA guidelines. Only full-text English-language articles published in a peer-reviewed journal were eligible. They had to compare obstacle crossing during walking by overweight or obese individuals with individuals of normal body weight. Five studies were considered eligible. All the studies assessed kinematics; only one assessed kinetics, but none investigated muscle activity or obstacle contact. Compared to normal individuals crossing obstacles, overweight or obese individuals exhibited lower velocity, shorter step length, lower cadence, and less time spent in single-limb support. They also exhibited increased step width, more time spent in double support, and greater trailing leg ground force reaction and centre of mass acceleration. Overall, the small number of included studies did not allow us to draw any conclusions. However, being overweight or obese seems to have a potentially negative influence on the kinematics of gait parameters due to a tendency to trip, fall, and suffer severe fall-related injuries when negotiating obstacles on foot in real-life environments. OW and ed as OB , sudden ed as OB  and funced as OB . Among ted as OB ,8,9.Molina-Garcia et al.  recentlyIndeed, tripping during obstacle crossing represents one of the main causes (up to 53%) of falls among healthy older adults . Over thThis systematic review’s protocol was registered with the International Prospective Register of Systematic Reviews (PROSPERO) (CRD#42021269949) and published in May 2022 ; it foll2 for adult studies or being at or above the 85th percentile of the Centers for Disease Control and Prevention’s Weight-for-recumbent-length Growth Charts for child studies [2). No other walking tasks were considered (comparison). We included studies reporting kinematic, kinetic, or electromyographic parameters, and the following outcomes were also extracted (outcomes): gait spatial and temporal stride parameters , kinetics , muscle activation outcomes  and obstacle contact. Randomised controlled trials, non-randomised controlled trials, and non-randomised, non-controlled trials (study design) were all included. Accordingly, study design inclusion criteria required original articles to have been published in English in a peer-reviewed scientific journal. Finally, we excluded case reports, abstracts, editorials, letters to the editor, case studies, reviews, meta-analyses, theses, grey literature , working papers, and government documents. The inclusion and exclusion criteria using the PICOS tool are described in A population, intervention, comparison, outcome and study design (PICOS) tool was used to select the eligibility, inclusion and exclusion criteria for the studies reviewed. Two reviewers (MC and TP) independently included studies based on samples of OW or OB individuals (together defined as having a BMI ≥ 25 kg/m studies ), excludTeam members developed and agreed upon the search strategy and selection criteria in line with the review questions. Following the recommendations in the PRISMA statement  and the(obes* OR overweight OR over-weight OR adipos* OR “body mass index” OR BMI) AND . The references of included studies were not checked for potential citations. No experts in the field were contacted.The same two reviewers (MC and TP) independently selected the studies, screened the titles, abstracts, and keywords identified via the search strategy, and applied the eligibility criteria. After this initial selection, full-length texts were subjected to the same procedure. In cases of disagreement, and if subsequent discussions between the two reviewers were inconclusive, a third reviewer (NV) was contacted to arbitrate until a consensus was found.(1)Study characteristics: first author, title, year of publication, journal name, country, study design, mention of any adverse events occurring during the study, and funding;(2)Sample description: sample size, age, sex, body weight, body height, body mass index, lower limb length, health status, fall status, and limb preference;(3)Obstacle-crossing protocol: task requirement, instrumentation, data acquisition methodology, and gait parameters assessed;(4)Main results obtained from gait measurement: spatial-temporal parameters, muscle activity, joint angle and moment, ground reaction force, and obstacle contacts.In line with the PRISMA guidelines , the numThe same two reviewers (MC and TP) independently performed a qualitative analysis of the selected studies . To do tGiven the small number of included studies, the wide range of age of the participants included in these studies, and the variety of experimental protocols and reported outcomes, we were unable to conduct meta-analyses of the extracted data. As an alternative, we provided a narrative synthesis of the available data regarding the influence of overweight and obesity on kinematic  and kineThe study selection process is presented in Our searches of PubMed, Web of Science, Scopus, and SportDiscus resulted in 33, 70, 83 and 6 records, respectively. One article was found via hand searching . After dThe studies retained were published from 2012 to 2021, with one study each published in 2012 , 2014 22 and 202Journal of Musculoskeletal Neuronal Interactions [Journal of Aging and Physical Activity [Research in Developmental Disabilities [American Journal of Physical Medicine and Rehabilitation [The articles were published in four different journals: two in the ractions ,22, and Activity , Researcbilities  and the litation .The studies originated from just two countries: four from the USA ,19,20,22The articles included adequately stated their objectives, provided an appropriate description of their participants , The basic demographic and anthropometric characteristics of each study’s participants are presented in Regarding sample size, a total of 132 OW/OB individuals and 82 NW individuals were included. Sample sizes ranged from 12 ,20 to 54Regarding sex, two studies only included female participants ,22, withRegarding age, two studies only included children ,20, withGill and Hung in 2012 reported their mean participant age as 8.58 ± 0.73 years for OW/OB individuals and 8.42 ± 1.00 years for NW individuals . The mea2 [2 [2 for NW individuals [2 [2 [2 [2 [Considering anthropometry, two studies reported the heights and weights of their adult participants ,21. Heig2  and 21.82 [2  for OW/Oividuals ,20. For ividuals ,21,22, B[2 [2 [2 .2. In Desrochers et al.’s 2021 study involving adults [2. In another study, three OW/OB groups were established according to their BMI: OW/Class I OB (25 kg/m2 ≤ BMI < 35 kg/m2); Class II OB (35 kg/m2 ≤ BMI < 40 kg/m2); and Class III OB: (BMI < 40 kg/m2) [Regarding obesity classification, four studies ,19,20,22g adults , OB indi2 and 17.2–26.2 kg/m2 for boys and girls, respectively), and those above the 95th percentile were classified as OB  [In the two studies involving children aged 4–13 years old ,20, partctively) .The obstacle-crossing tasks used in the retained studies are described in Regarding the walkway, four studies ,19,20,22Concerning the starting point, studies’ starting points and walking distances before arriving at their obstacles ranged from 203 cm  or two sFor the obstacle conditions and experimental conditions, all five studies performed an overground gait task and obstacle-crossing tasks ,20,21,22Regarding obstacle height and shape, Silva et al., 2018  used an As concerns instructions, in all five studies, participants were instructed to walk at a self-selected speed and cross an obstacle. In the study by Gill 2019) , partici , particRegarding practice and familiarisation, two studies ,21 reporAll five studies assessed kinematic parameters ,20,21,22Regarding kinematic parameters, the kinematic measurement tools used were Protokinetics LLC’s 610 cm long × 89 cm wide gait carpet , GAITRit-Step length, width, cadence, and single- and double-limb support phases ;-Stance and swing time and their coefficients of variation, and the coefficient of variation of velocity ;-Time from foot lift to maximum knee height and from maximum knee height to foot contact, maximum knee height , and ankle angle at foot contact ;-Leading- and trailing-leg toe clearance, hip and knee angles at maximum knee height in the sagittal and frontal planes, COM (centre of mass) anterior and posterior, and medial, lateral and vertical acceleration at the leading and trailing legs’ maximum knee height during crossing ;-Relative foot temporal data at initial and final foot contact and duration of contact of the HL , HM , MF (midfoot), M1-5 , T2-5 (toes), and T1  areas of the leading and trailing legs .The only kinematic parameter investigated by two studies was gait velocity during obstacle crossing ,22. The Regarding kinetic parameters, Gill and Hung, 2014 [2 ≤ BMI < 35 kg/m2), Class II OB (35 kg/m2 ≤ BMI < 40 kg/m2), and Class III OB (BMI > 40 kg/m2) [2 and <25 kg/m2) [Regarding gait velocity, both studies that investigated gait velocity ,22 repor5 kg/m2) ,22. ResuBeing OB led to a significantly lower crossing step length .The OB group showed a significantly lower cadence .OB individuals spent less time in single limb support .The OB group had a greater step width .OB individuals spent more time in the double-limb support phase .The OW/Class I OB group spent more time in stance than the NOB and Class II OB groups .The OW/Class I OB, Class II OB, and Class III OB groups had lower swing times during crossing .OB individuals showed faster COM anterior/posterior (A/P) acceleration when crossing the low obstacle (4 cm) during both-leg crossing . AdditioRegarding leg motion during crossing, OB individuals took longer from foot lift to maximum knee height and less time from maximum knee height to foot contact in the high obstacle (16 cm) condition, with obstacle conditions having no effects on maximum knee height .The OB group had a smaller ankle angle at foot contact during the low obstacle (4 cm) condition .Regarding vertical clearance, OB individuals showed greater trailing-leg toe clearance in the low (4 cm) obstacle condition than in the high one (16 cm), whereas the NOB group showed greater trailing-toe clearance in the high obstacle condition than in the low one. No influence of OB was observed for leading-leg toe clearance .The OB group’s leading-leg frontal hip angle at maximum knee height was greater in high (16 cm) obstacle conditions than in medium (11 cm) and low (4 cm) ones. No effects were observed for the NOB group .The OB group showed a greater trailing leg sagittal knee angle at maximum knee height than the NOB group for the low and high obstacle conditions, but no effect was observed for the medium obstacle condition .Regarding temporal foot data, there were no differences between the OB and NOB groups regarding their relative temporal data at initial and final contact or the contact duration of the plantar areas assessed  .Regarding the coefficient of variation of gait velocity, no differences were found between the OW/Class I OB and NOB groups . The ClaGill and Hung, 2014 investigated kinetic parameters during obstacle crossing [Regarding trailing-leg normalised ground reaction forces (GRF), the OB group showed a greater trailing-leg normalised A/P GRF during the low obstacle condition (4 cm) than in the medium (11 cm) and high conditions (16 cm) . The NOBFor leading-leg normalised GRF, no effects on the OB group were observed for any GRF .Five studies were considered eligible and were retained ,20,21,22This work aimed to systematically review and summarize all the available biomechanical-parameter data regarding the influence of OW and OB across the lifespan on obstacle-crossing tasks during walking tests. Our broad search and selection retained five studies for evaluation. Two studies involved children ,20, and 2 and <35 kg/m2) group had faster gait velocities and less variable velocities than those in the Class II OB (≥35 kg/m2 and <40 kg/m2) or Class III OB (>40 kg/m2) groups. In addition to slower velocity, Desrochers et al. (2021) [The most consistent between-group difference (NW vs. OB) in obstacle-crossing performance was the lower gait velocity in OB adults reported in two studies comparing them to NW adults ,22. One . (2021)  reported. (2021)  proposed. (2021) . It is n. (2021)  further All five studies assessed spatial outcome measures of gait ,20,21,22All five studies assessed temporal outcome measures of gait ,20,21,22The impaired gait control and stability observed during obstacle crossing due to OW/OB were reiterated in the kinetics results. NW and OB children exhibited significant differences in trailing-leg ground force reactions. Compared to the NW group, in the anterior/posterior direction, OB children had a higher normalised ground reaction force in low obstacle conditions than in medium or high obstacle conditions, and in the vertical and medial/lateral directions, their ground forces were significantly higher in the high obstacle condition than in the medium one  ,20,21,22Given the results, limitations, and strengths of the present review, several practical implications can be considered. It is presumable that the performance of an obstacle-crossing task depends on the participant’s height (and/or their lower limb length) and the height of the obstacle . AccordiDespite the small number of studies included, the present findings suggest that overweight and obesity do indeed affect kinematic and kinetic gait parameters during obstacle-crossing tasks executed by both children ,20 and a


