Arnica montana L. is a very important medicinal plant and simultaneously a European endemic endangered plant species. The morphological features and details of seed development and achene variability are poorly recognized. The aim of this study was to determine the impact of the achene position in the infructescence and the location of the inflorescence on the plant on the (i) morphological characteristics and germination ability of achenes, and (ii) recruitment of seedlings and their biometric features. Infructescences containing fully ripe achenes were randomly collected from A. montana individuals for the measurements and the germination experiment. Scanning electron microscopy, fluorescence microscopy, and light microscopy were used for characterization of flowers and achenes. The morphological traits of achenes and reproductive characteristics of A. montana were determined by the position of the achenes in the infructescence and the location of the inflorescence on the plant. The surface of arnica achenes is equipped with non-glandular and glandular trichomes, which is very rarely presented in species of the family Asteraceae. It is possible that the fluid-containing glandular trichomes are a source of essential oils. The peripherally located achenes were longer, thinner, and lighter. They were characterized by lower embryo weight, lower embryo/achene weight ratio, and lower germination capacity in comparison to the centrally located ones. The results presented in this article fill the gap in the knowledge of the morphology of achenes and the biology of the species, and provide information that can help in breeding programs, active protection, and field cultivation. Arnica montana (Asteraceae) exhibits such a variation in achene traits.The reproductive traits of vascular plants decline with time, and architectural constraints or resource limitation is responsible for the seasonal decreases in flower size, inflorescence size, and seed weight. Seeds or fruits that develop from early-pollinated flowers have access to a larger portion of the resource pool and are bigger. In turn, diaspores produced later are necessarily smaller due to the depleted resources . Within The formation of a capitulum inflorescence with two different types of florets is an interesting issue in floral biology and evolution. A frequent arrangement of the Asteraceae capitulum consists of peripherally located ray florets, which are highly specialized in pollinator attraction, and disc florets which serve the reproductive function ,12. PeriArnica montana L.) is an endangered medicinal plant species. It is endemic for Europe; in natural habitats, it grows in heathlands and pine forests [A. montana achenes are a rich source of interesting molecules characterized by antioxidant and anticancer activity [The mountain arnica  morphological characteristics and germination ability of achenes, and (ii) recruitment of seedlings and their biometric features.The possibilities for cultivation and reintroduction, and thus conservation, of arnica depend on the quality of achenes, which is measured by their germination capacity and vitality ,43,44,45o et al.  as an elo et al. . HoweverThe arnica flowers formed a single head inflorescence with peripherally located yellow female ray florets serving as an attractant. The central part of the head receptacle was occupied by tightly packed hermaphrodite disc florets. The disc florets had stamens located in the central part, which were fused with anther heads and formed a tube around the style. There were a greater number of disc florets in the head. The ray florets exhibited zygomorphic symmetry, while the disc florets were actinomorphic. The anthesis stage lasted approximately 7 days and was characterized by centripetal opening of individual florets A–D.The morphological studies of achenes were conducted using 25 randomly selected mature infructescences. The achenes in the infructescence differed, i.e., the white ones were sterile non-viable seeds E, whereaThe arnica pericarp was covered with numerous morphologically diverse trichomes . Two typThe FM observations conducted with the use of appropriate lignin-detecting filters revealed intense autofluorescence of the trichome walls. Blue autofluorescence was shown in the two-celled pointed non-glandular trichomes, indicating the presence of lignin in the cell wall of these structures. In contrast, the glandular trichomes emitted green fluorescence, indicating the presence of cellulose C. All trp < 0.05) (ACE), the percent share of achenes containing a developed embryo  (SACE), the receptacle area  (RA), and the achene density  (AD), but not on the total number of achenes  (TNA), and the number of empty achenes (EA) . The later developing inflorescence/infructescence on the branch stem was smaller and had a lower number of ACE, SACE, RA, and AD , length of the achene without pappus (LA), pappus length (PL), width of the achene (WA), thickness of the achene (TA), and the achene weight (AW) . The effThe values of such characteristics as LAP, LA, and PL increased from the proximal to distal area of the infructescence in the case of infructescences taken from both the main stem and the branch stem . The ranThe two-way ANOVA results showed a statistically significant impact of the achene position of the infructescence on the plant on the pericarp weight (PW)  and on tThe highest EW, EW/AW, and G values, i.e., 0.62 mg and 0.42, and 77%, respectively, were obtained in the case of achenes produced by the disc florets from the periphery of the receptacle (D3) and achenes