The termination of employment is not the end of an organization–employee relationship. As ex-employees can provide various benefits to their former organizations, and a large number of ex-employees have accumulated in enterprises because of increased employee mobility, research on ex-employees’ contribution behavior, and how it is generated are significant to organizations in making use of their ex-employees effectively and consequently improving organizational efficiency. Based on the research into organizational citizenship behavior, Study 1 extended the focus of organizational citizenship behavior research to include ex-employees, introducing the concept of ex-employee citizenship behavior. The measurement of ex-employee citizenship behavior was developed based on Hinkin’s tutorial. Using social identity theory, Study 2 discussed how ex-employee citizenship behavior is generated. A two-wave survey of 291 former employees was conducted. Hierarchical regression analysis and the bootstrap method were then applied to test the hypotheses. The results showed that legacy identification was positively related to ex-employee citizenship behavior. Furthermore, the interaction between perceived organizational prestige and perceived insider status was positively related to legacy identification. Perceived organizational prestige and perceived insider status were also indirectly and interactively related to ex-employee citizenship behavior through legacy identification. The positive relationship between legacy identification and ex-employee citizenship behavior was moderated by the cooperative relationship between the current and former organizations. Additionally, the indirect positive effect of the interaction between perceived organizational prestige and perceived insider status on ex-employee citizenship behavior through legacy identification is moderated by the cooperative relationship between the current and former organizations. The theoretical and practical implications of this study were discussed. Finally, the limitations of this study were presented alongside suggestions for future research. Historically, employment termination has been considered the end of the relationship between organizations and ex-employees . HoweverThe majority of previous research on ex-employees’ contributions has been conducted in the field of organizational strategy, based on social network and social capital theory. To fill these research gaps, this study extends the concept of organizational citizenship behavior (OCB) to include ex-employees, to capture the various behaviors that could benefit their former organizations. In line with This study contributes to the existing literature in several ways. First, it extends OCB to include ex-employees and proposes the concept of Ex-ECB to conceptualize the various behaviors of ex-employees. These behaviors are discretionary, non-contractual reward requirements that contribute to organizational effectiveness. Second, using SIT as a framework, this study considers an LI perspective to illustrate how Ex-ECB is generated, which provides a proper perspective to explain how Ex-ECB occurs. Third, premised on SIT, the interaction between POP and PIS is posited as the antecedent of LI and Ex-ECB, which provides a comprehensive picture of the factors relating to LI and Ex-ECB. Fourth, the study identifies the CR between current and former organizations as a moderator, which affects the relationship between the LI and Ex-ECB. The study also explores how the interaction between POP and PIS affects Ex-ECB through LI, enriching the boundary conditions of how citizenship behavior is generated. Organizational citizenship behavior has been widely studied because it helps improve organizational effectiveness . Given tThis study defines Ex-ECB as discretionary behaviors which benefit the former organization but are not contractually rewarded by it. The first component of this constitutive definition, “discretionary,” suggests that the behavior is not an executable requirement of the role or behavior description, but a matter of personal choice . As no rThe measure of Ex-ECB was developed following First, initial items were generated. To ensure the success of initial item generation, we carefully considered the development of a well-articulated theoretical foundation that indicates the content domain for the new measure. Our goal was to develop measures that would sample the theoretical domain of interest to demonstrate content validity . Based oNext, a content validity assessment was conducted. Content validity refers to how well a scale measures a given construct. We used a popular method, provided by The third step was item scaling. Likert-type scales are the most frequently used in survey questionnaire research, and they have high compatibility for use in behavioral research . As suchp < 0.001), indicating that the scale met factor analysis requirements. An exploratory factor analysis revealed that the six items were presented on one factor, with a contribution rate of variance reaching 72.6%. Each item’s factor loading was greater than 0.5, indicating that the internal structure was clear, the construction validity was high, and the contribution to the potential construction was ideal. All of these aspects conformed to the theoretical hypothesis of one-dimensional construction. Thus, all six items were retained.Next, the initial number of items was reduced. Once data were collected, factor analysis was conducted to further refine the new scales . The preInternal consistency was then assessed to determine the efficacy of our test measures. The higher the internal consistency, the more reliable the survey . Cronbac2/df = 1.38. These values were all at or above the recommended standards  with 84 participants in the pretest stage . We usedds e.g., . The iteFinally, convergent validity was assessed to test how closely an item was related to other items measuring the same construct. To calculate convergent validity, we calculated average variance extracted (AVE) and composite reliability. The results revealed an AVE of 0.73 (>0.5) and composite reliability of 0.94 (>0.7). Convergent validity is achieved when the correlations between items of the measures are sufficiently high ; thus, tNomological validity was assessed according to the extent to which systematic support exists for theoretically proposed connections between Ex-ECB and related variables . AccordiFirst, we found that LI was positively related to Ex-ECB. According to SIT, people are likely to generate behaviors consistent with their identity . The morTo test nomological validity, snowball sampling was used to collect data. Direct participants were obtained from the alumni groups of three universities in China. Eligible indirect participants were obtained through direct participants. Each participant was informed of the study’s purpose and procedures, and they received a compensation of 5 RMB for each wave. We then distributed surveys at two time points, using WeChat . Three questions were set in the beginning, to identify the eligible participants. Subjects were required to have been full-time employees at their former organizations, and their most recent termination must have been voluntary. In the first wave, 210 participants were recruited. They participated in a survey on LI, PCV, and nostalgia. Fifteen days later, a second wave was conducted, and 181 participants were obtained. They participated in a survey on Ex-ECB and demographic information. A unique WeChat ID was used to match the two-wave surveys. Three attention test questions and a final self-evaluation question on seriousness were used to screen out careless participants. A total of 172 participants were included in this study. Among them, 90 were women (52.3%) and 82 were men (47.7%). Two were aged 20 or below (1.2%), 95 were aged 21–30 years (55.2%), 62 were aged 31–40 years (36.0%), and four were aged 41 years or above (2.3%). Forty-eight had not achieved a bachelor’s degree (27.9%), 98 had a bachelor’s degree (57.0%), and 26 had a master’s degree or higher (15.1%).Participants were asked to score examples of Ex-ECB , LI  and nostalgia (r = 0.51), and it was negatively and significantly correlated with PCV (r = –0.49) at moderate levels. In addition, we conducted path modeling using structural equation modeling (SEM) to test the hypothesized relationships. We specified the effects of LI, PCV, and nostalgia on Ex-ECB. The path model fit the data well . As shown in p < 0.001), PCV , and nostalgia  were positively related to Ex-ECB.Correlation analyses showed that Ex-ECB was positively and significantly correlated with LI  and 152 were men (52.2%). Four were aged 20 years or below (1.4%), 165 were aged 21–30 years (56.7%), 100 were aged 31–40 years (34.4%), and 20 were aged 41 years or above (6.9%). Of these, 93 had not achieved a bachelor’s degree (32.0%), 181 had a bachelor’s degree (62.2%), and 17 had a master’s degree or above (5.8%).Perceived organizational prestige, PIS, and LI were measured using maturity scales written in English. Four doctoral students majoring in Human Resource Management conducted a “back translation” procedure to generate the Chinese version. Study 1 developed the Ex-ECB scale. All scales were revised based on a pre-test (84 participants). All scales adopted a 5-point Likert scale, with “1” meaning “very inconsistent” and “5” meaning “very consistent.” Finally, we used CR as a two-category moderating variable.To measure POP, we referred to the scale of To measure PIS, the To measure LI, The CR between the current and former organizations was a categorical variable. This was measured by one question: “Does your current organization have a CR with your former organization?” The answer options were “Yes” or “No.”The control variables were proposed based on previous studies. First, the former organizational tenure and time-lapse might affect the LI . TherefoDiscrimination validity and common method biases were first calculated. Descriptive statistics and correlation analyses were then performed. Finally, hierarchical regression, simple slope analysis, and the bootstrap method were applied to test our hypotheses.To test the discrimination validity of each variable, AMOS17.0 was used for CFA. To reduce common method bias, process and statistical controls were performed. Process control was ensured by the anonymity of our data collection methods, as well as the inclusion of reverse questions and screening questions. To ensure statistical control, Harman’s single-factor test was performed in the data analysis stage. The first factor explained 34.86% of the variance, which was less than 40% . Thus, nr = 0.35, P < 0.001), POP was positively correlated with Ex-ECB , PIS was significantly positively correlated with LI , PIS was significantly positively correlated with Ex-ECB , LI was significantly positively correlated with Ex-ECB , and CR was significantly positively correlated with Ex-ECB . In addition, the interaction between POP and PIS was significantly positively correlated with LI , and the interaction between LI and CRs was significantly positively correlated with Ex-ECB .The descriptive statistical analysis results are shown