These data were correlated with inflammatory parameters associated with the clinical severity of COVID-19. Additionally, we utilized RNAseq analysis to examine the gene expression of enzymes involved in the SL pathway. Our analysis revealed the presence of thirty-eight SL species from seven families in the plasma of study participants. The most profound alterations in the SL species profile were observed in patients with severe disease. Notably, a predominant sphingomyelin (SM d18:1) species emerged as a potential biomarker for COVID-19 severity, showing decreased levels in the plasma of convalescent individuals. Elevated SM levels were positively correlated with age, hospitalization duration, clinical score, and neutrophil count, as well as the production of IL-6 and IL-8. Intriguingly, we identified a putative protective effect against disease severity mediated by SM (d18:1/24:0), while ceramide (Cer) species (d18:1/24:1) and (d18:1/24:0)were associated with increased risk. Moreover, we observed the enhanced expression of key enzymes involved in the SL pathway in blood cells from severe COVID-19 patients, suggesting a primary flow towards Cer generation in tandem with SM synthesis. These findings underscore the potential of SM as a prognostic biomarker for COVID-19 and highlight promising pharmacological targets. By targeting sphingolipid pathways, novel therapeutic strategies may emerge to mitigate the severity of COVID-19 and improve patient outcomes.SARS-CoV-2 infection triggers distinct patterns of disease development characterized by significant alterations in host regulatory responses. Severe cases exhibit profound lung inflammation and systemic repercussions. Remarkably, critically ill patients display a “lipid storm”, influencing the inflammatory process and tissue damage. Sphingolipids (SLs) play pivotal roles in various cellular and tissue processes, including inflammation, metabolic disorders, and cancer. In this study, we employed high-resolution mass spectrometry to investigate SL metabolism in plasma samples obtained from control subjects ( The ongoing global pandemic known as the 2019 coronavirus disease (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While the majority of patients with mild or moderate symptoms have a positive prognosis . IndividUnderstanding the underlying reasons for the significant variation in individual responses to SARS-CoV-2 infection is essential for the development of effective therapies . HoweverThe metabolic processes involving SLs are highly complex. Ceramide (Cer) holds significant importance in the SL metabolic pathway and can be generated through the breakdown of sphingomyelin (SM) or through de novo synthesis . The iniEmerging evidence suggests that SLs play a crucial role in modulating SARS-CoV-2 infection ,18. StudIn light of previous findings documenting changes in SL patterns among COVID-19 patients, our research takes a step further by integrating targeted mass spectrometry-based sphingolipidomics with the measurement of specific inflammatory mediators, clinical parameters, and the gene expression of SL-metabolizing enzymes. Notably, our study includes patients who have recovered from COVID-19, providing a comprehensive understanding of the disease trajectory. Through this multifaceted approach, our primary objective is to identify biomarkers that can serve as diagnostic and prognostic indicators, while also unraveling the intricate role of SL in the pathophysiological pathways of COVID-19. In line with this, our study emphasizes that SARS-CoV-2 infection induces a notable increase in SM levels in the plasma of patients with severe illness. The implications of these alterations warrant careful consideration, as they could potentially influence disease progression and outcomes.n = 204) through the analysis of nasopharyngeal swabs using a genomic RNA assay with RT-PCR  or serology-specific IgM and IgG antibody tests , and control subjects  who tested negative for SARS-CoV-2. Children under 18 years of age and pregnant or lactating women were excluded. Blood samples were collected from patients classified as asymptomatic to mild (n = 36), moderate (n = 60), severe (n = 67), or critical (n = 41). The clinical classification criteria were determined at the time of sample collection, based on a modified statement from the Novel Coronavirus Pneumonia Diagnosis and Treatment Guideline (7th edition) . The regression model, which demonstrated statistical significance (p < 0.001) and yielded high values for R2MacFadden (0.8) and R2Nagelkerke (0.9), along with a specificity of 0.93, sensitivity of 0.89, and an AUC of 0.98, suggested a possible protective effect of SM (d18:1/24:0) against disease severity  (p < 0.001), with R2MacFadden = 0.6, R2Nagelkerke = 0.5, specificity = 0.91, sensitivity = 0.64, and an AUC of 0.92, indicated that the Cer species (d18:1/24:1) and (d18:1/24:0) may be linked to mortality, with odds ratios of  and   in severe COVID-19 patients when compared to those with mild symptoms and convalescent individuals. Interestingly, severe patients exhibited a higher proportion of SM in their overall SL output compared to patients with moderate symptoms and critical cases. This observation may indicate a pivotal point in the immunopathogenesis pathway of COVID-19, suggesting a bias towards SM synthesis in Cer metabolism.SPTLC1, SPTLC2, and SPTLC3) were upregulated in severe and critical forms of the disease, potentially contributing to the increased production of sphinganine and dHCer. Additionally, the expression of desaturase (DEGS1) was enhanced, leading to enhanced Cer production [SGMS1 and SGMS2) [ASAH1), upregulated in critical illness, could deacylate Cer to form Sph [CERS2, CERS4, and CERT1) responsible for the synthesis of Cer from Sph [SMPD2 and SMPD3), which catalyze the formation of Cer from SM [To gain insight into the underlying mechanisms of SL production during COVID-19, we analyzed gene expression patterns of a comprehensive panel of enzymes involved in the SL pathway in patients’ peripheral blood cells, as the biochemical activity assays for these enzymes are yet to be developed. Overall, we identified altered expression of enzymes associated with de novo SL synthesis. Notably, serine palmitoyltransferases (oduction . The Cerd SGMS2) , which cform Sph . Notablyfrom Sph  were upr from SM , tended SM is the primary SL present in cell membranes. Acid sphingomyelinase activity (aSMase), an enzyme found in lysosomes and in the cell membrane, is responsible for the conversion of SM to Cer. The aSMase/Cer system actively participates in host defense responses ,41,42. Sp < 0.001) have the potential to identify clinically severe symptoms. In fact, our findings demonstrated that SM (d18:1/16:0), (22:1), (23:0), (23:1), (24:0), (24:1), (25:0), (25:1), (26:0), and (26:1) may serve as predictors of a poor prognosis (AUC > 0.7) for COVID-19. Additionally, the results demonstrated a moderate positive correlation between total SM and inflammation markers in COVID-19, with a high disease clinical score. It is crucial to identify the mechanism by which SARS-CoV-2 increases SL levels. SM has the ability to bind with cholesterol and form lipid rafts, potentially facilitating virus entry into cellular surfaces [Our findings also indicate a decrease in total SM counterparts and an increase in sphinganine, Sph, and Cer species in convalescent patients, consistent with reports of elevated levels of these SL species in symptomatic individuals compared to asymptomatic ones . This susurfaces ,54,55. Csurfaces . On the surfaces ,22,23,57p < 0.001). In fact, elevated levels of SM (24:1) were positively correlated with clinical and inflammatory indicators in COVID-19, including the clinical score, hospitalization days, and neutrophil count, as well as the production of IL-10, IL-6, and IL-8. On the other hand, the correlation between Cer (24:1) and these indicators was weaker. These systemic SM species hold promise as potential indicators or targets for preventing disease progression and exploring new treatments. They may have direct effects on regulating inflammation, coagulopathy, and the cytokine storm, which are defining characteristics of severe COVID-19 [We have identified novel potential SM biomarkers that could predict the progression of COVID-19 towards severe symptoms. Specifically, the SM species (d18:1/24:0) and (d18:1/24:1) were evaluated as potential severity biomarkers in our study. To account for confounding variables such as age, sex, comorbidities, body mass index (BMI), hospitalization days, glycemia, and the neutrophil–lymphocyte ratio (NLR), we used multivariate binomial logistic regression to assess their association with disease severity. Interestingly, our analysis revealed a tendency toward a protective effect mediated by SM (24:0) against severe forms of COVID-19, as well as a risk factor mediated by Cer (24:1), based on a significant regression model (AUC = 0.98; COVID-19 ,59. FurtCOVID-19 ,61.Our study distinguishes itself from previous research by including a larger and more extensively characterized cohort of COVID-19 patients, encompassing healthy controls and individuals in the convalescent phase. However, it is crucial to acknowledge certain limitations that should be addressed in future investigations. Firstly, we have not accounted for the impact of chronic disorders and secondary infections, which could potentially contribute to the dysregulation of SL metabolism. These factors may influence the observed SL patterns and should be considered in future studies. Additionally, it is important to note that our sample is regionally limited, which may introduce geographic and demographic biases. To establish the predictive effect of SL biomarkers in critically ill individuals with COVID-19, it is necessary to conduct cohort studies with broader representation and diverse populations. Furthermore, to gain a comprehensive understanding of SL dynamics, it would be advantageous to assess the SL profile over the course of the disease. This longitudinal analysis would allow us to examine how SL patterns evolve and whether early alterations can serve as predictive markers for disease progression and outcome. Such investigations could provide valuable insights into the pathogenesis of COVID-19 and aid in the development of targeted interventions. By considering additional factors, expanding the scope of the study population, and conducting longitudinal analyses, we can enhance our understanding of the predictive potential of SL patterns and their implications for patient management and therapeutic interventions.