produced by the ray florets (R) from infructescence on the main stem B–D. Achep < 0.01) (p < 0.01) (p = 0.052), (p = 0.194), (p = 0.094), (p = 0.053), (p = 0.610) did not differ between the groups  D and sur < 0.01) C were co 0.052), A, seed g 0.194), B, stem l 0.094), E, cotyle 0.053), F, and roe groups G.The observations of the arnica achenes showed that the pericarp cover was uneven, notched, and had numerous depressions. On this surface, morphologically diverse trichomes were present. A very large group was composed of secretory trichomes. The glandular trichomes were more varied, but all had a fluid-filled head at the apex. In angiosperms, trichomes may occur on leaves, petals, stems, petioles, peduncles, and seed coats, depending on the species. Trichomes are outgrowths of protodermal origin that can exist in all morphological parts of a plant ,49. SuchGossypium hirsutum L. are necessary to maintain the correct shape of flower buds. Thanks to the mechanical entanglement of hairs from adjacent corolla petals, the proper architecture of the cotton flower is preserved. The silencing of the master regulator of petal trichome development caused flower buds to have abnormal shapes, exposing the developing anthers and pistils to desiccation damage [The observation of the arnica achenes showed the presence of another type of these structures, i.e., mechanical/non-secretory trichomes, which were pointed and directed towards the distal part. Non-secretory trichomes serve different functions: they act as a mechanical barrier against insects ,55, refln damage . In thisIn the present study, the value of TNA per infructescence was 102 on the main stem and 92 on the branch stem, respectively. Maurice et al.  reportedA. montana L. In research on a natural population originating from the Carpathians, Ukraine, Balabanova et al. [In Europe, some studies investigated the possibility of cultivation of a et al.  showed ga et al.  compareda et al.  exhibiteIn the case of natural arnica populations, the germination capacity, seed weight, and percentage of seeds containing a developed embryo are positively correlated with the population size depending on the environmental condition . It is nA. montana. Variations in morphological and functional characteristics of flowers grouped in inflorescences are commonly observed [In the case of some Asteraceae plant species, achene morphology affects the time of seedling emergence . This deobserved ,69. The observed ,73,74,75Some studies have investigated relationships between the reproductive success and the within-inflorescence spatial position and time of flower opening (early or late). Plant species with acropetal or basipetal inflorescence development have higher fruit and seed set in early-opening flowers than in late-opening flowers ,78,79,80According to the non-uniform pollination hypothesis, the relatively low fruit or seed sets of certain flowers on the inflorescence may be attributable to variations in pollen receipt over the inflorescence flowering period  ,82,83,84A. montana achenes. The patterns of the variation in most variables show a decline from the lateral to central position. The decline was observed in the infructescence of the main stem and the branch stem. Such an intra-inflorescence variation is a common pattern in plants [Lavandula stoechas, early-opening flowers were characterized by higher fruit set than later opening flowers [The present results show that there is a significant intra-infructescence variation in the traits of n plants ,87,88,89n plants ,89. IntrSeed mass is regarded as an important aspect of the reproductive strategy . In the In the present study, the mean embryo weight/achene weight ratio ranged from 0.33 to 0.41, whereas Strykstra et al.  reportedA. montana achenes are adapted to wind dispersal, thus the achene shape and mass and the pappus size play a key role in spatial dispersion. Strykstra et al. [In the present study, the values of such characteristics as the achene weight, embryo weight, pericarp weight, and pappus length are similar to data presented from a natural population . A. monta et al.  conducteA. montana fruiting individuals growing on experimental plots . The seed material was divided into two equal parts.Fifty infructescences located on the main stems and fifty infructescences located on the branch stems containing fully ripe achenes intended for the research were randomly collected from fifty 3-year-old 2 atmosphere, sprayed with gold, and viewed in an LEO1430VP electron microscope with a 15k acceleration potential. Documentation was made using INCA-Mapping software . The samples were analyzed using a scanning electron microscope (LEO1430VP) with an accelerating potential of 15 kV.Samples for SEM observations were prepared according to the method described by Talbot and White . Fresh mThe slides were analyzed under a Nikon Eclipse Ni-U fluorescence microscope without the use of fluorochromes, but with the use of appropriate cut-off filters. The cell wall components were visualized using the autofluorescence method in the fresh material. Lignin and cellulose autofluorescence can be observed using the following filters: an excitation wavelength 330–380 nm and an emission wavelength over 450–480 nm (UV). Lignin shows blue fluorescence while cellulose emits green fluorescence . PhotogrSeeds were imaged using a Nikon SMZ 74ST stereoscopic microscope, and photographic documentation was made with the use of the Delta Pix program.