in p < 0.001). Thus, H1 was verified.A hierarchical regression analysis was conducted using SPSS 22.0 to verify H1. As demonstrated by Model 3 in p < 0.05). To better interpret the moderating effect, following Hierarchical regression and simple slope analyses were conducted to verify H2. The interaction term was calculated after the two variables were mean-centered to eliminate collinearity. Model 2 in via LI is dependent on PIS. The pattern of the results for H1 and H2 offers tentative support for this complex association. To corroborate this conclusion, we used the bootstrap method to verify the mediation effect. As shown in SE = 0.02, 95%CI = 0.003, 0.08). These results indicate that PIS moderates the indirect association between POP and Ex-ECB via LI. As shown in B = 0.15, SE = 0.05, 95%CI = 0.07, 0.25). These findings supported H3.H3 proposes a conditional indirect relationship , wherebyp < 0.001). Furthermore, the simple slope analyses shown in A hierarchical regression analysis was used to verify H4. The results of the analysis are presented in SE = 0.03, 95%CI = 0.01, 0.13). These results indicate that the CR between the current and former organizations moderated the indirect association between the interaction term (POP and PIS) and Ex-ECB via LI. As shown in B = 0.15, SE = 0.06, 95%CI = 0.05, 0.28; Mean: B = 0.24, SE = 0.06, 95%CI = 0.14, 0.37; +1 SD: B = 0.33, SE = 0.09, 95%CI = 0.18, 0.53) than for ex-employees with non-CRs . These findings supported H5.H5 proposed that the positive effect of the interaction between POP and PIS on Ex-ECB through LI is moderated by the CR between the current and former organizations, such that the mediating effect is stronger when a CR is present. The pattern of results from H1, H2, H3, and H4 offers tentative support for this complex association. To corroborate this conclusion, the bootstrap method was used to verify this effect. As shown in The termination of employment is not the end of the organization-employee relationship. Ex-employees can provide various benefits to their former organizations . MoreoveFirst, most research on ex-employees’ contributions exists in the field of organizational strategy . HoweverFurthermore, the interaction between POP and PIS was posited as an antecedent of LI and Ex-ECB. These findings concur with SIT, showing that the identification resulted from neither organizational prestige nor social interaction and interdependence, such as PIS . InsteadWe also found that the CR between current and former organizations strengthened the relationship between LI and Ex-ECB, as well as the indirect positive effect of the interaction between POP and PIS on Ex-ECB through LI. These findings concur with previous research showing that identification is not related to an individual’s behaviors, but rather the relationship between identification and behaviors depends on identity salience . CRs, asFirst, this study calls on organizations to monitor Ex-ECB. Although some excellent enterprises’ management practices lead to theories, many companies remain unaware of beneficial ex-employee behaviors; some do not even include ex-employees in their human resource management. This study explores the phenomenon of Ex-ECB and identifies six forms . Thus, this study may educate companies about the benefits of Ex-ECB.This study also found that POP and PIS influence Ex-ECB through LI. Therefore, to improve LI and finally improve Ex-ECB, enterprises should pay attention to improving the POP and PIS of ex-employees. To improve POP, enterprises may institute efforts such as hiring high-profile leaders , being cFinally, to improve the efficiency of ex-employee management, enterprises should establish a robust external cooperation network and pay attention to the relationship between ex-employees’ current and former organizations. When a CR exists, an ex-employee has a more salient LI with their former organization, and they are more inclined to return resources. If a CR is not present, employers may suppress an employee’s LI with former organizations.First, this study explains how Ex-ECB is generated from LI. This is an important perspective in explaining Ex-ECBs and is lacking in the social network of former organizations, which has irreplaceable significance for how Ex-ECB is generated. In addition, further studies may want to explain Ex-ECB in the context of other theoretical perspectives, such as mourning  and sociMoreover, like OCB, ex-employees who carry out citizenship behaviors are also faced with the distinction of being a “good soldier” or “good actor” . AlthougFurthermore, this study identifies CRs as the boundary condition of the relationship between LI and Ex-ECB. It also considers the existence of a CR between the current and former organizations as an important factor. However, the relationships between former and current organizations can vary and become complicated. For example, they may cooperate in some fields but be competitive in other domains. Our measurement captured whether any CR existed between the current and former organizations. However, this definition failed to capture more complicated features of this concept, such as the degree and aspect of the cooperation. We will pursue this research direction, and we anticipate exploring the complexities of the CR between current and former organizations in the following studies.Additionally, it is worth studying how Ex-ECB influences current organizations. Role conflict theory holds that individual participation in one role activity will inevitably lead to the reduction of time and energy to participate in another role activity; therefore, Ex-ECB would consume time and energy that would otherwise be contributed to the current organization . In contWe also conducted scale development strictly according to Finally, the data used in this study were obtained from a single source. Although this study adopted a two-wave approach, it is still at risk of common method bias. In future research, multisource evaluation methods may be adopted; this approach would also reduce the problem of social approval in self-reported data.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval were not required for the study on human participants, in accordance with the local legislation and institutional requirements. The subjects provided their written informed consent to participate in this study. Written informed consent was obtained from the individuals for the publication of any potentially identifiable images or data included in this article.ZT, QY, SQ, and YG: research design and revising the manuscript. ZT: data collection and writing of the original draft. ZT and SQ: data analysis. All authors contributed to the article and approved the submitted version.


Euphorbia supina Rafin (ESR), has been used for centuries as a folk medicine to treat bronchitis, hepatitis, hemorrhage, and skin inflammation. Flavonoids, terpenes and tannins are enriched in ESR, and although ESR has proven antioxidative activity, its biological activities are largely unknown. Here, we investigate whether and how ESR protects keratinocytes against DPM-mediated apoptosis. We found that ESR-extracts (ESR-Ex) protect keratinocytes from DPM-induced apoptosis by inhibiting NOX activation in keratinocytes in response to DPM. We also demonstrated that ESR-Ex suppresses NOX activation via a blockage of the aryl hydrocarbon receptor (AhR) activation-mediated transcription of neutrophil cytosolic factor 1 (NCF1)/p47phox, a subunit of NOX. Our study reveals previously uncharacterized biological activity of ESR-Ex; i.e., its inhibition of Ahr and NOX activation. Thus, because the inhibition of NOX has already been developed to treat NOX-mediated diseases, including various types of cardiovascular diseases and cancers, initiated by air pollutants and because AhR activation contributes to the development of chronic inflammatory diseases, our study provides further advantages for the medical use of ESR.Air pollutants contribute to the development of diseases such as asthma, chronic obstructive pulmonary disease (COPD), pulmonary cancer, cardiovascular problems, and some skin diseases. We recently found that a major air pollutant, diesel particulate matter (DPM), induces apoptosis in human keratinocytes by increasing a proapoptotic lipid mediator, ceramide. DPM activates nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX), which stimulates sphingomyelinase, leading to an increased conversion of sphingomyelin to ceramide. Interestingly, we characterized that although NOX is a reactive oxygen species (ROS) generator, the activation of sphingomyelinases by NOX is an ROS-independent mechanism. A Korean weed, prostrate spurge  Air pollutants are one of the main environmental factors in the development of diseases such as asthma, chronic obstructive pulmonary disease (COPD), pulmonary cancer, cardiovascular problems, some skin diseases (contact hypersensitivity and dermatitis), the impairment of skin barrier function and the acceleration of premature skin aging. Diesel particulate matter (DPM) is produced from diesel engines, is comprised of polycyclic aromatic hydrocarbons, benzene, formaldehyde, acetaldehyde, and 1,3-butadiene, and is a major air pollutant.Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs) are plasma membrane-bound enzymes that produce reactive oxygen species (ROS) . NOX is Our prior study demonstrated that DPM induces apoptosis in human keratinocytes (KC)(HaCat cells) via a pro-apoptotic lipid mediator, ceramide-dependent pathway. DPM activates NOX followed by sphingomyelinase activation, resulting in increased ceramide production via the hydrolysis of sphingomyelin, leading to ceramide-inducing apoptosis . InteresEuphorbia supina Rafin (ESR), has been used as a folk medicine to treat several types of diseases, including bronchitis, hepatitis, hemorrhage, and skin inflammation [A weed, the Korean prostrate spurge ammation . ESR extammation  and exhiammation . Severalammation  and melaammation  and induammation  have alrHere, we investigated whether and how ESR-Ex protects keratinocytes against DPM-mediated apoptosis in cultured human KC, and we discovered that ESR-Ex inhibits NOX activation via the blockage of Aryl hydrocarbon receptor (Ahr) activation-mediated increases in the transcription of neutrophil cytosolic factor 1 (NCF1)/p47phox, a subunit of NOX in KC in response to DPM exposure, resulting in suppressed DPM-mediated ceramide-inducing apoptosis.An LC-MS/MS analysis identified that ESR extracts (70% ethanol) (ESR-Ex) contain several flavonoids and tannins, such as quercetin, kaempferol, isorhamnetin, gallic acid and their derivatives .We first assessed that influence of ESR on cell viability and then investigated whether ESR-Ex suppresses decreases in cell viability in KC exposed to DPM. Up to 100 g/mL ESR alone does not affect cell viability, while 200 µg/mL modestly decreases cell viability . ConsistWe next examined the effect of ESR-Ex on NOX activation by DPM. Consistent with our prior study , DPM actWe next measured cellular ROS levels by a DCFDA agent. ROS production was significantly increased in KC incubated with DPM, while the elevation of cellular ROS was significantly