Diabetes is a global health problem which is accompanied with multi-systemic complications. It is of great significance to elucidate the pathogenesis and to identify novel therapies of diabetes and diabetic complications. Sestrin2, a stress-inducible protein, is primarily involved in cellular responses to various stresses. It plays critical roles in regulating a series of cellular events, such as oxidative stress, mitochondrial function and endoplasmic reticulum stress. Researches investigating the correlations between Sestrin2, diabetes and diabetic complications are increasing in recent years. This review incorporates recent findings, demonstrates the diverse functions and regulating mechanisms of Sestrin2, and discusses the potential roles of Sestrin2 in the pathogenesis of diabetes and diabetic complications, hoping to highlight a promising therapeutic direction. It is a type of disease in which defects in insulin production and activity lead to abnormal glucose metabolism. Continuous hyperglycemia leads to impaired cellular autophagy and oxidative stress response, which further induces inflammatory response and the stimulation of coagulation, and finally gives rise to the occurrence of complications in multiple organs and systems . In receAs an important member of the Sestrins (Sesns) protein family, Sestrin2 is a newly discovered stress-inducible protein widely distributed in animals. Sestrin2 gene was originally identified in human neuroblastoma cells as a hypoxia-activated gene . Sestrin2As a sensitive stress receptor, Sestrin2 is activated in stress conditions. A variety of adverse stresses could promote Sestrin2 expression, such as oxidative stress, ER stress, hypoxia, energetic stress, and age- and obesity-associated metabolic pathological conditions –10. Up-r2.1In response to stress, Sestrin2 could be regulated by various transcription factors, including tumor-suppressor protein p53, hypoxia inducible factor-1α (HIF-1α), nuclear factor erythroid 2-related factor-2 (Nrf2), nuclear factor-κB (NF-κB), activated transcription factor 4 (ATF4), c-Jun NH(2)- terminal kinase (JNK)/c-Jun, Foxhead box O3 (FoxO), activated protein 1 (AP-1), and CCAAT. A series of stress conditions, such as oxidative stress, ER stress, DNA damage, hypoxia and mitochondrial dysfunction, provoke the release of these upstream factors, and result in altered expression and activity of Sestrin2 , 16. The2.2After being induced, Sestrin2 thereafter mediates several signaling pathways, including adenosine monophosphate-activated protein kinase/mammalian target of rapamycin (AMPK/mTOR) pathway , Kelch-l2.2.1Comprising of two distinct protein complexes (mTORC1 and mTORC2), mTOR functions as a crucial sensor for energy, nutrient, and redox states, and thereafter regulates protein synthesis and autophagy . PersistSestrin2 exerts antioxidant and apoptosis-associated effects in a variety of diseases through the inhibition of mammalian target of rapamycin complex 1 (mTORC1) and/or the activation of AMPK –23. Sest2.2.2As a member of a family of basic leucine transcription factors, Nrf2 is involved in a serious of important cellular events including redox regulation, DNA repair, metabolic homeostasis, and apoptosis prevention . Nrf2 acThe Nrf2 activators up-regulate the expression of Sestrin2 in a time- and dose-dependent manner and the Nfr2-ARE pathway activation seems to be necessary for Sestrin2 induction. In turn, Sestrin2 can function as a positive regulator of Nrf2 signaling, activate the Nrf2 pathway by augmenting autophagy-directed degradation of Keap1, which targets and breaks down Nrf2 , 53. Ses2.3Autophagy is a distinct type of programmed cellular death. Autophagy helps maintain cell survival and tissue stabilization by degrading misfolded and aged intracellular proteins and dysfunctional organelles during stress. The process of autophagy is regulated by various pathways and involves diverse organelles such as mitochondria, ER, ribosomes, peroxisomes, and lysosomes. The dysfunction of autophagy is related to a myriad of diseases, such as diabetes, cardiovascular diseases, cancer, neurodegenerative diseases, liver diseases, and inflammatory diseases –59. AutoAutophagy activation is required for the antioxidant effects of Sestrin2 . After a2.4Oxidative stress is considered to be an imbalance in redox properties in certain cellular environments, and plays a crucial role in the development of numerous human diseases, such as diabetes, obesity, and myocardial injury , 68. OxiSestrin2 is essential for the maintenance of cellular homeostasis under oxidative stress. In various types of diseases, Sestrin2 is up-regulated and is important for the resistance to oxidative stress injury. Under oxidative stress, Sestrin2 is activated by various transcription factors, including NF-κB, activator protein-1 (AP-1), CCAAT-enhancer-binding protein beta (C/ERPβ), forkhead box O3 (FOXO3), and p53 . Sestrin2.5ER stress is provoked when unfolded or misfolded proteins accumulate in the endoplasmic network lumen in pathophysiological conditions . Many phIncreasing evidence has shown that Sestrin2 is activated under ER stress , 80. How2.6Mitochondria are the prime organelle which not only offers energy substrates to cells but also controls the fate of cells via mediating diverse cellular processes such as autophagy, apoptosis, cellular mobilization and metabolism , 85. Mitin vivo and in vitro  is a typical chronic microvascular diabetic complication and is a major cause of chronic kidney disease and end-stage renal disease worldwide . About 3The activation of Sestrin2 could inhibit AMPK/mTOR signaling, promote autophagy and reduce the susceptibility of renal cells to diabetes-related damage. The potential therapeutic role of Sestrin2 in DKD was initially found in a human proximal tubule cell line (HK-2) model, illustrating that overexpression of Sestrin2 represses DKD-induced renal epithelial tubular cell epithelial-to-mesenchymal transition and ER stress, but its mechanism is still unclear. The researchers further found that administration of microRNA-4756 regulates DKD-induced renal tubular epithelial cell damage by the interaction with Sestrin2 . Later, TGF-β1 is a decisive regulator of renal fibrosis and overactivation of TGF-β1 could cause progressive renal injury . HyperglThe Hippo pathway, a kinase cascade that regulates cellular proliferation, differentiation, and tissue homeostasis, is inhibited in diabetic conditions. The Hippo pathway has been indicated to be involved in the development and progression of DKD , 158. PI4.2Diabetic heart disease (DHD) is a major cause of death in patients with diabetes. It refers to abnormal heart structure and manifestations in patients with diabetes in the absence of other cardiac risk factors. DHD is a conglomeration of coronary artery disease, heart failure, diabetic cardiomyopathy (DCM) and diabetic cardiac autonomic neuropathy, and is characterized by molecular, structural, and functional changes in the myocardium , 160. Thepigallocatechin gallate, or cannabidiol, possibly due to the inhibitory effects on TGF-β signaling  drives cardiac hypertrophy , 163. Alignaling –174. TSPignaling . The effignaling . Downregignaling , 178.(rare). Previously, Sestrin2 is considered to be cardioprotective in several models of cardiovascular diseases, including myocardial infarction and cardiac dysfunction induced by ER stress or lipopolysaccharide, via AMPK/mTOR signaling cascade , cataract, diabetic papillopathy, glaucoma, and ocular surface diseases . DR is aBased on its pleiotropic modulating effects, Sestrin2 may have an impact on the pathogenesis of DR. So far there are several studies exploring the role of Sestrin2 in models of ocular lesions, but still no reports in diabetic ocular complications have been found. Previously, Hanus et al. demonstrated that upregulation of Sestrin2 protects retinal pigment epithelial cells from oxidative stress-induced necrosis . Sestrin4.4Diabetic neuropathy (DN) is another frequent chronic complication of diabetes, consisting of four major types including peripheral neuropathy, autonomic neuropathy, proximal neuropathy, and mononeuropathy . The pat5The exploration of Therapeutic strategies through mediation of Sestrin2 is now underway. Several natural products and medications in diabetes have been shown to alter the expression levels of Sestrin2, which lead to the possibility of novel treatments in diabetes and diabetic complications targeting Sestrin2 and the associated pathways . Initial6Diabetes is a condition causing multi-organ injuries and is a major global threaten for human health. As a stress-induced protein, Sestrin2 can be activated by diverse stresses and can exert pleiotropic effects. Sestrin2 can interact with various signaling perspectives involved in the development of diabetes. Increasing numbers of studies indicate a prominent role of Sestrin2 in the pathogenesis of diabetes and diabetic complications. Sestrin2 may functions in a multitude of ways and offer exciting prospects for the treatment of diabetes and diabetic complications, though currently the strong supporting evidence is limited. Despite of the protective roles of Sestrn2 in various conditions, the pros and cons of excessive activation or inhibition of Sestrin2 is yet to be confirmed. How to exert accurate mediation under different conditions also remains elusive. The modulation of Sestrin2 activity to effectively achieve homeostasis might be more appropriate. Further researches are needed to thoroughly reveal the relationship between Sestrin2 and diabetes which includes multi-organ injuries, to disclose associated signaling pathways and to explore potential treatment protocols.XZ: Writing – review & editing, Conceptualization, Writing – original draft. ZL: Writing – original draft. JL: Writing – original draft. YLin: Writing – original draft. YLi: Writing – original draft, Project administration, Resources, Software. WL: Writing – review & editing, Supervision, Validation.