−2 of the receptacle area) (AD).In the group of 25 infructescences located on the main stems and 25 infructescences from the branch stems, the following achene characteristics were measured: total number of achenes (TNA), number of achenes containing a developed embryo (ACE), number of empty achenes (EA), percent share of achenes containing a developed embryo (SACE), receptacle area (RA), and achene density (number of achenes per cmIn the other group (25 infructescences located on the main stems (MS) and 25 infructescences located on the branch stems (BS)), each simple infructescence was centrally divided into four sections. The following four categories were distinguished: D1–D3—3 categories of achenes formed from disc florets, and R—one category of achenes formed from peripherally located ray florets. Finally, eight categories were distinguished: MSD1, MSD2, MSD3, MSR, BSD1, BSD2, BSD3, and BSR. From each category, 100 achenes were chosen randomly, and next the following achene characteristics were measured: length of achenes with pappus (LAP), length of achenes without pappus (LA), pappus length (PL), achene width (WA), and achene weight (AW).−2 s−1, temperature regime (25/18 °C), and relative humidity 40–45% for 30 days in a growth chamber. During the experiment, the seeds were watered to maintain constant surface moisture. The experiment lasted 30 days. The germination rate was registered throughout the experiment. When the seedlings formed the first leaf, they were transferred into pots filled with garden soil and placed in a chamber. At the same time, the pericarp was taken, dried, and weighed, and finally the pericarp weight (PW) was measured. The embryo weight (EW) was calculated as the difference between achene weight and pericarp weight and the embryo weight/achene weight ratio (EW/AW) was calculated.After conditioning, the achenes were sown into Petri dishes (10 Petri dishes x 8 categories) with moist vermiculite a surface and incubated with simulation of a day/night cycle (16 h light/8 h dark) under light-emitting diodes at photosynthetic photon flux density of 150 μmol mA. montana seedlings from each category (eight achene categories) with two mature leaves were collected. The measurements included the following parameters: seedling weight, seedling stem length, seedling cotyledon length, and seedling root length. The shape index (I) was calculated based on the measurement results of the length and width of the achenes without pappus. The measurements were taken using analytic scales RADWAG, model XA 52.4Y with an accuracy d = 0.01 mg and an electronic caliper ARTPOL with an accuracy of 1 mm.Cultivation was conducted in a growth chamber in the conditions described above for 30 days. In order to conduct the biometrical analysis, 30 randomly selected t-test was used to determine if there is a significant difference between the means of two groups. The results were expressed as means and SD, and the differences were considered significant at p < 0.05. The statistical analyses were conducted using Statistica 12.0 software . Principal component analysis (PCA) was applied to explain the relationships between the presented parameters and to show the variability of factors. Prior to the PCA, the data were centered, and log transformed. The analyses were conducted using the statistical package (MVSP) program version 3.1 [After testing the data for normality (Shapiro–Wilk test) and homoscedasticity (Levene’s test), analysis of variance of different sets of data was performed, followed by subsequent Tukey’s test or the nonparametric Kruskal–Wallis test, and the subsequent U Mann–Whitney test. In turn, a sion 3.1 .A. montana were determined by the location of the achene in the infructescence and the location of the inflorescence on the plant. The patterns of the variation in most variables show a decline from the lateral to central positions in the infructescences. The peripherally located achenes were longer with longer pappus, thinner, and were characterized by lower achene weight, lower embryo weight, lower embryo weight/achene weight ratio, and lower germination capacity in comparison to the centrally located achenes. Research on declining arnica populations often requires the use of achenes for propagation, access to which is significantly limited by nature protection institutions for obvious reasons. Therefore, in such situations, achenes located peripherally in the infructescence of the main stem characterized by the greatest mass and germination capacity should be used in propagation, which will contribute to reproductive success. The proposal to use appropriate types of achenes is also relevant for sowing and seedling production. The results presented in this article fill the gap in the knowledge of the morphology of achenes and the biology of the species and provide information that can help in breeding programs, active protection, and field cultivation.The morphological traits of achenes and the reproductive characteristics of