suppressed by the coincubation of DPM with ESR-Ex, Apo or NAC B. We furSince our prior study shows that an increases in a pro-apoptotic lipid mediator, ceramide, increases apoptosis in KC following exposure to DPM , we nextExcess ceramide is pro-apoptotic, while the metabolic conversion of ceramide to sphingosine-1-phosphate (S1P) (via ceramide hydrolysis by ceramidase followed by phosphorylation by sphingosine kinase) rescues cells from apoptosis . We founThese results suggest that ESR-Ex and Apo suppress an increase in proapoptotic short chain ceramide production and maintain a metabolic rescue mechanism (ceramide-to-S1P), thereby reducing cell toxicity in KC following DPM. Moreover, although NAC inhibits ROS production C,D, NAC We demonstrated that sphingomyelinase is activated by NOX activation and results in increased ceramide production by sphingomyelin hydrolysis . We nextp-dioxin and benzo(a)pyrene that activate AhR [DPM contains chemicals such as 2,3,7,8-tetrachlorodibenzo-vate AhR ,14, leadvate AhR ,16. Becavate AhR ,18 (TablWe first investigated the activation of AhR in KC exposed to DPM (assessed by AhR phosphorylation). We confirmed AhR activation in our cultured system using an established AhR ligand, FICZ  Figure . We thenWe next investigated levels of p47phox protein of which production is transcriptionally regulated by AhR . WesternFinally, we found that ESR-Ex suppresses DPM-induced apoptosis. ESR-Ex diminished DPM-induced increases in the pro-apoptotic regulator, BAX and an inducer of apoptosis, caspase 3, and conversely decreased anti-apoptotic regulator Bcl-2 expression in KC . These rWe investigated the ability of ESR-Ex to protect KC from DPM (a major air pollutant)-induced apoptosis. We also characterized that ESR-Ex inhibits NOX activation by DPM, thereby blocking ceramide-induced apoptosis via the suppression of NOX-dependent sphingomyelinase activation .2.5. ESR-Ex suppresses NOX activation via blockage of AhR-induced p47phox production), chemicals contained in DPM increase the transcription of p47phox via AhR activation [How does ESR-Ex inhibit DPM-mediated NOX activation? As described above in the Results section  DPM activates AhR followed by increased transactivation of p47phox (a subunit of NOX required for NOX activation); (2) Increases in p47 activate NOX; (3) NOX activates sphingomyelinase by a ROS-independent (unidentified) mechanism, leading to increases in ceramide production by sphingomyelin hydrolysis; and (4) Increased ceramide induces apoptosis in KC.ESR-Ex has been used as a folk medicine for centuries, and its anti-oxidative activities are well known. Here, we have shown previously unrealized biological activities of ESR-Ex; i.e., as an AhR inhibitor and a NOX inhibitor. Our study could lead to new clinical indications for ESR-Ex; i.e., its use in prevention of DPM-mediated diseases, including asthma, chronic obstructive pulmonary disease (COPD), pulmonary cancer, and cardiovascular diseases initiated by environmental air pollutants and cigarette smoke. Since air pollutants are involved in accelerating premature skin aging ,23. ESR-In conclusion, ESR-Ex protects KC from DPM-induced apoptosis by inhibiting AhR activation-mediated NOX activation, followed by a blockage of increases in an apoptotic lipid mediator (ceramide production) by activating sphingomyelinase and sphingomyelin hydrolysis .Diesel particulate extract used in the present study is Standard Reference Material 1975 (SRM 1975), purchased from the National Institute of Standards and Technology (NIST) . Nicotinamide, NADPH, NADP, and 2, 7-dichlorofluorescin diacetate were obtained from Sigma-Aldrich . Apo, N-acetylcystine. C17-S1P, S1P (d18:1), sphingosine, ceramides , C17-ceramide (d17:1/C18:0), and C12-sphingomyeline sphingosine, ceramides  were obtained from Avanti Polar Lipids . Organic solvents for sphingolipid extraction or LC-MS/MS analysis were purchased from Merck .ESR was provided by the National Institute of Biological Resources . The dry stem and leaf of ESR-Ex (200 g) were extracted with 70% ethanol reflux for 90 min at 70 °C three times. The extracts were filtered with Whatman NO.3 filter paper  and concentrated by a vacuum evaporator . Subsequently, the concentrated extract was freeze-dried  to obtain a dry powder sample (ESR-Ex) with a yield of about 18% (36 g).The extracts were separated using a ExionLC™ Series UHPLC system . Furthermore, chromatographic separation was performed on a Waters ACQUITY UPLC HSS T3 C18 column  and a thermos tatted at 40 °C was used and then pre-equilibrated in solvent A (0.1% formic acid in water). Extracts were eluted with increasing percentages of solvent B (acetonitrile with 0.1% formic acid) at a flow rate of 0.5 mL/min. The elution gradient steps were as follows: 0–1 min, 0% B; 1–4 min, gradient to 70% B; 4–6 min, 100% B; 6–6.2 min, gradient to 0% B; 6.2–10 min, 0% B. The injection volume was 5 μL and the effluent was connected to an ESI-triple quadrupole-linear ion trap (QTRAP)-Mass spectrometry .m/z 200 and 1000. Phenolic acids and flavonoids in ESR-Ex extracts were identified by comparing the retention times and mass spectra with the reference standards.Mass detection was performed in an API5500 QTRAP equipped with an electrospray ionization (ESI) source and a triple quadrupole-ion trap spectrometer controlled by the Analyst 1.7.1 software. The negative ionization mode was applied, and the optimized instrument settings were as follows: curtain gas (CUR): 30.0 psi; collision gas (CAD): medium; ion spray (IS) voltage: −4500 V; source temperature: 500 °C; GS1: 60 psi and GS2: 50 psi; The mass detector was programmed to perform two consecutive modes: enhanced mass scan (EMS) and multiple reaction monitoring–information-dependent acquisition–triggering enhanced product ion (MRM-IDA-EPI) mode analysis. EMS was employed to obtain full scan spectra, so as to provide an overview of all the ions in the sample. The settings used were: declustering potential (DP) −120 V, entrance potential (EP) −9.5 V, collision energy (CE) −30 V. Spectra were recorded in negative ion mode between Immortalized, non-transformed (HaCaT) human keratinocytes (KC), derived from human epidermis , were grown, as described previously ,27. BrieCell viability was measured by the water-soluble tetrazolium salt (WST) method using the cell counting kit-8 (CCK-8) assay kit  in accordance with the manufacturer’s instruction. Cytotoxicity was assayed using in vitro Toxicology assay kit, lactic dehydrogenase based (Sigma-Aldrich).Activity of NADPH oxidase cell membrane fractions was measured by the lucigenin chemiluminescence assay kit using N, N′-Dimethyl-9,9′-bicridium dinitrate and 1 μM NADPH (Sigma-Aldrich), in accordance with the manufacturer’s instruction.Activity of NADPH oxidases was measured by the ratio of NADP+ to NADPH using LC-ESI-MS/MS  by multiple reaction monitoring (MRM) mode, as described previously 2−) and hydrogen peroxide (H2O2), the oxidant-sensing probe 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA)  was used, as described previously [To detect the production of cellular reactive oxygen species (ROS), including superoxide (Oeviously . Brieflym/z) were 510→264 for C14-ceramide, 538→264 for C16-ceramide, 552→264 for C17-ceramide, 566→264 for C18-ceramide, 594→264 for C20-ceramide, 648→264 for C24:1-ceramide, 650→264 for C24-ceramide, 676→264 for C26:1-ceramide, and 678→264 for C26-ceramide, respectively. The S1P MS/MS transitions (m/z) were 366→250 for C17 S1P as an internal standard and 380→264 for C18 sphingosine 1-phosphate, respectively. Data were acquired using Analyst 1.5.1 software (Applied Biosystems). Sphingolipid levels are expressed as pmol per mg protein.Cells were treated with or without NOX inhibitor  or ROS generation  for 30 min and then further incubated with DPM for 24 h, followed by extraction of lipids as we reported previously ,27. Cera3: CH3OH . The organic phases were dried and were resolved in MeOH, and then analyzed by LC-MS/MS. The activities of both sphingomyelinase are expressed as pmol (C12-ceramide) per mg protein per min.Activities of acidic or neutral sphingomyelinase were measured as described previously . BrieflyProtein levels of BAX, BCL-2, caspase 3 and β-actin were assessed using Western blot analysis, as described previously ,27. BrieAhR activation was assessed by AhR phosphorylation using Phospho-AHR Cell-Based Phosphorylation ELISA  in accordance with the manufacturer’s instructions. A pharmacological ligand of AhR FICZ was purchased from Sigma-Aldrich.p values were set at either *** <0.001, ** <0.01, or * <0.05.Results were expressed as the means ± standard deviation (SD). Statistical analyses were performed using the GraphPad Prism 8.0.1 software . Significance between groups was determined by a one-way analysis of variance (ANOVA) coupled with Dunnett’s multiple comparison test. The


Radix clematidis (TSC) have a clear therapeutic effect on RA, but the specific mechanism has not yet been clarified. Literature screening and previous research suggest that the lncRNA OIP5-AS1/miR-410-3p/Wnt7b signaling pathway exerts a regulatory effect on the pathogenesis of RA. In this study, we examined whether the TSC treatment of RA affects the lncRNA OIP5-AS1/miR-410-3p/Wnt7b pathway.  Rheumatoid arthritis (RA) is the most common autoimmune disease and affects multiple joints. Previous studies have shown that total saponins of β-catenin, c-Myc, cyclin D1, GSK-3β, and SFRP4 mRNA were detected by real-time-qPCR, the expression of Wnt7b, β-catenin, c-Myc, cyclin D1, GSK-3β, and p-GSK-3β (Ser9) protein were detected by immunofluorescence and Western blot.  