Hereditary cancer syndromes account for nearly 10% of cancers even though they are often underdiagnosed. Finding a pathogenic gene variant could have dramatic implications in terms of pharmacologic treatments, tailored preventive programs, and familiar cascade testing. However, diagnosing a hereditary cancer syndrome could be challenging because of a lack of validated testing criteria or because of their suboptimal performance. In addition, many clinicians are not sufficiently well trained to identify and select patients that could benefit from a genetic test. Herein, we searched the available literature to comprehensively review and categorize hereditary cancer syndromes affecting adults with the aim of helping clinicians in their daily clinical practice through a visual tool. Hereditary cancer syndromes (HCSs) account for nearly 10% of cancers even though they are often underdiagnosed . ArisingGenetic variants, the molecular basis of HCSs, describe DNA sequence variations within an individual’s or population’s genome that can influence gene expression or function to varying degrees. The American College of Medical Genetics and Genomics (ACMG) has established a framework to classify genetic variants into five categories based on their pathogenicity: Pathogenic (P), Likely pathogenic (LP), Uncertain significance (VUS), Likely benign (LB), and Benign (B) . This clFinding a gene variant predisposing to cancer in a patient through a genetic test could have at least three profound implications for his/her clinical scenario: BRCA1/2 for PARP inhibitors or the mismatch repair system for immune checkpoint inhibitors. In such instances, a genetic test could dramatically increase the therapeutic armamentarium for that patient [First, some gene variants are predictive of cancer treatment efficacy; for example,  patient ,5.Second, finding a HCS in a patient allows a tailored preventive program to be offered that might increase his/her lifespan with a favorable cost-to-benefit ratio, such as organ specific surveillance and prophylactic surgeries ,7.Third, the same gene variant can be searched for in every family member, expanding the benefit burden of therapeutic and preventive strategies discussed above ,9.However, the diagnosis of a HCS could be challenging because of a lack of validated testing criteria or because of their suboptimal performance ,11. In aHerein, we searched the available literature to comprehensively review and categorize HCSs affecting adults . We discRenal cancers can be associated with genetic syndromes, the most relevant of which are von Hippen–Lindau Syndrome (VHLS), hereditary papillary renal carcinoma syndrome (HPRC), hereditary leiomyomatosis and renal cancer cell syndrome (HLRCC), and Birt-Hogg-Dubé syndrome (BHD) .VHLS is caused by inactivation of the VHL protein (pVHL), whose best characterized function is the targeting of hypoxia inducible factor (HIF) for proteosomal degradation. Although rarely the first presentation of disease, up to 80% of patients with VHL develop renal cell carcinoma  . Classicfumarate hydratase (FH) gene that encodes an enzyme required in the Krebs cycle to convert fumarate to malate. HLRCC predisposes to various renal tumors, such as type 2 papillary renal cell cancer, collecting duct renal cell carcinoma, clear cell renal cell carcinoma, and Wilm’s tumor, which is present in 19% of all cases and often characterized by being unilateral and solitary but with exceptional aggressiveness. Other malignancies that characterize HLRCC syndrome include cutaneous and uterine leiomyosarcomas, Leydig cell tumors, gastrointestinal stromal tumors (GIST), and breast cancer. The outstanding feature of HLRCC, however, is the occurrence of multiple cutaneous leiomyomas, originating from the hair erector pili muscles and typically localized in limbs, trunk, and face [HLRCC is a rare disorder caused by a germline mutation in the and face . Other band face . FLCN , is associated with renal tumors of various histologic types in up to 19% of cases: hybrid oncocytic/chromophobe tumor, chromophobe tumor, oncocytoma, clear cell carcinoma, papillary carcinoma, and mixed-type carcinoma. Numerous are the potentially associated clinical manifestations. At the skin level, numerous characteristic benign lesions are present, such as fibrofolliculomas, skin tags (acrochordons), angiofibromas, and oral papules, although the risk of developing cutaneous (including multiple desmoplastic melanomas) and choroidal melanoma is not negligible. More than 80% of patients develop multiple bilateral lung cysts, usually asymptomatic or associated with cough or exertional dyspnea but predisposing to spontaneous pneumothoraxes with a high recurrence rate [BHD, caused by germline mutations in the tumor suppressor gene nce rate . Rarer iMET gene, that predisposes patients to bilateral and multifocal papillary type 1 renal tumors.Hereditary papillary renal cell carcinoma (HPRC) is another inherited renal cancer syndrome, associated with pathogenic variants of the MUTYH-associated polyposis (MAP), and hereditary diffuse gastric cancer syndrome (HDGC) with up to 10% lft.Other syndromes with less frequency predispose to renal tumors, including the Tuberous Sclerosis Complex (TSC) with a 4% lifetime risk (lft), Li–Fraumeni syndrome (LF), Bloom syndrome (BSyn), Fanconi Anemia, BAP1 tumor predisposition syndrome (BAP1) with up to 10% lft, Lynch syndrome (LS) with up to 16% lft, Among the genetic syndromes associated with thyroid tumors, Multiple endocrine neoplasia type 2 (MEN2A and MEN2B) and Cowden syndrome (CS) are the most relevant .RET proto-oncogene, which encodes a tyrosine kinase receptor whose activation promotes cell growth, proliferation, and differentiation. MEN2 is clinically divided into two entities: MEN2A and MEN2B. Medullary thyroid carcinoma (MTC), present in 90% of MEN2A and 100% of MEN2B cases, is often the first manifestation of disease, with a mean age of diagnosis ranging from 19 to 33 years [Multiple endocrine neoplasia type 2 (MEN2) is an inherited cancer syndrome associated with germline variations of the 33 years . A typical feature of MEN2A is the occurrence, with a prevalence of 17–42%, of both unilateral and bilateral pheochromocytoma. More easily bilateral than sporadic, typical symptoms of pheochromocytoma are hypertension, headache, sweating, and tachycardia [MEN2B is characterized by early and aggressive development of MTC, often associated with metastatic disease at an early age . Its manAdenomatous Polyposis Coli (APC) gene) can also be associated with the occurrence of thyroid tumors in up to 12% of patients. However, its prominent feature remains the appearance, during the second decade of life, of hundreds to thousands of colorectal adenomas which almost inevitably progress to carcinoma [Although less frequently than the above syndromes, Familial adenomatous polyposis , DICER1, and the Carney complex (CNC), predispose to thyroid tumors.There are numerous genetic syndromes that result in an important predisposition to the occurrence of melanoma: Familial atypical multiple mole melanoma (FAMMM), Xeroderma pigmentosum (XP), BAP1 tumor predisposition syndrome (BAP1), Shelterin complex genes hereditary cancer syndrome (Shelterin), MC1R polymorphism (MC1R), and DICER1 .CDKN2A gene, involved in cell cycle regulation), clinically characterized by the presence of atypical nevi and a total body nevi count > 50, have a very high risk of melanoma, ranging from 28% to 67%. Other potentially associated typical malignancies include pancreatic cancer , astrocytoma, breast cancer, lung cancer, head and neck, and gastroesophageal cancer, the latter three apparently only in smoking patients. Patients with FAMMM , caused by alterations in genes involved in the nucleotide excision repair system and associated with skin cancer  in up to 65% of cases. XP is clinically characterized by increased sensitivity to the sun, typically associated with severe sunburn with blistering, persistent erythema, xerosis, and poikiloderma, and by sunlight-induced ocular involvement, such as photophobia, keratitis, and atrophy of the skin of the lids. In about 25% of patients, there is progressive neurodegeneration with sensorineural hearing loss, acquired microcephaly, progressive cognitive impairment, and diminished deep tendon stretch reflexes. Other malignant diseases that characterize Xeroderma pigmentosum include oral cancer, sarcoma, hematological diseases such as myelodysplastic syndrome and acute leukemia, colorectal and non-small cell lung cancer, glioma, and others see .BAP1 tumor predisposition syndrome (BAP1) is a newly recognized cancer syndrome that predisposes to uveal and cutaneous melanoma . BAP 1 is a deubiquitinating enzyme that acts as a tumor suppressor protein, regulating cell cycle, cellular differentiation and DNA damage repair. Uveal melanoma, which is the most frequently found cancer in these patients, has a mean age of onset of 51 years, lower than the 61 in the general population, and is typically more aggressive, with a high risk of metastasis and reduced overall survival . In contACD, TERF2IP, TERF1, TERF2, TINF2, and POT1), overall collected in the Shelterin complex genes hereditary cancer syndrome (Shelterin), can cause hereditary melanoma [Germline mutations in the Shelterin family of genes  has been shown to be predisposing to melanoma, with a 3–5-fold increase in associated risk compared with the normal population [MITF, clinically characterized by darker hair, fair skin, and non-blue eye color [pulation ,22. IndiTERT, considered to be one of the earliest secondary mutations following BRAF and NRAS driver mutations [Other syndromes that are less frequently associated with the occurrence of melanoma include the LF syndrome and the TERT hereditary cancer syndrome (TERT), the latter associated with somatic promoter mutations of utations .TP53) [BRCA1- and BRCA2-associated hereditary cancer syndrome  [STK11-associated hereditary cancer syndrome or Peutz–Jeghers syndrome (PJS)  [PALB2-associated hereditary cancer syndrome  [ATM-associated hereditary cancer syndrome or Ataxia telangiectasia  [CDH1) [BARD1-associated hereditary cancer syndrome  [MEN1) [CHEK2-associated hereditary cancer syndrome  [PTEN) [RAD51C-associated hereditary cancer syndrome  [RAD51D-associated hereditary cancer syndrome  [NF1) [POLE, POLD1) [NTHL1 tumor syndrome (gene: NTHL1) [BLM) [MLH3-associated polyposis (gene: MLH3) [Considering the hereditary breast cancer landscape, there are several genetic syndromes correlated to it . The mosTP53) ,25; BRCABRCA1/2) ,27,28,29: STK11) ,31,32; P: PALB2) ,36,37,38ne: ATM) ,40; here) [CDH1) ,42,43; B: BARD1) ; Multipl) [MEN1) ,46; CHEK: CHEK2) ,48,49; C) [PTEN) ,50,51; RD) [NF1) ; FanconiD) [NF1) ,57,58,59, POLD1) ,61,62,63: NTHL1) ,65,66; B1) [BLM) ; and MLHe: MLH3) .Beyond genetic analysis, differential diagnosis among these syndromes is possible, considering the different characteristic phenotypes of each syndrome. In this context, we could look for particular features corresponding to diagnostic criteria (often divided into major ones and minor ones for each syndrome) but also findings suggestive of or possibly associated with a specific syndrome. NTHL1 tumor syndrome, CS, HDGC, MLH3-associated polyposis, NF1, MEN1, LF, and BSyn. In this syndromes group, we could find Ataxia telangiectasia, PSJ, FA, Polymerase proofreading-associated polyposis, ATM, which encodes ATM, a serine/threonine protein kinase involved in DNA damage detection. In