Its use to enhance individual's physical activity (PA) participation should be examined. This research reviews the effectiveness and successful features of Facebook for influencing PA behaviors in young adults (Study 1), and empirically examines the efficacy of the features purported in increase PA In Study 1, PubMed, Medline, SPORT Discus, ERIC, and Embase were searched for articles that identified successful features and effectiveness of Facebook PA interventions published between January 2005 and February 2022. In Study 2, a 4-week Facebook PA intervention with University students was conducted using features identified in Study 1. The PA behaviors with objective (ActiGraph) and subjective (questionnaire) measures, perceived PA level, stage of readiness, effectiveness, and efficiency of Facebook were examined.Study 1 concluded that the most effective strategies for producing significant PA changes in young adults using a PA Facebook intervention included the following: Adding behavior modification , using influence agents, recruiting members of an existing network with the snowball technique, being attentive to group size, enhancing social support with motivational quotes, interactive posts, opinion polls, increasing tailored feedback, and providing educational information. Study 2 found no significant difference in PA between the intervention and the control groups, as measured objectively, but the subjective reporting of PA behavior was higher in the intervention group. Compared to the control group, the Facebook PA intervention group reported more positive change in perceived stage of readiness in PA participation, commuting type, sport type, sport venue, sport emotion, and fast breathing or sweating. When features were ranked by the Facebook PA intervention group, motivation (supports from your friends) and tailored feedback  were the top two ranked features.The use of influence agents in the Facebook PA intervention could address exercise preference and facilitate higher program engagement. Significant differences related to commuting type, sport types, sport venue barriers, and exercise intensity across groups were noteworthy and warrant additional investigation in the future. Insufficient physical activity (PA) represents a major contributing factor to morbidity and mortality across the world . PhysicaTraditionally, interventions designed to increase PA participation in young adults are implemented through face-to-face delivery. Most of these interventions are effective, however, there are limitations in terms of reach, maintenance accessibility, and cost . The US Researchers who have examined increasing PA through social media have concluded that Facebook is effective and efficient in terms of recruiting participants, follow-up investigation, social support, health information delivery, and intervention delivery –13. ThesThe rapid growth of social media use in our youth, along with the current COVID-19 pandemic (which has changed the way humans interact), provide a natural forum for online social networks to deliver health behavior change interventions. Recently, the researchers have focused on developing online social networks, as their use has been significantly growing from 90 min/day in 2012 to 147 min/day in 2022 . Some ofRecently, the researchers have begun to examine using Facebook to connect social media users to online social networks. Several studies have compiled information, made programming recommendations, and presented suggestions for future research. A systematic review by Maher et al.  examinedn = 7 out of 10) reported significant within- or between-group differences for at least 1 PA outcome. A more recent review by Goodyear et al.  intervention for young adults.Although some research has been done, there are many unanswered questions. To gain a more comprehensive view of Facebook's potential contribution to changing PA behavior in a specific, impressionable population , this research consisted of the following two studies: Study 1 reviewed the literature and identified the features of successful Facebook interventions to increase PA. Study 2 was an empirical study that tested the efficacy of the features purported to increase PA PubMed, Medline, SPORTDiscus, ERIC, and Embase were searched from January 2005 to February 2022 using keywords “Facebook/OR social network/OR social media/OR Twitter/OR web /OR internet” combined with “physical activity/OR physical fitness.” The eligibility criteria were as follows: (1) Experimental studies and systematic reviews reported in peer-reviewed journals or as peer-reviewed full conference papers, (2) studies using an online social network either wholly or in part to increase PA, (3) participants were adults and children regardless of their health status, and (4) the intervention had to target PA level and measure related outcomes.Of the 1,300 initial search results, 45 met the eligibility criteria, and 38 of those were original research studies or study protocols, and 7 were literature reviews .Of the 38 original studies, 31 (81.6%) showed an increase in self-reported (subjective) PA level; conversely, few of these studies showed an increase in objective PA as measured by a device such as an accelerometer. Specifically, Fjeldsoe et al.  reportedA second finding was that positive results mainly occurred in light intensity PA or walking time compared to vigorous PA. For example, in a Facebook-based PA intervention for insufficient active adults, Maher et al.  found thThird, in a couple studies, participants' awareness, motivation, perceived behavior control, and satisfaction increased after a 3–4-months intervention , 27. SpeTaken together, these findings indicate that social media may have an impact on the adoption and maintenance of light intensity PA, and on the psychological aspects of PA adherence and engagement.The results were mixed relative to the impact of Facebook PA interventions on young adult's PA behaviors although most of the findings were similar to what was found in Section The effectiveness of social network interventions on PA behavior. Most studies showed increases in self-reported PA as a result of participating in a Facebook PA intervention. For example, in a pilot RCT designed to increase