Freund's complete adjuvant was used to create an adjuvant arthritis (AA) rat model with rat synovial cells being harvested and cultured. The experiment comprises a normal group, model group, TSC optimal-dose group, TSC optimal-dose group + lncRNA OIP5-AS1siRNA group, lncRNA OIP5-AS1 siRNA group, and lncRNA OIP5-AS1 siRNA + NC group. MMT was used to screen the optimal concentration of TSC. The level of lncRNA OIP5-AS1, miR-410-3p, Wnt7b, β-catenin, c-Myc, cyclin D1, and p-GSK-3β/GSK-3β mRNA/protein expression, whereas the miR-410-3p and SFRP4 mRNA/protein expression levels were significantly upregulated. Our data suggest that TSC can inhibit the excessive proliferation of FLS to treat RA, the mechanism of which may be closely related to regulation of the lncRNA OIP5-AS1/miR-410-3p /Wnt7b signaling axis and the Wnt signaling pathway. We found that TSC inhibits the proliferation of RA FLS, TSC significantly reduced lncRNA OIP5-AS1, Wnt7b,  When in (TNF-α) , 3. The Previous studies have shown that an imbalance between excessive proliferation and apoptosis of FLS is the pathological basis of RA . The conLong noncoding RNAs (lncRNAs) are nonprotein-coding RNA transcripts with a length >200 nucleotides . LncRNAsClematis chinensis is composed of the dried roots and rhizomes of Clematis chinensis Osbeck, a plant in the Ranunculaceae family. It has the effect of curing rheumatism, promoting blood circulation, and eliminating blood stasis [Radix clematidis (TSC) is the primary component responsible for the antiarthritis effects of Clematis chinensis.d stasis . Total sIn previous studies using an adjuvant arthritis (AA) rat model, we confirmed that TSC has a clear anti-inflammatory effect, and its therapeutic mechanism may be related to regulation of the JAK2/STAT3 signaling pathway and regulation of lipid, amino acid, and energy metabolism . TherefoIn this study, Sprague Dawley rats  were purchased from Anhui Experimental Animal Center. According to the method described in the previous study, rats were reared adaptively for 1 week. Except for the normal group, 0.1 mL of Freund's complete adjuvant  was intradermally injected into the left hind toe of each rat. After 18 hours of modeling, the inflamed part began to swell. After about 10 days of modeling, rats developed secondary lesions. The paws on the opposite side of the inflamed side of rats began to swell, and knots appeared on the ears and tails of rats. The peak period of inflammation occurred 20 days after modeling . Therefo2 using the tissue explant method. Under an inverted fluorescence microscope, the cells swimming from the edge of the tissue block were observed, and the morphology and characteristics of primary and passaged synovial cells were recorded. Vimentin was identified using an immunofluorescence staining kit according to the manufacturer's instructions [The hind limbs of rats were separated, and synovial layer tissue was removed. FLS were cultured in complete Dulbecco's Modified Eagle Medium with 20% fetal bovine serum Gibco, 1715752) at 37°C and 5% COructions , 17.15752 at μg/mL). Synovial cells in the logarithmic growth phase were inoculated into a 96-well culture plate and incubated for approximately 48 h. The cells were then observed under an inverted fluorescence microscope to determine when they reach ≥80% confluence. Preprepared concentrations of TSC were then added to the wells, and the cells were incubated for another 24 or 48 h. After culture, MTT solution was added to each well  and incubated for 4 h. Next, 200 μL of dimethyl sulfoxide was added to each well, and the plate was shaken at room temperature for 10 min to allow the reagent to dissolve. The absorbance of each well was then recorded at 490 nm using a microplate reader (BioTek) to determine the effect of different concentrations of TSC on FLS proliferation.The 3--2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the effect of different concentrations of TSC on the proliferation of synovial cells at 24 and 48 h after exposure. Rats were divided into normal, model, and different TSC concentration groups  targeting lncRNA OIP5-AS1 were synthesized and purchased from Hefei Delpu Biotechnology Co., Ltd. . Sequences of the constructed lncRNA OIP5-AS1 interference fragments are shown in μg of total RNA was used as a template for reverse transcription to synthesize cDNA . Quantitative fluorescent PCR  was used to determine the relative expression of lncRNA OIP5-AS1, miR-410-3p, Wnt7b, β-catenin, c-Myc, cyclin D1, GSK-3β, and SFRP4 mRNAs. The poly(A) tailing method was used for miR-410-3p tailing and cDNA synthesis. The primers used are listed in TRIzol  was used to extract total RNA from FLS according to the manufacturer's instructions. A total of 2 The downstream primers for miR-410-3p were directly provided in the miRNA tailing kit.β-catenin ; c-Myc ; cyclin D1 , p-GSK-3β(Ser9) ; SFRP4  was added, and samples were placed in a humid box and incubated overnight at 4°C in the dark. Then, the corresponding second antibody was added and incubated at room temperature for 50 min. DAPI  was used to stain the cell nucleus.FLS were fixed in 4% paraformaldehyde for 20 min, after which 0.5% Triton X-100 diluted in phosphate-buffered saline  was added to the wells and incubated for 20 min. Sections were rinsed three times with PBS, and 5% bovine serum albumin was added to these sections and blocked for 30 min. Diluted primary antibody: Wnt7b ; μL of protein lysate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to separate total proteins, which were then transferred onto polyvinylidene fluoride (PVDF) membranes, which were blocked in TBST . PVDF membranes were then incubated overnight at 4°C with the following primary antibodies: β-actin (1 : 50000); p-Wnt7b (1 : 5000); p-β-catenin (1 : 6000); p-c-Myc (1 : 2000); p-cyclin D1 (1 : 1000); p-GSK-3β (Ser9) (1 : 1000); and p-SFRP4 (1 : 500). The membranes were then washed three times with TBST and incubated for 2 h at room temperature with secondary antimouse or antirabbit antibody conjugated with horseradish peroxidase (1 : 10000). Proteins were detected using an enhanced chemiluminescence kit. Each protein band shown was representative of three replicates. Western blotting data were quantified using an ImageJ software.FLS were washed with cold PBS three times and disrupted to prepare 100 t-test was used for comparisons between the two groups. Differences were considered statistically significant at P < 0.05.All data were analyzed using SPSS version 23.0, and data are shown as mean ± standard deviation. One-way analysis of variance was used for comparisons between the multiple groups, and the μg/mL. Therefore, in the follow-up experiment, the action time of the drug was selected as 48 h, and 62.5 μg/mL was selected as optimal drug concentration.The MTT assay was used to evaluate the effect of TSC on the proliferation of FLS at different times of action and different drug concentrations. As shown in P < 0.01). Compared with the model group, the TSC + lncRNA OIP5-AS1 siRNA optimal-dose group and the lncRNA OIP5-AS1 siRNA group, the expression level of lncRNA OIP5-AS1 mRNA decreased significantly, and the expression of miR-410-3p mRNA increased significantly. These differences were statistically significant (P < 0.01). Compared with the model group, there were no significant changes in the mRNA expression of lncRNA OIP5-AS1 or miR-410-3p in the lncRNA OIP5-AS1 siRNA + NC group  were primarily expressed in the cytoplasm  protein and mRNA in the model group and the OIP5-AS1 siRNA + NC group was higher. Compared with the model group, the protein and mRNA expression levels of Wnt7b, β-catenin, c-Myc, cyclin D1, GSK-3β, and p-GSK-3β (Ser9) in the TSC optimal-dose group, a TSC optimal dose + lncRNA OIP5-AS1 siRNA group, and a lncRNA OIP5-AS1 siRNA group were significantly reduced (P < 0.01). Compared with the model group, Wnt7b protein and mRNA expression levels in the lncRNA OIP5-AS1 siRNA + NC group were not significantly different.The results of immunofluorescence experiments showed that Wnt7b, P < 0.01). Compared with the model group, there was no significant change in the protein and mRNA expression levels of SFRP4 in the lncRNA OIP5-AS1 siRNA + NC group.The results of immunofluorescence analyses showed that SFRP4 was primarily expressed in the cytoplasm . RT-qPCRRA is a systemic chronic autoimmune disease, and its etiology is complex and affects multiple systems. RA is characterized by synovial inflammation, which can cause gradual destruction of bone and cartilage and lead to disability in severe cases . The comLncRNA not only regulates cell proliferation, differentiation, and apoptosis but also mediates inflammation and immune processes of various diseases and plays an important role in the process of cell growth and development , 21. Lncα and IL-6 and promote the proliferation of FLS while inhibiting apoptosis [miRNAs are short, noncoding, single-stranded RNAs that play important roles in the pathology and biology of many diseases. miRNAs are posttranscriptional regulators of gene expression and have been widely studied as oncogenes in variety of cancers . Many stpoptosis . Many repoptosis . In thisβ-catenin signaling pathway regulates cell proliferation, migration, invasion, and inflammation [β-Catenin is the key gene in the classical Wnt signaling pathway, playing a central role in signal transduction that is directly related to the abnormal proliferation of RA synovial cells. Activation of the Wnt signaling pathway can lead to an increase in the level of β-catenin in the synovium of RA patients, thereby promoting stable activation of the synovium of the joint [β-catenin has a strong inhibitory effect on the proliferation of RA-FLS cells. C-Myc and cyclin D1 are direct downstream targets of the β-catenin/Wnt signaling pathway. C-Myc is a major proto-oncogene that regulates activation, transcription, and suppression of other genes [The Wnt signaling pathway is an important regulator of cell growth and development, and the classic Wnt/ammation . The Wnthe joint , 28. Yoshe joint  confirmeer genes . Cyclin α and GSK-3β. GSK-3β is located upstream of the β-catenin/Wnt signaling pathway, and the level of β-catenin is negatively regulated by GSK-3β–mediated phosphorylation or other degradation pathways [β is inhibited, and the phosphorylation and degradation of β-catenin are blocked, thereby positively regulating the β-catenin/Wnt signaling pathway.GSK-3, a serine-threonine kinase with a structural activity, is involved in the regulation of multiple signaling pathways . Two subpathways . When Seβ-catenin pathway, SFRP attenuates Wnt signaling, antagonizing the Wnt signaling pathway [β-catenin, c-Myc, and cyclin D1 were significantly reduced after TSC drug intervention, whereas the mRNA and protein expression levels of the negative regulatory protein SFRP4 of the Wnt pathway were significantly increased. There was no significant change in the expression levels of GSK-3β among the groups, and the mRNA and protein expression levels of p-GSK-3β (Ser9)/GSK-3β were significantly reduced. In the TSC + lncRNA OIP5-AS1 siRNA and lncRNA OIP5-AS1 siRNA groups, the expression levels of key factors downstream of the Wnt pathway, including p-GSK-3β (Ser9)/GSK-3β, were significantly downregulated, whereas the expression of SFRP4 was significantly upregulated. These data suggest that downregulation of Wnt7b expression inhibits the expression of downstream β-catenin and also inhibits activation of the Wnt signaling pathway.As a negative regulator of the Wnt/ pathway . This stThe adverse effects of RA lower the quality of patients' life and bring a heavy burden to the society. At present, main drugs for the treatment of RA include nonsteroidal anti-inflammatory drugs, immunosuppressants, and glucocorticoids, but all have certain side effects for a long-term use, so it is very urgent to find a new alternative. This study further explained the pathogenesis of RA, clarified the therapeutic effect of TSC on RA, provided experimental support for TSC in the treatment of RA patients, and provided new ideas for the treatment of RA. This study also enhances our confidence in the treatment of RA with traditional Chinese medicine.TSC intervention significantly inhibited the proliferation of FLS. At the optimal dose, TSC significantly reduced the expression of lncRNA OIP5-AS1, the expression of miR-410-3p was significantly upregulated, and that of its target gene, Wnt7b, was significantly downregulated. The expression of key factors in the classic Wnt signaling pathway was also inhibited. Our data suggest that the inhibitory effect of TSC against the proliferation of FLS is related to regulation of the lncRNA OIP5-AS1/miR-410-3p/Wnt7b signaling axis and inhibition of the Wnt signaling pathway activation.