the biallelic form, it is characterized by neurological problems such as progressive cerebellar ataxia or progressive dystonia, while in heterozygosis, coronary heart disease is a frequent manifestation [Ataxia telangiectasia could present into three different forms: heterozygotic/monoallelic, classic ataxia telangiectasia , and the non-classic form (attenuated phenotype). In this syndrome, the altered gene is estation ,40. STK11 gene encodes the Serine/Threonine Kinase 11 (STK11), which is involved in several functions: DNA damage response, apoptosis, cellular metabolism, and polarity. It acts as a tumor suppressor protein, controlling the activity of AMP-activated protein kinase (AMPK) family members [The presence of multiple hamartomatous polyps, especially gastrointestinally, is peculiar to PSJ. In addition to this, people with PSJ present with freckling or hyperpigmentation on the lips, buccal mucosa, genitalia, fingers, and toes. In this case, the  members ,31,32.Considering FA, more than 20 responsible genes have been correlated with it . FA-related genes encode proteins which work together in “the FA-pathway”, a DNA damage response pathway. FA can typically be identified by congenital abnormalities such as those included in the VACTERL-H  association. In addition, Fanconi Anemia is often characterized by endocrine disorders, skin pigmentation alterations, growth deficiency, skeletal and ophthalmic malformations, central nervous system alterations, and hematological abnormality ,57,58,59PTEN gene, which encodes the protein PTEN, a phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase. This protein negatively regulates the PI3K/AKT/mTOR pathway [CS is associated with epithelial thyroid cancer  in up to 10% of cases. Other malignancies that characterize CS include genitourinary cancer , endometrial cancer (10%), colorectal cancer (9%), and melanoma (6%). The CS phenotype is peculiar for cerebellar dysplastic gangliocytoma, macrocephaly, mucocutaneous lesions, hamartomatous intestinal polyps, and genitourinary malformation. CS is characterized by alterations in the  pathway ,50,51. BLM, encoding the DNA helicase RecQ protein that is involved in several pathways for the maintenance of genome stability. BSyn has a typical phenotype: proportionate small stature, facial and skin pigmentation alterations , immunodeficiency, diabetes mellitus, variable intellectual impairment, hypogonadism, and premature menopause [BSyn is caused by a mutation in the gene enopause . NF1 (Neurofibromin 1) gene, which encodes neurofibromin, a protein belonging to a family of guanosine triphosphate hydrolase (GTPase)-activating proteins (GAPs) that negatively regulates the Ras signal transduction pathway [NF1’s typical presentations are cutaneous features , ocular alterations , neurologic alterations, musculoskeletal disorders, and vascular and cardiac problems. NF1-responsible alterations occur in the  pathway ,55. MEN1 (Menin 1) which encodes menin, a scaffold protein that functions in epigenetic gene regulation. MEN1 is known for three main characteristics , which may also include skin alterations as well as thyroid disorders [The MEN1-related gene is isorders ,46. CDH1 (Cadherin 1), a gene encoding CDH1, a calcium-dependent cell adhesion protein. CDH1 regulates the proliferation and mobility and of epithelial cells and cell–cell adhesions [Cleft lip/palate is a particular feature of hereditary diffuse gastric cancer syndrome; in breast cancer, this syndrome typically is associated with a specific histotype, namely, lobular breast cancer. This syndrome is caused by alterations in dhesions ,43,54,69NTHL1 tumor syndrome (1–100 cumulative polyps) [MLH3-associated polyposis (10–100 cumulative polyps) [POLD1) [POLD1 encodes the catalytic subunit of DNA polymerase delta, while POLE encodes the catalytic subunit of DNA polymerase epsilon; both these proteins are involved in genome replication [NTHL1 tumor syndrome, the altered gene is NTHL1 (Nth Like DNA Glycosylase 1), encoding a DNA N-glycosylase (endonuclease III family) involved in base excision repair (BER) [MLH3 (MutL Homolog 3) is the altered gene in MLH3-associated polyposis, and the encoded protein MLH3 plays a role in maintaining genomic integrity during DNA replication and after meiotic recombination [TP53, also known as the “guardian of the genome”. The encoded protein is a tumor suppressor protein involved in inducing cell cycle arrest, DNA repair, senescence, apoptosis, and changes in metabolism [Multiple colonic adenomas are characteristic of PPAP (0–100 cumulative polyps) ,61,62,63 polyps) ,65,66, M polyps) , and LF  polyps) ,25. In P [POLD1) ,61,62,63lication ,61,62,63ir (BER) ,65,66. Mbination . LF is rtabolism ,25.NTHL1 tumor syndrome also presents with duodenal adenomas, meningiomas, skin alterations, ovarian and hepatic cysts, and breast papillomas [The pillomas ,65,66.BRCA1/2, CHEK2, PALB2, RAD51C, RAD51D, and BARD1 do not present specific phenotypes, but correlate with a higher risk in developing neoplasms. Alterations in these genes could induce the relative associated hereditary cancer syndrome. All these genes encode proteins involved in the maintenance of genome stability and DNA repair, mainly in the homologous recombination pathway for double-strand DNA repair [On the other hand, genetic syndromes with alterations in A repair ,27,28,29A repair . BRCA1- and BRCA2-associated hereditary cancer syndromes, there is a higher risk of prostate cancer and gastrointestinal cancers , while the risk of melanoma, colorectal, and lung cancer is still a matter for debate [In the r debate ,27,28,29CHEK2-associated hereditary cancer syndrome could include gastrointestinal tumors (colon and gastric cancer), prostate cancer, renal cancer, thyroid cancer, sarcomas, and hematological neoplasms (leukemia and plasma cell neoplasms) [TP53. It has been demonstrated that there is an association of germline CHEK2 variants with breast and prostate cancer but some of the data  is heterogenous [The oplasms) ,47,48,49rogenous . PALB2-associated hereditary cancer syndrome is also associated with pancreatic cancer [BARD1-associated hereditary cancer syndrome is associated with colon cancer [The c cancer ,36,37,38n cancer ,38,44. BRCA2 (FANCD1), PALB2 (FANCN), RAD51 (FANCR), and BRCA1 (FANCS) [BRCA2 (FANCD1), BRIP1 (FANCJ), PALB2 (FANCN), RAD51C (FANCO), BRCA1 (FANCS) [However, in the FA spectrum, unique phenotypes are associated with specific alterations in  (FANCS) ,57,58,59 (FANCS) .BRCA1- and BRCA2-associated hereditary cancer syndrome  [STK11-associated hereditary cancer syndrome or Peutz–Jeghers syndrome (PJS)  [MLH1, MSH6, MSH2, PMS2) [MUTYH) [RAD51D-associated hereditary cancer syndrome  [RAD51C-associated hereditary cancer syndrome  [BRIP1-associated hereditary cancer syndrome  [PALB2-associated hereditary cancer syndrome  [ATM) [POLE, POLD1) [BLM) [DICER1) [Hereditary ovarian cancer is associated with several genetic syndromes : BRCA1- BRCA1/2) ,27,28,29: STK11) ,31,32; L2, PMS2) ,72,73,74 [MUTYH) ; RAD51D- RAD51D) ,52,53; R RAD51C) ,52; BRIP: BRIP1) ; PALB2-a: PALB2) ,36,37,382) [ATM) ,40; PPAP, POLD1) ,61,62,631) [BLM) ,77, DICE[DICER1) .An overlap exists among the following hereditary syndromes associated with a higher risk of developing both breast and ovarian cancer: Ataxia telangiectasia ,38,39,40DICER1 tumor predisposition, LS, and MAP-associated polyposis. As for the previous hereditary cancer syndromes related to breast cancer, there are some hereditary cancer syndromes related to ovarian cancer which are characterized by a typical phenotype: DICER1 tumor predisposition could present suggestive features such as macrocephaly, retinal abnormalities, multinodular goiter, renal alterations , nasal chondromesenchymal hamartoma, and dental anomalies. The hereditary syndrome is also associated with other neoplasms aside from ovarian sex cord–stromal tumors: pleuropulmonary blastoma, medulloepithelioma, differentiated thyroid carcinoma, embryonal rhabdomyosarcoma of the cervix, and central nervous system cancers . In this case, the mutated gene is DICER1, which encodes a double-stranded RNA endoribonuclease involved in gene suppression but which also acts as a strong antiviral agent against RNA viruses [ viruses ,79. MLH1 (MutL Homolog 1), MSH6 (MutS Homolog 6), MSH2 (MutS Homolog 2), and PMS2 (PMS1 Homolog 2). The encoded proteins are involved in DNA mismatch repair, creating MLH1–PMS2 and MSH2–MSH6 heterodimers. [LS represents a well-known hereditary cancer syndrome mainly in gastrointestinal neoplasms and endometrial cancer, but it could also be responsible for an increased risk of developing ovarian cancer (endometrioid) as well as breast cancer, though less frequently. In addition, colon adenomas, mostly with high grade dysplasia and/or villous histology, are peculiar features of LS, similar to skin tumors . LS is caused by a mutation in at least one of the following genes: odimers. ,72,73,74Among the hereditary cancer syndromes characterized by colonic polyposis, together with PJS and Polymerase proofreading-associated polyposis, MAP-associated polyposis is discussed below ,75.BRCA1/2, CHEK2, PALB2, RAD51C, RAD51D, and BRIP-1 are associated with an increased risk of developing ovarian cancer, without a specific phenotype [BRIP-1-associated hereditary cancer syndrome could also include a higher risk of developing breast cancer. The encoded protein BRIP-1 is involved in the DNA double-strand break repair by homologous recombination [Considering hereditary ovarian cancer, as mentioned earlier, alterations in henotype ,48,49,76bination ,76.BRCA1- and BRCA2-associated hereditary cancer syndrome (gene: BRCA1/2) [ATM) [Considering the syndromes predisposing to prostate cancer, several overlaps with breast or ovarian cancer syndromes emerge : BRCA1- BRCA1/2) ,27,28,292) [ATM) ,39,40, aHOXB13-hereditary cancer syndrome, associated with mutations in the HOXB13 gene, is closely related to prostate carcinoma. HOXB13, a member of the homeobox family, codes for a protein with an important role in urogenital development and which retains high levels of prostate expression even in adults, although the molecular mechanism by which HOXB13 promotes prostate cancer development remains unknown.TP53) [BRCA1- and BRCA2-associated hereditary cancer syndrome  [PALB2-associated hereditary cancer syndrome  [ATM-associated hereditary cancer syndrome or Ataxia telangiectasia  [STK11-associated hereditary cancer syndrome  or Peutz–Jeghers syndrome (up to 36%) [Similar to prostate cancer, pancreatic cancers are also associated with predisposing genetic syndromes in the mammaric/ovarian sphere: LF (gene: TP53) ,25; BRCARCA1/2,) ,27,28,29: PALB2) ,36,37,38ne: ATM) ,40; STK1 to 36%) ,31,32.PRSS1 gene, which encodes a cationic trypsin [SPINK I, CFTR, and CTRC. SPINK I encodes an acute-phase protein that functions as a trypsin inhibitor, preventing autodigestion of the pancreatic parenchyma by inhibiting the activation of digestive enzymes. The CFTR mutation is associated with several symptoms, including sinusitis, respiratory distress, male infertility, and constipation. CTRC, on the other hand, encodes a protease that can degrade trypsin and trypsinogen. Hereditary pancreatitis, on the other hand, is a syndrome predisposing specifically to pancreatic tumors. It is commonly characterized by an autosomal dominant inheritance pattern, typically associated in up to 80% of patients with mutations in the  trypsin . ClinicaWhile less common, it is noteworthy that