low-active young adult's PA, significant improvements were found over time on self-reported weekly leisure–time PA, although no interaction was observed between groups or on objectively measured outcomes . Pope etvs. control group using Facebook among female college freshmen. Compared to the control group, the walking intervention participants reported a significant increase in objectively measured (pedometer) walking  and a decrease in waist circumference. Social support was highlighted as a pivotal element in this successful intervention  nitoring , 30, 31.Influence agents or a small group of individuals who promote behavior change and provide support when/where needed, may be effective for facilitating behavior change. Van Woudenberg et al. (2) g et al.  concludesnowball method should be used to recruit “influence agents” into an intervention group as it may improve effectiveness (3) The tiveness , 33–35. tiveness  concludetiveness . Other ftiveness  specifieSocial support defined as enhancing companionship, esteem, and encouragement (4) ragement , is at tragement . Irvin eragement  stated tragement . Goodyearagement  suggesteTailored feedback consists of providing participants with individualized information based on their preferences and motivational incentives. Nishiwaki, et al. (5) , et al.  designedEducational information, which consists of various behavior management skills including PA information links , goal setting strategies, setting individual expectations, overcoming barriers and maintaining behaviors, is another essential strategy for a successful social network PA intervention (6) rvention , 38, 40.A 4-week Facebook PA intervention was designed using the features recommended in Study 1. The intervention was implemented with college students. Intervention effectiveness was evaluated using several measures, collected pre- and post-intervention, including objective PA (accelerometer), subjective PA , and psychological aspects of PA participation . The snowball sampling was used to recruit participants. In the initial stage, physical education (PE) major students were selected as the influence agents, also known as the captains. The captains then invited their friends to join his/her group. In total, 62 young adults, aged 18–25 years agreed to participate in this study after signing the consent form. There were 5 intervention groups between 5 and 9 participants, and 3 control groups between 10 and 11 participants.The PA level of the participants from intervention and control group was measured at baseline (pre-intervention) and upon completion of the intervention (post-intervention). During the intervention, each group used one non-public Facebook Page which could only be seen by captain and the group members, as well as related research staff. The intervention group members received education links and intervention messages on the Facebook page posted by the captains, and were required to complete a 14-item questionnaire on the page each day. Captions could have a personalized page name and photos could be added to the page to make it more attractive. In addition, intervention group captains participated in discussions among his/her group members about intervention contents, and they were encouraged to share their experiences and feelings. Intervention messages were sent on a daily basis, and were designed to promote PA levels through goal setting, peer support, motivational quotes, and tailored feedback. The participants from the control group received no such intervention messages and had no group captain or discussion; they only finished the questionnaires.Two types of measurements were used in the study as follows: Objective measurements by ActiGraph and subjective measurements by self-reported questionnaire, which were collected on the Facebook page.The PA level at baseline and post-test was measured using an activity monitor from ActiGraph . The models GT3X, GT3X+, and wGT3X-BT were used, and initialized at an epoch of 5 s (GT3X) and a sampling rate of 30 Hz . Each participant carried the same accelerometer over a 2–3-week period. At least 3 valid weekdays and 1 valid weekend day were required for an eligible wearing. For each valid day, a wearing time of more than or 480 min was required. A non-wear period was defined as 60 min minimum length with a spike tolerance of 2 min. Vector magnitude was also applied. For setting of cut points and MVPA, there were four categories: Sedentary (0–99 cpm); light ; moderate ; and vigorous . The participants were asked to wear the accelerometers for an additional time if their first wearing data were unqualified.To collect additional objective PA information, and verify step count stated in the questionnaire, a free version mobile App named Accupedo pedometer was used after getting approval from the company. The App was selected due to the fact that it was one of the most downloaded pedometer applications, and has shown good accuracy and compatibility with different mobile phones .via their Facebook account. A pre-post questionnaire covering psychological aspects of PA  was also conducted to the both groups.The self-constructed and self-report questionnaire aimed to collect daily physical activity-related information such as commuting type, sport type, sport venue, sport duration, and intensity. The participants were asked to complete this questionnaire daily t-tests were used for continuous variables, and Chi-squared tests were used for categorical variables; p < 0.05 was used as the significance level.The ActiGraph data were analyzed by ANCOVA with