Nakamurella sp. PAMC28650 was isolated from a glacier of Rwenzori Mountain, Uganda, which species belonged to Nakamurella genus based on 16S rRNA phylogeny, ANI (average nucleotide identity), and BLAST Ring Image Generator (BRIG) analysis among Frankineae suborder. We conducted the whole genome sequencing and comparative genomics of Nakamurella sp. PAMC28650, to understand the genomic features pertaining to survival in cold environment, along with high UV (ultraviolet) radiation. This study highlights the role of polysaccharide in cold adaptation, mining of the UV protection-related secondary metabolites and other related to cold adaptation mechanism through different bioinformatics tools, and providing a brief overview of the genes present in DNA repair systems. Nakamurella sp. PAMC28650 contained glycogen and cellulose metabolism pathways, mycosporine-like amino acids and isorenieratene-synthesizing gene cluster, and a number of DNA repair systems. Also, the genome analysis showed osmoregulation-related genes and cold shock proteins. We infer these genomic features are linked to bacterial survival in cold and UV radiation.The mechanisms underlying the survival of bacteria in low temperature and high radiation are not yet fully understood. The online version contains supplementary material available at 10.1007/s10142-022-00937-6. Nakamurella, a genus of the Nakamurellaceae family, was first highlighted by Yoshimi et al., with the illegitimate genus name “Microsphaera”  database).Nakamurella species have been isolated from different sources . The 16 s rRNA of Nakamurella sp. PAMC28650 was subjected to EzBioCloud database . The quantity and purity were determined by spectrophotometry . Also, we processed the genome to decide the quality by using A260/A280. The extracted genomic DNA was checked by agarose gel electrophoresis to evaluate its quality and stored at − 20 °C. The strain grew enough cells at 15 °C and then provided genomic DNA.We received Nakamurella sp. PAMC28650 was obtained by PacBio sequel single-molecule real-time (SMRT) sequencing technology . SMRTbell library inserts (20 kb) were sequenced using SMRT cells. Results were generated from raw sequence data of 5,629,850 bp that were assembled de novo by using the hierarchical genome assembly process (HGAP) protocol . The complete genome sequence was deposited in the GenBank database under the GenBank accession number CP060298.1.Complete genome sequencing of Nakamurella species was performed using the Rapid Annotation Subsystem Technology (RAST) server . First, the relationship of PAMC28650 with other Frankia suborder species was determined by comparing the value of average nucleotide identity (ANI), calculated using an OrthoANI in the Orthologous Average Nucleotide Identity Tool (OAT)   was also an important factor to withstand the extreme surrounding, and therefore, PAMC28650 has the highest proportion of genes as compared to other Nakamurella species.The complete genome sequence of Nakamurella panacisegetis. The phylogenetic position of Nakamurella sp. PAMC28650 was analyzed using 16S rRNA sequence of 31 species belonging to Frankineae suborder, including our strain. We included 16S rRNA of all the complete and partial genomes of Nakamurella species. All the Nakamurella species fell in the same clade with a common ancestor, while the closest relative of Nakamurella sp. PAMC28650 was found to be Nakamurella panacisegetis strain P4-7. We can say that this strain belongs to the family Nakamurellaceae and phylum Actinobacteria. Figure The result obtained from EzBioCloud database for 16 s rRNA analysis is shown in Table Nakamurella sp. PAMC28650 strain, by comparison with the complete nucleotide sequences of the species from Frankineae suborder. ANI values were calculated using OrhtoANI in the Orthologous Average Nucleotide Identity Tool (OAT) with cutoff value (95 to 96%)  isolated from activated sludge acclimated with sugar-containing synthetic wastewater and Nakamurella antarctica s14-144 (69.81%) from soil . We had only included Nakamurella multipartita DSM44233 and Nakamurella antarctica s14-144 among the Nakamurella species for ANI calculation, as complete nucleotide sequences of other Nakamurella species were not available in the NCBI. The ANI method is the common method for bacterial comparative genomics. This calculates the average base similarity between two different microbial genomes of homologous fragment at the nucleotide level like the DNA–DNA hybridization method, to identify the relatedness between bacterial species. We also compared the whole genome sequences of the Nakamurella sp. PAMC28650 as a reference strain including Nakamurella multipartita DSM44233 and Nakamurella antarctica s14-14 using BLAST Ring Image Generator (BRIG). BRIG analysis represents the circular genomic map showing the identity between genome of Nakamurella sp. PAMC28650 against Nakamurella antarctica s14-144 and Nakamurella multipartita DSM44233 in a circular ring, colored depending on the BLAST identity  is another way to determine the relatedness of ity Fig. . This anNakamurella sp. PAMC28650 strain. The CAZyme-related genes predicted in this strain are categorized into 6 groups, namely, glycoside hydrolases (GH), glycosyltransferases (GT), polysaccharide lyases (PL), carbohydrate esterases (CE), carbohydrate-binding modules (CBM), and auxiliary activities (AA). Among the 117 CAZyme genes predicted by dbCAN database in PAMC28650 strain, the maximum numbers of genes annotated are in GH families (57), 28 genes in CBM family, 25 genes in GT family, and 2 in AA family   and a cellulose-binding domain , with cellobiose serving as the key hydrolysate. Similarly, the role of Cel5G, a psychrophilic cellulase from the Antarctic bacterium Pseudoalteromonas haloplanktis, was studied through protein engineering, where the unusually long flexible linker region was found to enhance catalytic efficiency in the cold region  and BetB  for the synthesis of glycine betaine. BetA and BetB genes are present in both Nakamurella sp. PAMC28650 and Nakamurella antarctica s14-144 strains, but absent in Nakamurella multipartita DSM44233. Similarly, three different SOX related to sarcosine oxidase subunit genes are also present in both Nakamurella species except Nakamurella antarctica s14-144. Glycine betaine transporters OpuA three genes are present in our strains; however, Nakamurella multipartita DSM44233 contains one ProU gene. Overall, the number of genes is higher in Nakamurella sp. PAMC28650 compared to other Nakamurella strains  synthesize them. Some bacteria and actinomycetes are also known to produce mycosporine. Nakamurella sp. PAMC28650 signifies its environmental adaptation strategies, but they also carry biotechnological importance. Isorenieratene may ease age-related macular degeneration (AMD) , nucleotide excision repair (NER), and mismatch repair (MMR), are the major repair systems in various microorganisms that function by producing glycosylases and polymerases. Other mechanisms include recombination repair, mutagenic repair, and apoptosis. The photolyase-mediated DNA repair system is responsible for the protection of bacterial cells released during photoactivation , or UV radiation or strong alkylating agents , which help to recognize the damage, cleave the target site, and carry out the completion of the repair system. The genes like UvrA and UvrB are the energy-independent factors that recognize the lesion, and UvrC and UvrD play the role of the removal of damaged DNA, showing nuclease and helicase activity, respectively. Nakamurella sp. PAMC28650 has all the homologous genes related to UvrABCD repair systems and may play a role in the UV-resistant mechanism , and MutH (incision of mismatch strands) genes are mainly responsible for the repair of distorted DNA. However, these three genes are absent in our PAMC28650 strain; however, UVRD helicase II, Exo VII exonucleases, SSB, and DpoIII that play a role in DNA resynthesis, and finally Lig for the ligation of DNA strand, are present.Acinetobacter baumannii RecA protein played a role in SOS mutagenesis and protection from UV radiation, as well as other stresses, such as oxidative agents and different types of antibiotics  and mycosporine-like amino acid (MAA), the major secondary metabolites that help in the protection against UV radiation, were found in Nakamurella sp. PAMC28650. Along with it, the strain also holds the genes responsible for osmotic stress regulation, which is one of the major stress conditions in a cold environment. Overall, we expect that the information provided in this study provides a research scope for understanding of the bacterial ability to withstand extreme cold environment, UV-resistant mechanisms, and osmotic stress with respect to polysaccharide metabolism, secondary metabolite biosynthesis, and osmoregulation. In addition, the highlighted features and genes also show the biotechnological importance of the strain Nakamurella sp. PAMC28650.This study presents the complete genome analysis of Supplementary file1 (XLSX 15 KB)Supplementary file2 (DOCX 47 KB)Below is the link to the electronic supplementary material.