the FAMM syndrome, which has already been extensively discussed in the melanoma section, can predispose up to 17% of patients to pancreatic cancer.Neuroendocrine tumors are linked to several genetic syndromes , includiMEN1 tumor suppressor gene that encodes menin [MEN1, an autosomal dominant disorder, is caused by germline mutations in the es menin ,83, a ubes menin ,85,86. Ies menin ,88. MEN1CDKN1B, which codes for the cell cycle-regulating protein p27 that inhibits cyclin/cyclin-dependent kinase complexes. While the malignant component is unknown, approximately 25% of MEN4 patients may develop neuroendocrine tumors, including gastrinomas potentially leading to peptic and gastric ulcerations, insulinomas, glucagonomas, VIPomas, and somatostatinomas. MEN4 is also linked with carcinoids and adrenocortical carcinomas secreting cortisol or aldosterone. Up to 80% of MEN1 patients develop primary hyperparathyroidism associated with parathyroid adenomas and with a female predominance [MEN4 is a recently discovered syndrome caused by germline mutations in the tumor suppressor gene ominance , while up < 0.005) [The von Hippel–Lindau syndrome, as discussed in the section on renal tumors, is an autosomal dominant disorder that predisposes individuals to a range of malignant and benign tumors, including renal cell carcinoma (clear cell subtype), pheochromocytoma, CNS hemangioblastomas, and retinal angiomas. Up to 12% of patients with vHL syndrome may develop pancreatic neuroendocrine tumors (PNETs), although most pancreatic lesions associated with the vHL syndrome are cysts or serous cystadenomas . In the < 0.005) .Gastric and esophageal cancers display a peculiar geographic, ethnic, and socioeconomic distribution, with higher incidence in Asian and developing countries . Well-esWith regard to gastric cancer (GC), the hereditary component is mainly related to two major syndromes: hereditary diffuse gastric cancer (HDGC) and gastric adenocarcinoma and proximal polyposis of the stomach (GAPPS), with different pathogenetic mechanisms. CDH1) gene, which encodes the cell adhesion protein E-cadherin [linitis plastic) without forming a mass [CDH1 testing should be offered in the presence of [HGDC is caused by germline mutations in the cadherin 1  represents a rare, autosomal dominant with incomplete penetrance variant of familial adenomatous polyposis (FAP) . APC gene that reduce gastric APC expression [APC gene is an oncosuppressor that encodes a protein involved in the control of cell adhesion though the negative regulation of β-catenin and E-cadherin interaction, thus preventing uncontrolled cell growth and cell migration [GAPPS is caused by specific point mutations in promoter 1B regions of the pression . The APCigration . Typicaligration ,98,99. Tigration . Neverthigration ,101. MolGastric cancer is also associated with a wide range of several cancer-associated syndromes . LS is oSTK11 [BRCA1/2 mutation carriers [A large case series also described a rate of approximately 5% of GC cases with both diffuse and intestinal-type histology in LF patients ; of thesSTK11 ; a lifetSTK11  and apprcarriers . Extremely rare genetic conditions are linked to esophageal cancers (EC). In particular, the Howel–Evans syndrome is characterized by distinctive cutaneous features such as hyperkeratosis of the palms of the hands and soles of the feet (tylosis), and it is strongly associated with esophageal squamous cancer, favored by traditional risk factors . BRCA2, ATM, STK11, FA, and FAMM may confer an increased risk of EC.Moreover, hereditary deleterious variations of Genetic predisposition due to the presence of germline pathogenetic mutations has been implicated in approximately 5–6% of colorectal cancer cases (CRCs) . AccordiAPC tumor suppressor gene with an autosomal dominant pattern of inheritance [Among polyposis syndromes, the most frequent are familial adenomatous polyposis (FAP) and its variants, such as the Gardner syndrome and attenuated familial polyposis syndrome (AFAP), which are all caused by germline mutations in the eritance . APC-mutation carriers also display a higher risk of duodenal and periampullary cancer, brain tumors (Turcot syndrome), thyroid papillary carcinoma, hepatoblastoma, pancreatic cancer, and desmoid tumors [Classic FAP is characterized by the presence of more than 100 colorectal adenomas, with a lifetime risk of CRC of >90% and a median age of CRC onset in untreated individuals of 39 years. It is responsible for ≤1% of all CRC diagnoses . Extracod tumors . Attenuated familial adenomatous polyposis (AFAP) is a phenotypic variant of FAP and is characterized by the presence of 10–99 colonic adenomas, delayed polyp development, and subsequently older age at cancer diagnosis compared with the classic form . In AFAPAPC-associated polyposis condition should be suspected if any of the following criteria is present: at least 10–20 cumulative colorectal adenomatous polyps, a family history of multiple colorectal polyps, and having a known APC variant and/or typical extracolonic features [An features . MUTYH, which encodes a DNA glycosylase belonging to the base excision repair complex [MUTYH alterations can develop CRC even without polyposis [Other less frequent polyposis syndromes include rare entities which represent a clinical challenge in terms of diagnosis and management as consequence of their low incidence and some overlapping features . MAP is  complex . The MUT complex . The cli complex . Of noteolyposis . The freolyposis . Large solyposis . HoweverMSH3 and MLH3, mismatch repair genes not associated with LS. The presence of biallelic variants is implicated in polyposis syndromes with an AFAP-like phenotype  called MSH3/MLH3-associated polyposis [Current multigenic panels also include olyposis ,120. PJS and Juvenile Polyposis syndrome (JPS) are characterized by an increased CRC risk caused by the presence of hamartomatous gastrointestinal polyps. Both syndromes are also associated with extracolonic cancers: breast, pancreatic, and gynecological cancers for PJS and upper GI cancers for JPS . Colonic serrated polyps are typical of sessile serrated polyposis cancer syndrome (SSPCS), with an overall CRC risk of approximately 20% . NTHL1 tumor syndrome (NTHL1), polyps’ histology may vary with adenomatous, hyperplastic, or serrated polyps. The NTHL1 gene encodes a protein that excises DNA base damage caused by reactive oxygen species [In the  species . The CRC species . Other rare causes of inherited polyposis include the PPAP syndrome or hereditary mixed polyposis .MLH1, MSH2, MSH6, and PMS2), leading to the loss of expression of their respective proteins [Among nonpolyposis syndromes, the Lynch syndrome has a central role. LS is responsible for nearly 3% of all colon cancers and is associated with a germline mutation in the mismatch repair (MMR) genes .A limitation of our work is the paucity of data for some syndromes and the weak or hypothetical sociation of some phenotypes with some HCSs. However, there are ongoing international studies that aim to level these gaps of knowledge —which quantify an individual’s risk of developing a particular disease through the evaluation of a score derived from single nucleotide polymorphisms present in a genome—will allow for personalized risk assessments, which are beneficial for prevention, early diagnosis, and disease management. Juxtaposing clinical knowledge with technological advances is and will be indispensable to best manage the complexity of hereditary cancer syndromes.


Prior studies demonstrated that encapsulation in poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) enhanced the delivery of enzymes used for replacement therapy (ERT) of lysosomal storage disorders (LSDs). This study examined how the copolymer lactide:glycolide ratio impacts encapsulation, physicochemical characteristics, stability, and release under lysosomal conditions. Hyaluronidase, deficient in mucopolysaccharidosis IX, was encapsulated in NPs synthesized using 50:50, 60:40, or 75:25 lactide:glycolide copolymers. All NPs had diameters compatible with cellular transport (≤168 nm) and polydispersity indexes (≤0.16) and ζ-potentials (≤−35 mV) compatible with colloidal stability. Yet, their encapsulation efficiency varied, with 75:25 NPs and 60:40 NPs having the lowest and highest EE, respectively (15% vs. 28%). Under lysosomal conditions, the 50:50 copolymer degraded fastest (41% in 1 week), as expected, and the presence of a targeting antibody coat did not alter this result. Additionally, 60:40 NPs destabilized fastest (<1 week) because of their smaller diameter, and 75:25 NPs did not destabilize in 4 weeks. All formulations presented burst release under lysosomal conditions , with 50:50 and 60:40 NPs releasing an additional small fraction after week 1. This provided 4 weeks of sustained catalytic activity, sufficient to fully degrade a substrate. Altogether, the 60:40 NP formulation is preferred given its higher EE, and 50:50 NPs represent a valid alternative, while the highest stability of 75:25 NPs may impair lysosomes. These results can guide future studies aiming to translate PLGA NP-based ERT for this and other LSDs. Lysosomes play a central role in maintaining cellular homeostasis by intersecting with multiple biological functions, including cellular metabolism, recycling, signaling, defense, and cell death ,2,3,4. AThe first-line treatment for LSDs is enzyme replacement therapy (ERT) using intravenous (i.v.) infusion of recombinant enzymes to restore deficient function ,12. ThisVarious approaches are being explored to help with these aspects. One strategy aims to provide recombinant enzymes with targeting elements to enhance their transport to disease sites, including the CNS, while another strategy aims to protect, hide, and/or control the release of enzymes by encapsulation ,16. The For these reasons, several studies have explored PLGA NPs for lysosomal ERT, including formulations loaded with acid sphingomyelinase for treatment of Niemann–Pick disease types A or B, hyaluronidase (HAse) for MPS type IX, iduronate-2-sulfatase for MPS type II, palmitoyl-protein thioesterase-1 for neuronal ceroid lipofuscinosis 1, galactosylceramidase for Krabbe disease, and acid α-glucosidase for Pompe disease ,39,40,41Among these factors, PLGA’s lactide:glycolide ratio is of key relevance since it is known to impact the overall copolymer hydrophobicity and, thus, physicochemical features such as NP encapsulation efficiency, size, stability, degradation, and cargo release ,45,46,47Therefore, the present study focused on exploring the role of PLGA copolymer, particularly, the lactide:glycolide ratio, on the encapsulation efficiency and physicochemical properties, as well as the stability and release under lysosomal conditions, of NPs encapsulating an enzyme for lysosomal ERT. We used PLGA copolymers with either 50:50, 60:40, or 75:25 lactide:glycolide ratios to formulate NPs encapsulating HAse, the enzyme deficient in MPS type IX OMIM 601492), as a model ,49,50. T2, as a m125Iodine (125I) were from Thermo Fisher Scientific . BioSpin Tris Columns were from BioRad . HAse Type I-S from bovine testes, 1000–2200 kDa hyaluronic acid (HA), Pluronic F68, ethyl acetate, trichloroacetic acid (TCA), and all other reagents were from Sigma-Aldrich . Gel permeation chromatography (GPC)-grade tetrahydrofuran (THF) was from Scharlab S.L. .PLGA 50:50 ) was purchased from Evonik . PLGA 60:40  and PLGA 75:25  were purchased from PolySciTech . Mouse anti-human ICAM-1 (clone