group (intervention and control) as the factor and baseline PA as the covariate. Between-group mean difference and its 95% confidence interval (CI) were calculated after adjusting the baseline difference between intervention and control groups. The differences of subjective outcomes between intervention and control groups were tested week by week. Independent A sample of 56 young adults aged 18–25 years joined this study and 49  of the participants completed the post-test. Of this group, 24 participants were in the intervention group and 25 were in the control group. The average age was 21.5 ± 1.68 years. No gender or age differences were found between the intervention and control groups.No significant difference was found in the objective/ActiGraph measurements between intervention and control groups , but parp < 0.001) or for the total step counts from the whole week .Finally, more positive changes in perceived stage of readiness in PA participation were reported from the pre-contemplation to action/maintenance stages in the intervention compared to control group . These rSignificant differences in commuting type were observed between two groups in the first and second weeks. The intervention group reported higher levels of public transport use .For sport type, there were significant differences between the two groups each week. Compared to the control group, the intervention group reported higher level of walking, jogging and other sport types.For sport venue, there were also significant differences between two groups across all 4 weeks. The intervention group reported lower levels of sport venue problems—which means they were able to more effectively access sport venues to participate in PA.vs. 37.4% in control in week 2; 49.3% in intervention vs. 29.4% in control in week 4).Except during week 3, participants from the intervention group reported more positive emotion compared to the control group  considered Facebook an effective way to enhance their PA behavior, compared to only 25.8% (8 out of 31) in the control group .Finally, the participants were asked to rank the most important features in Facebook that facilitated their PA participation. Motivation (supports from your friends) and tailored feedback  were the top two features listed by the intervention group, whereas motivation (supports from your friends), rewards (number of “Likes” you received) and personal goals [announcing your goal (s)/ progression to your friends] were the top three features in control group .via a Facebook intervention.The most important findings from Study 1 were that behavior modification strategies , influence agents, snowball grouping and group size, social support , tailored feedback, and educational information are effective approaches for producing changes in PA outcomes in young adults vs. the control group . After a 4-week intervention, no significant differences were found between the intervention and control groups in PA level, as measured objectively with an ActiGraph. Other findings, including pedometer readings and more subjective assessments of PA were more favorable for the intervention group. This finding is consistent with previous studies that concluded that objective PA level did not increase significantly  25, 32,,32, 42. ol group . This fiol group , who fouol group , 45.From this study, the finding of subjective and positive perception of behavioral change in PA level indicated that the participants in the intervention group did “Feel” they have changed and increased their PA level but the “Do” stage has not been achieved yet. In the CBT triangle, “Think-Feel-Do” is a reciprocal relationship which reinforces each other in the cycle. The perceived change “Feel” is a reflection of what the participants attributed, and this attribution could act as an important determinant to actualize their behavior “Do” . DijksteWith regards to the categorical variables included in this study, we found that compared to the control group, the intervention group participated in a higher level of public transport in the first 2 weeks. The intervention group also reported higher level of walking, jogging, and other sport types, and significantly higher level of breathing fast and sweating in all of the 4 weeks. No previous studies have investigated the relationships between an individual's commuting pattern, sport types, sport venue, barriers, and exercise intensity and the use of a Facebook social network group. As a whole, these four variables may represent an individual's PA lifestyles and choices. Further investigation of how Facebook influences these lifestyles and choices may shed light on how to facilitate more objective changes in PA behavior.Compared to the control group, the effectiveness and efficiency of using Facebook to enhance participant's PA behavior was rated much higher in the intervention group (77.4% vs. 25.8%). The different rating of effectiveness and efficiency may originate from the daily intervention message  delivered to the intervention group by the captain. Rayward et al.  assessedBoth intervention and control groups considered motivation (supports from your friends) as the most important feature when using Facebook to promote PA participation. Our findings confirm that peer support plays an important role during young adult's PA intervention. Allen  and GreeAlthough many important findings have been reported, some limitations are worth mentioning. First, the sample size is small, which may affect the statistical power to detect an intervention effect. Since reachability is one of the advantages of