The in vivo function of PRMT5 in gastric tumorigenesis, however, is still unexplored. Here, we showed that Prmt5 deletion in mouse gastric epithelium resulted in spontaneous tumorigenesis in gastric antrum. All Prmt5-deficient mice displayed intestinal-type gastric cancer within 4 months of age. Of note, 20% (2/10) of Prmt5 mutants finally developed into invasive gastric cancer by 8 months of age. Gastric cancer caused by PRMT5 loss exhibited the increase in Lgr5+ stem cells, which are proposed to contribute to both the gastric tumorigenesis and progression in mouse models. Consistent with the notion that Lgr5 is the target of Wnt/β-catenin signaling, whose activation is the most predominant driver for gastric tumorigenesis, Prmt5 mutant gastric cancer showed the activation of Wnt/β-Catenin signaling. Furthermore, in human gastric cancer samples, PRMT5 deletion and downregulation were frequently observed and associated with the poor prognosis. We propose that as opposed to the tumor-promoting role of PRMT5 well-established in the progression of various cancer types, PRMT5 functions as a tumor suppressor in vivo, at least during gastric tumor formation.Previous studies have demonstrated the Gastric cancer is the fifth most common type of cancer and the fourth leading cause of cancer mortality globally PTEN and Smad4 in the gastric antral Lgr5+ stem cells, we observed the invasive intestinal-type gastric cancer in double mutant mice within as early as 3 months post induction Gastric cancer is caused by the combination of genetic and environmental factors. Although many genes and related signaling pathways are altered in human gastric cancer, studies using genetically engineered mouse models verified that a few genetic mutants can initiate gastric tumor formation. Our previous study showed that 100% of mice with gastric epithelium specific loss of PTEN, which is frequently deleted in human gastric cancer, developed spontaneous gastric tumorigenesis as early as 2 months of age  in vitro studies  in vivo.Protein arginine methylation, a post-translational modification, is catalyzed by a class of enzymes called protein arginine methyltransferases (PRMTs) Prmt5 knockout mouse line. Unexpectedly, all Prmt5 mutants developed spontaneous gastric tumor at antrum within 4 months of age, accompanied by the hyperactivation of Wnt/β-catenin signaling. Thus, we provided the first critical in vivo evidence for the causal relationship between PRMT5 downregulation and gastric tumorigenesis.In the present study, we generated a gastric-specific PRMT5 gene in 434 human primary gastric cancer samples from TCGA database  PRMT5 deletion, including shallow deletion and deep deletion, occurred in 25.1% (109/434) of human gastric cancer samples, which was more prevalent than gain 11.8% (51/434) and amplification 0.2% (1/434), indicating that PRMT5 loss might be implicated in gastric cancer formation and progression . Furthermore, we discovered that the decreased PRMT5 expression was linked to a poor outcome for gastric cancer . This indicated that PRMT5 may act as a tumor suppressor in gastric cancer.We analyzed the alterations of P < 0.01).In parallel, we examined PRMT5 protein level in a human tissue microarray representing 94 patients with intestinal-type gastric cancer and adjacent normal tissues. To better exhibit the gastric epithelium, we performed co-immunofluorescence staining (Co-IF) for PRMT5 (green) and E-cadherin (red). The protein level of PRMT5 was calculated by the mean optical density values of immunofluorescence. The ratio of tumor versus adjacent normal tissue > 2 was defined by “upregulation” and the ratio < 0.5 was defined by “downregulation”. We found that 40.4% (38/94) of paired samples showed a lower expression of PRMT5 in the gastric cancer compared to the adjacent normal tissues Figure D. We als in vivo, we generated gastric-specific Prmt5 knockout mice. Strategically, Prmt5-conditional knockout mice (flox/floxPrmt5 mice) were crossed with SP-A-Cre transgenic mice, where Cre-mediated recombination begins from embryonic day 16.5 (E16.5) Prmt5 in fl/+SP-A-Cre;Prmt5 (here after control) and fl/flSP-A-Cre;Prmt5 (here after mutant) mice. In control mice, PRMT5 was mainly restricted to the lower region of antral glands , Western blot, and immunohistochemical (IHC) analyses Rosa26-LoxP-Stop-LoxP-tdTomato  of n Figure . To examant mice .Prmt5 mutant mice developed hyperplasia as early as 2 months of age , immunohistochemical staining showed that the expression of both surface mucous cells marker ulex europaeus agglutinin type 1 lectin (UEA-I) and mucous neck cells marker trefoil family factor 2 (TFF2) were comparable between control and mutant antral glands  through further breeding with Lgr5-eGFP-IRES-CreERT2 knock-in mice + cells were significantly increased in Prmt5 mutant epithelium compared to that of control mice at 2 months of age  and sclerostin domain containing 1 (SOSTDC1), two negative regulators of Wnt/β-catenin signaling s Figure B-C. SOSTe Figure B-D.Prmt5 deletion-induced gastric cancer exhibited the hyperactivation of Wnt/β-catenin signaling, recapitulating the molecular alterations in human gastric cancer.Taken together,  in vivo role of PRMT5 in suppressing gastric cancer formation. This seems to be contrary to the notion that PRMT5 can promote tumor growth and progression in a variety of human cancer cells including gastric cancers in vitro experiments of cancer cell lines PRMT5 gene deletion was found in approximately 25% of human gastric cancer samples. We propose that in these gastric cancer samples, low level of PRMT5 caused by gene deletion leads to or contributes to gastric tumorigenesis. However, it cannot be ruled out the possibility that PRMT5 overexpression can also lead to gastric tumorigenesis although it is needed to investigate using in vivo mouse models. Nevertheless, since a few PRMT5 inhibitors are currently being tested in clinical trials  Prmt5 knockout mouse models.Herein, we provide the first criticalPrmt5 mutant antral epithelium as evidenced by several lines: the alterations in RNA level of c-Myc, Cyclin D1, CD44, Gsk3β, Lgr5, and Sostdc1, all of which led to the corresponding protein changes; the increase in the protein level of SOX9; the upregulation of non-phosphorylated β-catenin at Ser45 and Ser33/37/Thr41 sites. Therefore, we demonstrate that PRMT5 could inhibit the hyperactivation of Wnt/β-catenin signaling in normal antral epithelium. Previous in vitro studies from various cancer cell lines, however, suggest that PRMT5 could promote the activation of Wnt/β-catenin signaling + stem cells and its direct progeny transient proliferating cells localize  Prmt5 deletion led to the extensive expansion of proliferating cells upward, which was accompanied with hyperactivation of Wnt/β-catenin signaling. Therefore, we assumed that PRMT5 might be a brake of Wnt/β-catenin signaling in antral epithelium. In the future, it is needed to investigate how PRMT5 inhibits Wnt/β-catenin signaling in gastric epithelium. A clue came from the cellular localization of PRMT5. We found that PRMT5 was localized to both the nuclei and cytoplasm of antral epithelium. Given that the roles of cytoplasmic and nuclear PRMT5 are pleiotropic, it is proposed that, in normal antral epithelium, PRMT5 can inhibit the hyperactivation of Wnt/β-catenin signaling through multiple mechanisms directly or indirectly.In the present study, we found that the hyperactivation of Wnt/β-catenin signaling in fl/flPrmt5mice, SP-A-cre mice were described earlier ERT2Lgr5-eGFP-IRES-Cre and Rosa26-loxP-stop-loxP-tdTomato mice https://www.alenabio.com).Mouse gastric tissues were fixed in 4% paraformaldehyde (PFA) overnight and then embedded in paraffin. Five-micrometer-thick slices were cut from paraffin blocks and placed on coated slides for the following hematoxylin and eosin (H&E), immunohistochemistry (IHC) and immunofluorescence (IF) staining. As for the tissue microarray of human gastric cancer, it was purchased from Xi'an Best Biotechnology Ltd., Co.  was used to unmask the antigen, followed by blocking with goat serum for 1 hour at 37°C to decrease nonspecific antibody binding. Immunohistochemical staining for BrdU requires additional diluted hydrochloric acid solution treatment for 20 minutes. Then the slices were incubated overnight at 4°C with the following primary antibodies: PRMT5 , H4R3me2s , E-cadherin , CDX1 , CDX2 , Villin , RFP , BrdU , TRITC-labelled UEA-I , TFF2 , GFP , Non-phospho-β-cateninSer45 , Non-phospho-β-cateninSer33/37/Thr41 , c-Myc , Cyclin D1 , CD44 , SOX9 , SOSTDC1 . Then we added the secondary antibodies (Beijing Zhongshan Golden Bridge Biotechnology Co.) and incubated at room temperature for 1 hour. IHC was observed by DAB  and hematoxylin counterstaining, while IF staining was performed by Tyramide signal amplification (TSA) method with DAPI reverse staining and observed by confocal microscope .After deparaffinization and rehydration, H&E staining was performed using standard techniques. As for IHC and IF staining, 3% HFor quantification analyses of PRMT5 protein in human tissue microarray, the integrated optical density (IOD) and area of immunofluorescence were measured by Image-Pro Plus 6.0 software, and the mean optical density (MD) was calculated. In addition, the average number of TFF2-positive cells and GFP-positive cells in single gland were counted from three sections per mouse and repeated in 4 independent mice.Trizol Reagent  was used to extract total RNAs. 2μg total RNAs were reversely transcribed into cDNAs using a reverse transcription kit . Then the cDNAs could then be used as a template for RT-qPCR utilizing the SYBR-Green Master PCR Mix  and unique primers on a 7500 Fast Real-Time PCR System (Applied Biosystems). Hprt served as the internal control. Table RIPA lysis buffer  with phosphatase inhibitors  and complete mini protease inhibitors  were used to lyse mouse gastric tissues. Protein concentrations were measured with the Pierce BCA Protein Assay Reagent . Immunoblotting by Western blot was performed according to standard procedures, using the following antibodies at a concentration of 1:1000. PRMT5 , H4R3me2s , CD44 , Cyclin D1 , GSK3β , SOSTDC1 . Beta Actin  was served as the internal control. The immunoblot signal was detected and imaged using Enlight Western blotting detection reagents  and Image Quant LAS 4000 mini .http://www.cbio portal.org) https://kmplot.com/) The TCGA gastric cancer data were downloaded from the cBioPortal, including the DNA alterations and mRNA expression ( P < 0.05, ** P < 0.01 and *** P < 0.001 were used to determine statistical significance for all results.The unpaired, two-tailed Student's t-test was used to evaluate difference between two groups. Survival curves were plotted by Kaplan-Meier estimates and compared by log-rank test. The relative protein level of PRMT5 in human tissue microarray was analyzed by one-way ANOVA test. GraphPad Prism software 8 was used for statistical analysis and data visualization. Data were represented as mean ± SEM. *Supplementary figure.Click here for additional data file.