R6.5) was affinity purified from a hybridoma obtained from American Type Culture Collection . Bovine serum albumin (BSA) and phosphate buffer saline (PBS) were from Thermo Fisher Scientific . Pierce Iodogen and 125I, as shown in [125I for 10 min at room temperature (RT) using Pierce Iodogen. Then, unconjugated 125I was removed using BioSpin Tris Columns with centrifugation at 1000× g for 4 min. Next, incubation in 15% v/v TCA containing 2.5% BSA was used to precipitate protein, which was separated with centrifugation at 2300× g for 5 min. Following this, the 125I-HAse or 125I-anti-ICAM-1 contents were measured as counts per minute (CPMs) using a gamma counter , and the respective protein concentrations were determined using a Bradford assay [125I-HAse or 125I-anti-ICAM-1 specific activities (CPMs/mass), from which the number of 125I-HAse or 125I-anti-ICAM-1 molecules can be obtained using 55 kDa or 150 kDa as the known MWs for HAse or anti-ICAM-1, respectively. In all cases, 125I and 125I-labelled materials were handled in an approved facility, under proper supervision and monitoring, and in compliance with all institutional regulations.Radiotracing was used to determine enzyme encapsulation and release described below, for which HAse was labeled with shown in . Alternashown in . For third assay . Data weg for 15 min at RT using an Avanti J-26 XP centrifuge with a J-12 rotor . PLGA NPs encapsulating HAse were manufactured using either 50:50, 60:40, or 75:25 glycolide:lactide copolymers, as described above . A previously described double emulsion and solvent evaporation technique was used . BrieflyThe NPs described above were either used without a targeting coat or further coated with an antibody that targets ICAM-1. For this purpose, NPs were mixed at RT for 2.5 h with 1 mg/mL anti-ICAM-1, as in our prior study . This coAll NPs were characterized by dynamic light scattering (DLS) using the Zetasizer Ultra instrument and analyzed with the respective ZS XPLORER software . NP hydrodynamic size and polydispersity index (PDI) were measured by diluting samples in PBS, while ζ-potential was measured by diluting samples in deionized water. For visualization using scanning electron microscopy (SEM), NPs were spread over slabs, dried under vacuum, coated with a gold layer in a cathodic evaporator, and finally observed using an SEM instrument from JEOL .125I-anti-ICAM-1, the 125I content was measured using a gamma counter to render the number of antibody molecules in the NP preparation, as described above in For NPs coated with 125I-HAse were centrifuged at 10,000 rpm for 10 min to separate the enzyme in the pellet. The 125I-HAse content of this pellet was then measured using a gamma counter after the removal of the supernatant and EE was calculated as a percentage, as follows: 125I-HAse specific activity (CPMs/mass), described in The encapsulation efficiency (EE) of PLGA NPs was determined using the direct method, as described . For thi125I-HAse, either with or without anti-ICAM-1 coating, were incubated for 30 min to 4 weeks at 37 °C under gentle shaking in artificial lysosomal fluid (ALF). The ALF was pH 4.5 and contained 0.525 mM magnesium chloride, 54.93 mM sodium chloride, 0.5 mM disodium hydrogen phosphate, 0.275 mM sodium sulfate, 0.871 mM calcium chloride dihydrate, 0.262 mM sodium citrate dihydrate, 150 mM sodium hydroxide, 108.3 mM citric acid, 0.786 glycine, 0.391 mM sodium tartrate dihydrate, 0.759 mM sodium lactate, and 0.782 mM sodium pyruvate. Similar to a previous study [125I-HAse in the supernatant. The pellet was dissolved in THF and analyzed using GPC to determine polymer MW and discern its degradation compared to control polymers. GPC measurements were run at 30 °C and 1 mL/min eluent flow through a PLgel 5 µm MIXED-D 300 × 7.5 mm column on an Agilent 1260 Infinity II instrument . This instrument was equipped with a dual-angle light scattering detector, a refractive index detector, and a viscometer. Calibration was performed with the Agilent EasiVial polystyrene calibration standard series. Finally, 125I-HAse release from NPs was quantified in the supernatant using a gamma counter, before and after TCA precipitation, as described in 125I content measured before and after TCA precipitation corresponds to the presence of free 125I and short, non-precipitable 125I-HAse-derived peptides, which are is interpreted as 125I-HAse degradation. PLGA NPs encapsulating us study , at indiExperiments similar to those described in n ≥ 3, and statistical significance was determined as p < 0.05. ANOVA followed by Tukey’s test was used for multiple comparisons, while two-group comparisons were assessed using Student’s t-test. All statistics were calculated using GraphPad Prism® version 8 .Data were calculated as mean ± standard error of the mean (SEM) from Our recent publication compared enzyme encapsulation vs. enzyme coating using 50:50 PLGA NPs . The resAs shown in Based on these differences, it is difficult to speculate which of the three formulations would be preferred. All NP formulations were monodispersed and had a markedly negative ζ-potential, which are favorable parameters regarding colloidal stability ,53. Theit-test but not using ANOVA. Interestingly, no fluctuations in these parameters were seen when we examined a similar formulation under physiological conditions [We next studied the fate of NPs under lysosomal conditions, which encompassed incubation at 37 °C and pH 4.5 in ALF, as this is the intended destination for these formulations. Regarding 50:50 PLGA NPs , similarnditions . This manditions ,63,64. Hnditions .Interestingly, the copolymer MW was found to significantly decrease before this time , with a Therefore, it appears polymer degradation starts much earlier than NP main destabilization observed after one week. This has been observed in prior studies showing a faster hydrophilic attack in internal spaces compared with the NP surface ,67. ThisFurthermore, we examined the stability under lysosomal conditions of the other two formulations . As for Instead, we observed that NPs made from the 60:40 PLGA copolymer behaved more similarly to those fabricated from 50:50 PLGA in terms of changes in hydrodynamic size a. They fAltogether, 75:25 PLGA NPs showed slower polymer degradation and NP destabilization, which may render more sustained enzyme release and, thus, require less frequent administration compared to the other two formulations. This is important because, typically, lysosomal ERTs are administered every two weeks in specialized medical centers, which increases the cost and burden of these treatments ,12. HoweAs stated, our previous study on PLGA NPs encapsulating lysosomal enzymes also involved NP coating with an antibody that recognizes ICAM-1 . This prWe used NPs fabricated using the 50:50 lactide:glycolide copolymer as an example . The antUpon incubation under lysosomal conditions, antibody-coated NPs presented slightly higher changes in size by day 2 and up to 1 week a compare125I-labeled HAse (The data described above would suggest that, for 50:50 PLGA NPs, HAse may be released under lysosomal conditions in a biphasic manner: fast first, as polymer degradation starts and pores open in particles, and then when enzyme molecules remaining in the structure are released upon full NPs destabilization. We assessed this by radiotracing led HAse . The resled HAse . This woled HAse a. For in125I-HAse from free 125I or 125I attached to short peptides, as described [Along with the release from NPs, we also inferred the possible occurrence of enzyme degradation using TCA precipitation to separate escribed . As showescribed , rather escribed . NeverthFinally, we investigated whether the enzyme levels observed would hold any catalytical significance, for which we focused on the 50:50 NP formulation given its lower enzyme release level . DespiteThese data demonstrate that HAse retains significant catalytic activity after encapsulation in PLGA NPs and is capable of sustaining activity for at least 4 weeks under lysosomal conditions, which agrees with previous results . DespiteHYAL1 gene, resulting in the progressive accumulation of the extracellular matrix glycosaminoglycan, HA [These results hold considerable relevance regarding ERT for MPS IX. This disease is caused by mutations in the ycan, HA ,49,50. Tycan, HA ,49,50. Pycan, HA ,49,50. Tycan, HA , suggestThe present study focused on the role of the PLGA lactide:glycolide ratio, particularly 50:50, 60:40, and 75:25, in the properties of NP encapsulating HAse, as a model for lysosomal ERT . All thr


One hundred and six candidate regions for salt tolerance were mapped on all of the chromosomes through BSA-seq using F2 in an extreme population. Based on the polymorphisms identified between both parents, we detected 77 genes. A large number of differentially expressed genes (DEGs) at the seedling stage under salt stress between these two inbred lines were detected using transcriptome sequencing. GO analysis indicated that 925 and 686 genes were significantly enriched in the integral component of the membrane of AS5 and NX420, respectively. Among these results, two and four DEGs were identified as overlapping in these two inbred lines using BSA-seq and transcriptomic analysis, respectively. Two genes (Zm00001d053925 and Zm00001d037181) were detected in both AS5 and NX420; the transcription level of Zm00001d053925 was induced to be significantly higher in AS5 than in NX420 (41.99 times versus 6.06 times after 150 mM of NaCl treatment for 48 h), while the expression of Zm00001d037181 showed no significant difference upon salt treatment in both lines. The functional annotation of the new candidate genes showed that it was an unknown function protein. (4) Conclusions: Zm00001d053925 is a new functional gene responding to salt stress in the seedling stage, which provides an important genetic resource for salt-tolerant maize breeding.