social media compared to traditional intervention delivery channels, additional studies with larger sample sizes are warranted. In addition, the duration of the study was of 4 weeks. It is possible that a longer intervention duration is necessary for more permanent behavior changes. A third limitation is that participants may have changed their behavior (or reported changing their behavior) because they were being observed . It is important to continue to test Facebook interventions and their impact on PA behavior with attention to minimizing the Hawthorne effect when conducting intervention research . Finally, we did not collect data related to level of social media engagement . These data are important so that more specific elements of effectiveness can be examined.Study 1 identified the important features of successful Facebook interventions to increase PA in the literature. Some of the components related to the successful interventions included behavior modification , influence agents, the snowball method of recruiting, social support , tailored feedback, and educational information.Study 2 designed, implemented, and evaluated a Facebook PA intervention for young adults. Study 2 found that objectively measured PA did not differ between the control and intervention group, but that subjective perception of PA level and positive emotion were reported more frequently by the intervention than the control group, as were differences in commuting type, sport type, sport venue, and exercise intensity. Not surprisingly, the effectiveness and efficiency of using Facebook to enhance participant's PA behavior was rated much higher in the intervention group, and strategies preferred were slightly different. Using influence agents shows promise for facilitating higher levels of program engagement.Although this study is focused on Facebook, the findings have meaningful implications for other online social network or e-health interventions. This study helps us understand more about using social networking technology to facilitate PA.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by Research Ethics Committee of Hong Kong Baptist University. The patients/participants provided their written informed consent to participate in this study.PL and JW designed the study, analyzed the data, and wrote the manuscript. PL collected the data. LR and LS and all other authors reviewed and revised the manuscript. All authors contributed to the article and approved the submitted version.Research funding was provided by the Fundamental Research Funds for the China Institute of Sport Science (Grant No. 20–14).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Background: mTOR inhibitors are a novel pharmacotherapy recommended for subependymal giant astrocytomas, refractory epilepsy, and the treatment of the other clinical manifestations of tuberous sclerosis complex (TSC). Clinical trials on everolimus proved it to be effective and safe in children. Despite its common use in clinical practice, the research on sirolimus is limited. This study is the first to determine and assess the severity of the adverse effects (AEs) of sirolimus administered to children with TSC under two years of age. Methods: We performed a bicenter retrospective data analysis of medical records of individuals with TSC who initiated therapy with sirolimus under the age of two. Results: Twenty-one patients were included in the study. At least one AE was reported in all participants. The most prevalent AEs were anemia, thrombocytosis, and hyperlipidemia. Infections and mouth ulcerations, often reported in the studies on older patients, were infrequent and of mild or moderate grade. Conclusions: Adverse effects associated with sirolimus use in infants and young children with TSC are frequent yet not life- or health-threatening. Further multicenter prospective clinical trials should determine the long-term safety of sirolimus. TSC1 at 9q34 (encoding hamartin) or TSC2 at 16p13.3 (encoding tuberin), affecting one in 6000 live births [TSC1 and TSC2 genes leads to the overactivation of the mTOR. Its selective activation results in a kinase signaling cascade that increases cell proliferation and growth; hence, multiple benign tumors are formed in different organs [Tuberous sclerosis complex (TSC) is a genetic disorder caused by a heterozygous mutation in either of the genes Epilepsy affects approximately 80% of patients with TSC, and in up to 30% of them, seizures become refractory to treatment ,5. The eNovel pharmacotherapy targeted at the metabolic pathway to inhibit the mTOR kinase has recently been approved in many countries, including EU members, US, and Japan . The mTORandomized clinical trials on everolimus have shown its safety profile in patients with TSC ,16,17,18This study aims to address the gaps in mTOR inhibitor therapy research. We assess the safety of sirolimus in young patients with TSC under the age of two years.The present study is a bicenter retrospective data review of children with TSC treated with mTOR inhibitors between 2014 and 2022.The data was collected from two hospitals in Warsaw, Poland: the Department of Neurology and Epileptology, The Children’s Memorial Health Institute, and the Department of Child Neurology, the Medical University of Warsaw. We searched the database with the alphanumeric coding of TSC in the ICD-10 list: Q85.1. The data was extracted from patients’ medical records in paper or electronic health records and put into a standardized spreadsheet. The data collection process ended on 30 June 2022.Clinical or genetic diagnosis