The ripeness of bananas is the most significant factor affecting nutrient composition and demand. Conventionally, cutting and ripeness analysis requires expert knowledge and substantial human intervention, and different studies have been conducted to automate and substantially reduce human effort. Using the Preferred Reporting Items for the Systematic Reviews approach, 1548 studies were extracted from journals and conferences, using different research databases, and 35 were included in the final review for key parameters. These studies suggest the dominance of banana fingers as input data, a sensor camera as the preferred capturing device, and appropriate features, such as color, that can provide better detection. Among six stages of ripeness, the studies employing the four mentioned stages performed better in terms of accuracy and coefficient of determination value. Among all the works for detecting ripeness stages prediction, convolutional neural networks were found to perform sufficiently well with large datasets, whereas conventional artificial neural networks and support vector machines attained better performance for sensor-related data. However, insufficient information on the dataset and capturing device, limited data availability, and exploitation of data augmentation techniques are limitations in existing studies. Thus, effectively addressing these shortcomings and close collaboration with experts to predict the ripeness stages should be pursued. Bananas are one of the most consumed foods in the world and the fourth most important food crop in terms of gross value of production, apart from wheat, rice, and maize. According to the Food and Agriculture Organization (FAO), they contribute about 16% of global fruit production . This reThe stage identification techniques are broadly categorized as destructive and non-destructive. The former is considered a reliable approach requiring the testing of bananas using peeling or cutting, whereas the latter does not require manual destruction. Specifically, the destructive approach deals with measuring dielectric parameters such as capacity, dielectric constants, resistance, and admittance changes throughout the ripening process of a banana ,5. The lFollowing the non-destructive approach, several studies have been conducted to predict the banana ripeness stages using imagery data ,6,9 or sIn the case of a banana ripeness review, this is the first work to remove selection bias using Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines  for the Different fruits have different colors, textures, aromas, and maturity levels for consumption. Therefore, mixing different fruits may change the outcome of the review. Consequently, this review focuses solely on banana ripeness, unlike Muthulakshmi and Renjith’s  study, wAlthough for humans visual, touch, and smell may be the primary senses to detect ripeness, for automation, different sensors, including cameras, can be used. However, due to the dominance of camera uses, a comparison between various sensors has not been made before. This review tries to fill that gap by including different sensors along with cameras .A statistical analysis, as well as quality assessment studies, was performed to examine the model performance.Finally, the review objectively reports on datasets, banana features, ripeness stages, data augmentation techniques, and on the shortcomings of the current state of the art.This systematic review overcomes the above-mentioned limitations by summarizing both image and sensor-based studies. Additionally, this review also identified a set of critical factors that was used for categorizing the existing studies. The contribution of this systematic review is as follows:In this systematic review study, This systematic review followed the new PRISMA guidelines . This seThe experimental studies on banana ripeness, based on feature extraction and classification of banana ripeness stages, have been included. However, the exclusion criteria are based on the full articles that are unaccessible for which the relevant author was contacted but did not reply and due to the articles where the English translation was unavailable. Articles that focus on other fruit’s ripeness, banana disease, grading studies as well as the articles that do not focus on banana ripeness stages were also excluded.The database selection covered Science Direct, Scopus, Institute of Electrical and Electronics Engineers (IEEE), and Springer. The final database search was completed on January 15th, 2022. The process of screening the articles based on keywords was followed for this review, and the employed words for the search in each database are presented in In the search strategy shown in The data collected for the study were gathered from reading the publisher’s full articles and journals, which were grouped by country and year of publication. The selection process was based on the banana ripeness stages using AI. In the two-phase selection process, first, the researchers Preety Baglat (PB) and Ahatsham Hayat (AH) independently performed the full-text screening, based on titles and abstracts, for the selected articles. In the second phase, four other authors, Ankit Gupta (AG), Fábio Mendonça (FM), Sheikh Shanawaz Mostafa (SSM) and Fernando Morgado Dias (FMD), applied the selection criteria to the full text of the selected articles. Finally, the final discussion for the selection of the articles was made by all the authors.For all the studies, the data were collected after scanning the complete articles. Therefore, PB collected the study by checking these parameters such as types of banana samples, varieties of banana categories, wavelength or spectrum, measurement tool camera or sensor, color space, features, classification stages, number of data samples, methods and results.The primary outcome of this review is the comparison of various banana ripeness detection methods using multiple imaging techniques with the respective capturing devices. The secondary outcome of this study is the evaluation of the performance of all the included studies by their relevant parameters and the analysis of practical significance and limitations by the performance metrics. Moreover, a similar performance metrics report was selected for all the included studies, and the median and mode were used for the study categorization.This review aims to find the available research on banana ripeness stages’ detection using machine learning methodologies. The quality assessment was categorized based on scores and is presented in the For the first parameter (capturing devices), a score of 1 was awarded if detailed information about the device was provided; otherwise, a score of 0 was given. Similarly, for the second parameter , the median of the quantitative value was calculated by combining the score of individual values from each study. A score of 1 was assigned if the total number of samples was greater than the median value; otherwise, the value 0 was assigned. In the following parameter (types of bananas ripening stages), the mode of the number of ripening stages was calculated by combining the score of individual values from each study. If the number of stages was greater than 3 , then a score of 1 was given; otherwise, the score value was 0. For the last parameter (results), the median of the quantitative value was calculated by combining the score of individual values from each study. A score of 1 was awarded if the result was greater than the median value and it was 0 otherwise. After this analysis, all individual score parameters were summed up for each study to attain the overall quality assessment.Finally, the summed-up score was categorized into three types: strong, fair and weak. A study was considered strong if the overall score was 4. A fair study occurred when the overall score was 3; lastly, a study was marked weak when the score was 2 or less.In this review, The proposed study penalizes missing information, which is helpful for the quality assessment study. This review provides a descriptive summary based on data collected from all the included studies for detecting banana ripeness stages. All the authors have gathered the data from individual studies and performed the quality assessment based on the collected data, yet the subgroup analysis was not performed. To support the effectiveness of the models, the proposed protocol calculates the median of the performance metrics.This section explains banana characteristics, which include the description of the grouping of the banana samples and the varieties of banana categories. Additionally, crucial parameters, such as capturing devices (camera or sensor), datasets, the different stages of banana ripeness, feature analysis, classification, and the feature-based methods and performance metrics, are useful for detecting and classifying banana ripeness stages.The terminology for banana analysis includes terms such as bunch, fingers, and hand. A single plant produces bunches of bananas. A single banana is called a finger, and a grouping of attached fingers of 10 or more bananas is called a hand . In thisFor the detection of banana ripeness stages, most of the studies have used Musa Cavendish varieties such as the Musa cavendish  group (Japan), the Musa Cavendish Ambon banana, Red, Musa basjoo Siebold var. Formosana, Musa acuminate, Musa Acuminata cv. Berangan, Embun, Mas and Berangan, Musa AAA group (Kluai Hom thong), the Musa AAA group (Brazil), and Egyptian species. Out of the 35 studies, most of the studies 21) have used Musa cavendish banana categories. Apart from that, three ,9,25 stu have useIn this section, the detection of banana ripeness stages using the ML pipeline is displayed in The non-destructive studies require camera or sensor-based devices for image acquisition. Hence, camera or sensor characteristics such as the camera type, the wavelength distance, and the sensor type are effective parameters for banana ripeness identification. Therefore, this section summarizes the relevant articles’ sensor types, the wavelength distance, and the color space distribution for the included studies. Out of the total considered studies 35), 18 [, 18 5,6,41,42,44A total of four of the 35 ,17,18,35While the rest of the studies (five of the studied 35) have used sensor-based devices, Altaf et al.  used a sFor the banana ripeness analysis, 28 of the 35 studies used a self-created dataset to prove the significance of their proposed techniques. Additionally, the remaining eight ,39,41,44Medians were calculated from all the included augmentation and sensor studies for the quality assessment analysis. Out of all 30 quality assessment studies, 12 ,38,41,44The banana texture alterations, starch breakdown, unexpected fragrance, pigment creation and abscission are all brought on by a series of biochemical changes that occur during the ripening process. The color of a banana peel changes as it ripens, going from green to yellow to brownish. The most common criterion by which consumers judge the actual and consumable quality of a banana is its peel color. Bananas lose some of their hardness when they ripen, which is also a sign of quality . TherefoThere are different stages used for the classification of banana ripeness. Among the 35 examined articles, 4 ,36,43 exThe remaining articles used a multi-stage analysis. Particularly, Zhu et al.  used thrThe banana ripeness stages identification can be conducted by external and internal characteristics, such as the color space  and HSV ), L*a*b, where L* values represent the brightness components of a banana that go from black (0) to white (100) values, a* (green to red) parameters represent the greenness and b* (blue to yellow) represent texture; SSC (Soluble Solid Content); a brown spot feature; chlorophyll; firmness; pH ; shape; the ratio of pulp to peel; light and ultra-white ration, etc. These attributes are the potential criteria for identifying various banana ripeness stages. Therefore, as the ripeness stages change from unripe, yellow-green, mid-ripe, and over-ripe, the hue color gradually changes from green to yellow; hence, it is essential to be undertaken. Taking the color space distribution into account, the hue of the banana ripens from fully green to yellow, and this serves as a crucial visual cue to distinguish between the various phases of ripeness . HoweverAlthough all RGB color space features simultaneously contain brightness and color information, the brightness information may not be useful for ripeness stage identification. Due to the independence of the color components in these spaces from the equivalent brightness components ,26, the Another inherent quality of a banana fruit surface is its texture. It describes how the gray area is distributed across the image area and the pixels, and it remains constant under changing lighting. The texture of a banana surface is the single aspect that can properly capture all its important characteristics. Nevertheless, obtaining high-level image representation using simply texture information is not yet achievable . HoweverColor space is essential for extracting accurate banana ripeness stages. Out of all 35 included studies, three ,38,41 usIn this review, all the combinations of feature studies were included. A total of three out of 35 ,18,35 stIn the remaining 26 studies, Zhuang et al.  utilizedChen et al.  employedMachine learning-based methods: Out of the 35 included articles, Zulkifli et al. [i et al.  used thei et al. ,18,30,37i et al. ,35 emploi et al. ,28 studii et al.  study usi et al.  used Pari et al.  used thei et al. ,39 used Choosing suitable methods for the classification and the feature creation for banana ripeness are essential in identifying the ripeness stages. Mazen and Nashat  used a cDeep learning-based methods: Furthermore, four [re, four ,23,34,41re, four ,42,44 stre, four  employedre, four ,32 studiMachine and deep learning-based methods: Only Zhu and Spachos [ Spachos  used the Spachos  used a c Spachos  used pri Spachos  article The performance analysis of the banana ripeness identification stages is based on two metrics, namely, accuracy and  studies ,29,32,42Different types of camera or sensor devices are used for the analysis and detection of banana ripeness. LLBI device studies ,17,18,35The sensor devices measure the temperature, the number of dots on a banana skin, the sweetness level, the humidity level, the ethylene concentration and the carbon dioxide level. However, sensor-based devices are not widely found for banana ripeness detection. The sensor devices have a more complex installation, and low-cost sensors do not provide information on the exact position. Secondly, most of the included studies have used a small number of data samples, formally decreasing the classification accuracy. Whereas other ,26,34,39Regarding the quality assessment analysis, four parameters were considered for identifying banana ripeness studies: namely, the capturing devices, the dataset, banana ripeness stages, and the results  device, which are powerful imaging techniques used to analyze banana