(1) Background: Salt stress is an abiotic factor that limits maize yield and quality. A highly salt-tolerance inbred AS5 and a salt-sensitive inbred NX420 collected from Ningxia Province, China, were used to identify new genes for modulating salt resistance in maize. (2) Methods: To understand the different molecular bases of salt tolerance in AS5 and NX420, we performed BSA-seq using an F2 population for two extreme bulks derived from the cross between AS5 and NX420. Transcriptomic analysis was also conducted for AS5 and NX420 at the seedling stage after treatment with 150 mM of NaCl for 14 days. (3) Results: AS5 had a higher biomass and lower Na Triticum aestivum L.), maize (Zea mays L.), rice (Oryza sativa L.), and cotton (Anemone vitifolia Buch), and cultivating salt-tolerant varieties can effectively improve these negative effects [As a major environmental abiotic stress, soil salinity affects more than 7% of the world’s land area, and the total area of saline soil in China accounts for 4.88% of the country’s total available land ,2. Large effects . The sal effects ,6,7, chr effects , and the+) evokes osmotic damage and ionic stress, and the uptake, translocation, and compartment of Na+ by the SOS pathway at cellular, tissue, and whole-plant levels are essential for plant salt tolerance [+ from the cytoplasm by transporting Na+ into the vacuole or out of the cell is conducted by SOS1, which is a plasma-membrane-localized NHX family Na+/H+ antiporter, and it is activated by the direct phosphorylation of SOS2 (a CBL-interacting protein kinase (CIPK) family kinase) [+ concentration is regulated is through ion transporters, especially those with a preference for Na+ or potassium (K+) ions, such as the Na+-selective transporters encoded by the Na+/H+ exchanger (NHX), high-affinity K+ transporter 1 (HKT1) family genes, and the Na+-selective transporter encoded by high-affinity K+ (HAK) genes [Among studies on osmotic, ionic, and oxidative stress under saline conditions, sodium chloride (NaCl) is the most studied worldwide due to its high solubility and ubiquitous distribution ,10. The olerance . The rem kinase) ,13. AnotK) genes ,14,15,16+ content in cells. For example, in a study, it was found that the wheat ancestral Na+ transporter gene NAX2 (Na+ Exclusion 2) could increase the yield by 25% under salinity [+ Content 1) promotes salt tolerance by regulating K+/Na+ homeostasis [+ variations were conferred by ZmNSA1  under NaHCO3 conditions [Cucumis sativus L.) fruit skin [Brassica napus [To broaden natural genetic diversities and to acquire more valuable sources for breeding salt-tolerant crops, dozens of quantitative genetic trait (QTL) genes and candidate genes from genome-wide association studies (GWAS) have been identified to be associated with natural variations in salt tolerance ,18,19,20salinity . In otheeostasis , the maieostasis , and shouit skin , the panca napus .Zm00001d053925 showed an inducible response at the transcriptional level upon NaCl treatment in NX420 and AS5, which were collected from Ningxia Province, China. Protein structure prediction indicated that Zm00001d053925 contained five transmembrane domains. These results indicate that Zm00001d053925 could be a new gene for salinity tolerance, with an unknown function.In this study, BSA-seq and RNA-seq were combined to find new candidate maize genes involved in leaf tolerance to salinity using two inbred lines, named “NX420” and “AS5”, which show differential tolerance levels to salinity. Two candidate genes were screened using the combination of RNA-seq from NaCl-treated inbred seedlings for 14 days and BSA-seq. + content and decreased leaf K+ content, which in turn caused an ion imbalance  [+ content was significantly lower in AS5 than that in NX420 leaves, and no differences were observed in the roots  caused a significant reduction in plant height and biomass at the seedling stage A–C. AS5  ratios) ,22,27. The roots D; the K+5 leaves E, which 0 leaves H. These 2 population derived from a cross between the salt-tolerant inbred AS5 and the salt-sensitive inbred NX420. This F2 population was used to identify the degree of salinity tolerance in the seedling stage in 150 mM of NaCl treatment for 14 days. Over 500 F2 seeds were analyzed, and 30 lines were screened to show salt tolerance (such as AS5 and LA1-30) and salt sensitivity (such as NX420 and LS1-30), using plant height to differentiate between the salt-tolerant lines and the salt-sensitive lines. Under 150 mM of NaCl treatment, the plant height survey and analysis of the two extreme populations showed that the plant height difference between the two extreme populations was significant, which was suitable for follow-up research , and the salinity-sensitive bulk  . A totalIn total, 6,516,313 SNPs were detected between the two bulks. There were a total of 657,086 SNPs on Chromosome 1, 639,709 SNPs on Chromosome 2, 878,652 SNPs on Chromosome 3, 668,120 SNPs on Chromosome 4, 642,954 SNPs on Chromosome 5, 598,968 SNPs on Chromosome 6, 690,312 SNPs on Chromosome 7, 625,423 SNPs on Chromosome 8, 598,375 SNPs on Chromosome 9, and 516,732 SNPs on Chromosome 10 . An assoTo understand the differentially expressed genes responding to salt stress, we also performed transcriptomic sequencing under the condition of normal growth  and salt stress (150 mM of NaCl treatment for 14 days) to establish which genes responded differently in AS5 and NX420. The RNA sequencing of the 12 cDNA  libraries was generated after filtering a total of 80.7 Gb clean bases; the average percentages for Q20 and Q30 were 96.2% and 87.7%, respectively . FurtherThe KEGG enrichment results showed the DEGs from the treatment with and without 150 mM of NaCl for 14 days in AS5 and NX420. For NX420 and AS5, the top 20 KEGG pathways were based on the rich factor B,C. CommA Gene Ontology (GO) analysis was performed to explore the biological process, the cellular component, and the molecular function related to salinity tolerance in NX420 and AS5 at the seedling stage. The top 20 GO terms were screened out based on enriched gene numbers D–I. CommZm00001d053925 and Zm00001d037181, and four genes in NX420, Zm00001d053925, Zm00001d037181, Zm00001d0039920, and Zm00001d045404. Among them, three genes were significantly enriched in the integral component of the membrane. In addition, Zm00001d053925 and Zm00001d037181 were both detected in AS5 and NX420  regions. With transcriptome sequencing, we detected a large number of differentially expressed genes (DEGs) at the seedling stage under salt stress. The overlap of BSA-seq and DEGs showed that were two genes in AS5, nd NX420 .Zm00001d037181 and Zm00001d053925, were finally determined through transcriptome and BSA techniques. To further confirm whether the candidate genes respond to salt stress and have different expressions in the two parents, 150 mM NaCl stress was applied to maize seedlings 10 days after sewing, and the leaves at 0 h, 24 h, and 48 h after treatment were taken for real-time PCR verification. The transcriptional levels of Zm00001d053925 in the AS5 leaves at 24 h were 7.83 times those at 0 h, while in NX420, its expression was only 1.46 times that at 0 h. After 48 h of treatment, its expression in AS5 was 41.99 times that at 0 h, while in NX420, its expression was only 6.06 times that at 0 h  [ZmHKT1 (chromosome 3) [ZmHAK4, is preferentially expressed in the root stele, and encodes a novel membrane-localized Na+-selective transporter that mediates shoot Na+ exclusion, probably by retrieving Na+ from xylem sap. Therefore, the function of the new gene may be different from that of ZmHAK4, which needs further functional analysis.The analysis of Nawo lines ,31. Simiwo lines ,16,31, te leaves . Howevered lines . We specosome 3) , and ZmHMaize salt tolerance is a complex trait which comprises distinct mechanisms, such as osmotic, ionic, and oxidative tolerance and other tolerances ,34,35, mZm00001d053925 and Zm00001d037181. Among them, three genes were significantly enriched in the integral component of the membrane. In addition, Zm00001d053925 and Zm00001d037181 were both detected in AS5 and NX420  or watered to soil saturation with water (Control). Ten seeds were planted in each pot and then grown in a glasshouse for 2 weeks; the leaves and roots were collected for the measurement of Na+ and K+ contents and to measure the height and biomass (fresh) of NX420 and AS5. The statistical analysis of the data used Graphpad prism9. The maize inbred NX420 and AS5 were collected from Ningxia Agricultural Institute, Ningxia Province, China. To measure the leaves and roots’ Na+ and K+ contents were determined with reference to the method described previously [+ and K+ concentrations were analyzed using a 4100-MP AES device .The Naeviously . In esseWe generated F1 seeds by crossing NX487 and AS5, and then we obtained the F2 population by selfing the F1 plants. The F2 plants were grown under 150 mM NaCl conditions for 14 days. Subsequently, we selected 30 salt-tolerant F2 plants showing a high height phenotype comparable with that of AS5 as a tolerant population, and 30 salt-sensitive F2 plants showed a tall height phenotype comparable with that of NX420 as a sensitive population.For the BSA-seq analysis, the young leaves of each line were sampled 14 days after being planted. The cetyltrimethylammonium bromide (CTAB) method was used to obtain high-quality genomic DNA . DNA fro2 population. We selected 30 plants that were extremely tolerant and sensitive to salinity to create an extreme population. Genomic DNA was extracted using a DNA extraction kit . The DNA quality was checked using a microplate reader and agarose electrophoresis. Library preparation was performed according to the manufacturer’s protocol; it can be found in https://www.yuque.com/yangyulan-ayaeq/oupzan/cfq29z, 13 September 2022, Whole Genome Sequencing Library Preparation (DNBSEQ) BGI-NGS-JK-DNA-001 A0. Genomic re-sequencing was conducted to generate paired-end 100-base (PE100) reads using the DNBseq platform, which was conducted by BGI . Clean reads were aligned to the reference genome sequences of the B73 genome (https://www.ncbi.nlm.nih.gov/assembly/GCF_000005005.2/ (accessed on 13 September 2022), Zea_mays.AGPv4.dna.toplevel.fa) using BWA software [BSA-seq was used to identify the genes regulating salt tolerance in the Fsoftware . SNPs ansoftware . We exclsoftware ,49. The Three replicated plants that were sampled and collected were the same as those in 4.1; the leaves of NX420 and AS5 were used for transcriptome sequencing. There were three independent RAN-seq libraries per treatment, namely, Control: AS5-1, -2, -3, and NX420-1, -2, -3, Salt treatment: AS5-1, -2, -3, and NX420-1, -2, -3. Total RNA samples were extracted using TRIzol reagent (Invitrogen) and then treated by RNAase-free DNAse I (NEB) in order to remove genomic DNA. mRNA libraries were created according to the standard protocols provided by BGI. mRNA quality, including the mRNA concentration and fragment size, was tested by using Qubit2.0 and Agilent 2100. mRNA was enriched using Dynabeads oligo (dT)  and a fragmentation buffer. Double-stranded cDNAs were produced using reverse transcriptase  and random hexamer primers and further purified using AMPure XP beads. The purified double-stranded cDNA samples were enriched by PCR to construct the final cDNA libraries for sequencing using Hiseq 2500 (150 bp paired ends), provided by BGI (China). Clean reads were also aligned to the reference genome sequences of the B73 genome. Gene expression differences among the different sample points were detected using the EBSeq package (vl.10.1). A fold change ≥ 2 and a false discovery rate (FDR) < 0.01 were set to act as the standards for screening the DEGs. Functional classification of the DEGs, including the Gene Ontology and KEGG pathways, was analyzed using the GOseq R package (Release2.12) and KOBAS software (v2.0).Zm00001d037181 and Zm00001d053925 transcription levels, 10-day-old NX420 and AS5 plants were treated with 150 mM NaCl solution; then, the samples (leaves and roots) were collected at 0 h, 24 h, and 48 h. Total RNA was extracted using an RNA pure plant kit ; 2.0 μg RNA was used to synthesize first-strand cDNA using M-MLV reverse transcriptase, and qRT-PCR analyses were conducted using TransStart Top Green qPCR SuperMix  on an ABI 7500 thermocycler . The maize GAPDH gene was provided as an internal standard. The primer sequences  are listed in −ΔΔCt-based calculation was used to quantify gene expression.To analyze the effect of salt stress on the Zm00001d053925, a new candidate gene involved in salt tolerance, which was identified with the combination of BSA-seq and transcriptome analysis based on the different phenotypes of two maize inbred lines. It encoded an unknown function protein which was predicted to contain five transmembrane domains, and its transcription levels were related to salt responses. Thus, a novel gene target for the development of salt-tolerant maize was identified, which provides a new gene resource for salt-tolerant maize research.In conclusion, our QTL approach obtained