quality. In one of the included studies, HIS was used to predict banana firmness, color space features, and the ripeness stages using the 380-1023 nm wavelength. The model’s performance was examined by the overall coefficient of determination of 0.795, and L*a*b* firmness and its residual predictive deviation values were 2.119, 2.234, 2.062, and 6.098, respectively . This teFew studies have used a laser light backscattering imaging device based on a monochromatic laser light source. LLBI comprises a series of monochromatic images captured at different wavelengths (532–1060 nm). When a monochromatic laser light source reaches the sample surface, some photons are reflected, while the rest of the light beam is scattered. Light absorption depends on chemical constituents such as chlorophyll, elasticity, and SSC. Scattering, which is a natural phenomenon, occurs due to the microstructural composition and cellular matrices. Therefore, LLBI is a preferable monitoring technique for banana ripening detection. These capturing devices displayed the potential to evaluate the ripening stages of bananas quickly and accurately without interrupting the composition. The included studies suggest that these innovative approaches can overcome the cumbersome, destructive, complex, and time-consuming sampling associated with traditional analytical tools. However, the limitation of these devices is their high cost and the need for more expertise for the experimental setup, which requires a high processing time to extract the important attributes from the spectral images. The condition and stability of the thermal behavior of the agricultural products, under various environmental factors, determine the accuracy of their results. Moreover, integrating these technologies with other sensing devices  may pave the way for a more accurate detection for the banana ripeness stages. Yet, the problem with these sensing devices is that they require a proper experimental setup with expertise.Furthermore, in most of the included studies, with relation to the other machine learning methods, ANN performed better for the classification and LDA performed better for the feature creation method (of the banana ripeness stages) when the dataset is small. When the dataset is large, it was observed that CNN models performed better. Regarding the sensor-based study, the SVM model was the most relevant. In terms of white box-based model performance, the Decision Tree model  has perfFor the model to perform better, identifying the correct number of banana ripeness stages is crucial. Most included studies have used three stages  to classify banana ripeness. However, only limited studies have used the standard seven stages of banana ripeness, which has helped the model classify the ripeness stages more accurately. In addition, there has not been any study that includes an independent dataset, and such a situation can arise in a real-world scenario when the dataset is completely new to the system , such asThere are challenges and limitations acquired while compiling this review. Out of all the included studies, five ,29,32,42This review identified the four key parameters for banana ripeness detection, which should be addressed while designing the review.The sample collection parameter affects the efficiency of the model. Future works should validate their works on larger datasets using standard machine learning methods reported in a standard notation. There is a gap in deep learning-based solutions that should be addressed, using large datasets that should avoid using too much augmentation. More work should be carried out on banana bunch detection in real-world scenarios, as most works aim to address only the finger-based analysis in perfect laboratory conditions.In addition, classifying the correct number of banana ripeness using four stages can aid the researcher in using the correct stages for extracting maximum information for building an accurate banana ripeness model. In most of the best results, four ripeness stages were used with color features  and the texture of the banana.The best performance was obtained using color-based classifiers, which was probably due to the significant differences in banana ripeness stages when compared to the other features. As a result, in the future, a low-cost method based on external features could be used to identify the automatic banana ripeness stages.It is important to identify the possibility of the mis-classification of banana ripeness stages by using other performance metrics such as confusion metrics. In our review study, most of the studies have used accuracy and This review study summarized the existing deep and machine learning techniques available for the identification of banana ripeness stages. Furthermore, the critical factors related to the data gathering, data pre-processing and classification such as capturing device/sensor, banana ripeness stages, appropriate features and classification methods are identified in this systematic review and can be used for designing a standardized study in the future for predicting accurate banana ripeness stages. The statistical analyses conducted under this review study can allow the researchers to compare their respective work with the performance metrics of existing techniques. It is visible from the existing studies that for the small dataset, ANN performed better for problems related to classification and LDA performed better for feature creation. Whereas, in the case of large datasets, CNN has performed significantly well. The performance of models varies with the change in data collecting devices/sensors: the SVM model works well when data are gathered by sensors instead of a camera. This review highlights the gaps in the existing banana ripeness stages identification techniques and the possible ways to fill those voids in future studies.Finally, to enhance the quality of future work, we proposed in this systematic review the following recommendations: use of unlabeled datasets; avoid a large extent of data augmentation; consider color features and appropriate stages; use other performance metrics.


The Internet of Health Things (IoHT), which is an extension of the Internet of Things (IoT) in healthcare, has provided a new type of telemedicine approach. In IoHT, wearable sensors are used to collect patient health data, and information is transmitted remotely to doctors who can develop accurate treatment plans and provide timely telemedicine services to patients. However, patient health data are transmitted over a public channel, which means that the privacy and medical data of patients are at significant risk of leakage and can be confronted by serious security problems. We proposed a lightweight authentication protocol known as LAP-IoHT for IoHT environments to overcome the various threats that are currently faced by IoHT. We verified the security of LAP-IoHT using a Real-or-Random model and demonstrated its significant performance advantage by conducting a comparative analysis with other similar protocols for a better adaptation to the IoHT environment. The rapid development of communication technologies has resulted in the extensive application of the Internet of Things (IoT) ,2,3,4. BFurthermore, the Internet of Health Things (IoHT) ,18, whicSecurity and privacy ,25,26,27This study proposed a lightweight authentication protocol (LAP) known as LAP-IoHT for IoHT environments. In LAP-IoHT, all participants, including the users and wearable sensors, are authenticated by the gateway. Subsequently, a shared session key is established for each communication session. LAP-IoHT encrypts the biometric features of the users to ensure anonymity. To demonstrate the security and reliability of this approach, we applied the Real-or-Random (ROR) model to analyze LAP-IoHT. The experimental results indicated that LAP-IoHT exhibits improved communication and computationally efficient performance.(1)To address the current security issues frequently encountered in healthcare IoT systems, we designed a three-factor IoHT-based protocol that incorporates authentication and key negotiation, thereby guaranteeing privacy and access control.(2)The introduction of biometrics, which protects the anonymity of users with unique information, can provide better user experience and privacy protection. In addition to using common one-way hash functions and simple XOR operations, we adopted asymmetric encryption and decryption in the protocol to provide higher security.(3)Based on a shared ROR model, we performed a formal security analysis to evaluate the security, soundness, and integrity of the session key and protocol. Moreover, the informal security analysis provided strong evidence that the protocol is resistant to currently known security attacks.(4)We conducted a comparative study and analyzed the performance of several protocols of the same type, taking into account the computational cost, time efficiency, and security properties. The results demonstrated that our protocol exhibits a significant performance advantage.The main contributions of this study are as follows:The remainder of this paper is organized as follows: IoT is widely adopted in healthcare monitoring systems. Onasanya et al.  proposedMoreover, several authentication protocols are available for IoHT. A summary of the applications of IoT in the medical industry is presented in (1)Wearable sensors are set on the bodies of patients. They can observe various body indicators, such as the electrocardiogram (ECG), electromyography (EMG), electroencephalogram (EEG), respiratory rate, pulse, blood pressure, blood glucose, and oxygen saturation. These wearable sensors should be registered with a gateway before being deployed to human bodies for precise management.(2)Users are organizations or groups of people who can view the health data of patients. For example, users may be hospital administrators, doctors, pharmacists, nurses, families of patients, data analysts, and drug trialists. If a person needs to enter the network and view patient medical data, the person must register with the gateway in advance and become a legitimate user with the appropriate authorities.(3)The gateway in our IoHT architecture acts as a trusted server. Prior to entering this network, all wearable sensors and users should register with the gateway. Subsequently, the gateway manages the list of all sensors and legitimate users.Assume that a user desires to obtain data from a specific wearable sensor. This user transmits a request to the gateway and the gateway forwards this request to the sensor. After receiving the request, the wearable sensor sends the data to the user with the help of the gateway. Since medical data are personal and private, all communications among the users, gateway, and sensors should be confidential. The most straightforward method for achieving this is to encrypt the transmitted data.The gateway can authenticate users and sensors using the proposed protocol. Moreover, a shared session key is established for each session.This section presents the proposed LAP-IoHT protocol for IoHT, which consists of three phases: user registration, sensor registration, and login and authentication. The notations and symbols are defined in Assume that user (1)(2)(3)(1)(2)b and calculates the pseudo-identity (3)A wearable sensor must also be registered before joining the network. Assume that sensor (1)M stored in the smart card. If (2)(3)L with his or her private key (4)(5)The computer of If This section first describes the capabilities that the attacker (1)(2)(3)(4)We consider the well-known Dolev–Yao (DY) adversary model  and assuIn LAP-IoHT, messages that are transmitted via a public channel have timestamps, such as Assume that Suppose that If We assume that the temporary random number N and If The ROR  model is a widely used security-proof method. The ROR model can obtain the probability of successfully breaking session key Our protocol comprises three entities: M to R and receive the response message from R.t represents the number of bits of biological information in the protocol.Theorem 1: In the ROR model, we use To prove the accuracy of Theorem 1, we performed four rounds of game b and does not perform any query operation. Therefore, we can obtainb is guessed, we obtainSince Using Equations –5) abov abov5) aUltimately, we can obtain We compare LAP-IoHT with other related protocols with similar architectures, such as those of Kumar et al. , Yu et aIn this section, we evaluate the performance of the proposed LAP-IoHT protocol by performing comparisons with other protocols, such as those proposed by Kumar et al. , Yu et aWe used different devices to obtain the computation time and communication cost required for the certification stage in the performance comparison. We used a mobile phone, laptop computer, and desktop computer to simulate the user, gateway, and sensor nodes, respectively. The relevant parameters for the three devices are listed in We use three devices to determine the computation time and communication cost. The times required to perform elliptic curve point multiplication, symmetric encryption/decryption, asymmetric encryption/decryption, single fuzzy extraction, and hash functions vary on different devices. Furthermore, the computation times required for the connection and XOR operations are insignificant compared to the other operations; thus, we ignore these in our evaluation.The computation times of the proposed protocol and other similar protocols are listed in We assume that the output of asymmetric encryption/decryption is 1024 bits; the length required for the elliptic curve point multiplication operation is 320 bits; each block for symmetric encryption/decryption is 256 bits; the hashed value and random number are 160 bits; the identity, password, and biometrics are all 128 bits in length; the timestamps require a length of 32 bits.According to Internet of Health Things (IoHT), which promotes intelligent healthcare, plays a pivotal role in the future e-healthcare environment. Due to its high sensitivity, the health data transmitted through a public channel should be protected from unauthorized access. This means that an authentication protocol is essential. This paper presented a more secure and reliable authentication protocol called LAP-IoHT for the Internet of Health Things. LAP-IoHT provides mutual authentication among users, sensors, and a gateway over a public channel. Moreover, a user and a sensor can establish a common session key after a protocol run. By using the ROR model and performing an informal analysis, it was proven that LAP-IoHT has adequate security and reliability as well as sufficient ability to resist various attacks. Furthermore, we compared LAP-IoHT with related protocols and found that our protocol is at the mid-to-upstream level in terms of time and communication costs, exhibiting a significant performance advantage. In summary, the proposed protocol offers specific practical value in the current environment and has more robust adaptability relative to the future development of IoHT.