of TSC;Treatment with oral sirolimus before the age of two years;A follow-up at least three months after the initiation of treatment.The inclusion criteria were:The patients were excluded if mTOR inhibitors were introduced after two years of age, the patient was not followed for at least three months, or the applied mTOR inhibitor was everolimus.TSC1 or TSC2 gene mutation;Genetic analysis results: Sex;Age at the initiation of mTOR inhibitor treatment;Age at the onset of epileptic seizures;Reason for mTOR inhibitor treatment;Whether antiepileptic drugs (ASMs) were included;If the ASM treatment was preventive.The recovered baseline data included:Age: from birth to 6 months of age, 6–12 months, 12–24 months, and 24–36 months of age.Treatment duration (months).We stored the results of laboratory blood tests, the incidence of infections or mouth ulcerations, sirolimus dose, sirolimus blood concentrations, the patient’s body mass, and the ASMs used. For each patient, the data was analyzed twice, depending on the following:Anemia was defined as either hemoglobin levels below the lower limit of normal (LLN) or red blood cell (RBC) count below the LLN. Hyperlipidemia was determined in case of the elevated blood level of at least one of the below: cholesterol, low-density lipoprotein cholesterol (LDL-C), or triglycerides. Thrombocytosis was considered “mild” for a platelet count (PLT) between 450,000 and 700,00/µL and “moderate” up to 900,000/µL . All theThe primary outcome of this study was to assess the safety profile of sirolimus in young children with TSC under the age of two. The data collected at the onset of treatment and then three, six, twelve, and twenty-four months after the initiation focused on the known AEs caused by mTOR inhibitors. Additionally, repetitive alterations in the laboratory results and general outcomes were recorded.Grade 1: MildGrade 2: ModerateGrade 3: Severe, of medical significanceGrade 4: Life-threatening consequencesGrade 5: Death related to AEThe AEs were graded based on their severity according to Common Terminology Criteria for Adverse Events (CTCAE) v5.0 :Grade 1:The interpretation of the laboratory test results was based on the norms provided by the laboratories and adjusted for age.Continuous variables were calculated; central tendency was reported as mean and median while the variation as standard deviation (SD) and minimum and maximum values, respectively. Quantitative variables were used for sirolimus serum levels calculation. Each AE was assigned a qualitative value (present or not present), and the results were reported as the count and frequency. Analyses were conducted using Statistica 13.3. for Windows.Twenty-five patients at both clinical centers started mTOR inhibitor therapy under two years of age. Twenty-one were treated with sirolimus and four with everolimus. One patient began the treatment with sirolimus. After three months, the pharmacotherapy was changed to everolimus due to their inclusion in the national program of everolimus treatment for patients with SEGA not qualified for surgical treatment.We included all 21 patients treated with sirolimus in this study. Seventeen out of twenty-one patients were treated in the Department of Neurology and Epileptology, The Children’s Memorial Health Institute in Warsaw . All individuals began pharmacotherapy in the years 2014–2022.TSC2 gene was found. In addition, three of them carried an additional mutation in the PKD1 gene , and thirteen were female . The median age at the onset of sirolimus therapy was 104 days (range 3–656 days). Genetic analysis was performed on 15 patients . In all 15 patients, a mutation in the KD1 gene .The treatment with mTOR inhibitors was initiated due to SEGA in twelve patients , drug-resistant epilepsy in seven , rhabdomyomas causing arrhythmia or obstruction of blood flow in five , and renal angiomyolipomas and retinal hamartomas in one each . Six patients presented with more than one reason for mTOR inhibitor introduction  .2/day and varied between 0.01–0.07 mg/kg/day. The dosing regimens were then adjusted according to the sirolimus plasma levels. The targeted levels of sirolimus ranged between 3–4 ng/mL. The median plasma levels of sirolimus throughout the study were 4.34 ng/mL (0.79–13.89 ng/mL).In all patients, sirolimus was administered once a day, orally, in the form of a solution of concentration 1 mg/mL. The protocol for mTOR inhibitor initial dose was 0.5 mg/mData was extracted from medical records collected during the routine check-ups performed at the treatment initiation and three, six, twelve, and twenty-four months after pharmacotherapy initiation.The clinical tests included laboratory testing, such as complete blood count, lipid profile, liver transaminases, ALT, AST, and creatinine. In some patients, fibrinogen, D- dimers, bilirubin, and gamma-glutamyl transferase (GGTP) were also analyzed. In addition, physical examination, echocardiography, and neuroimaging were performed.Eighteen patients  continued pharmacotherapy until the end of the follow-up. Nine of them were observed for two years, and nine continued the treatment until the end of data collection, yet the observation period was shorter than two years. Three individuals discontinued the treatment before the end of the follow-up . The median follow-up period was 12 months.The reasons for withdrawal were an unsatisfactory response to the treatment; four patients were qualified for neurosurgery, and one began the treatment with cannabidiol. None of the individuals discontinued the treatment due to the AE.During the follow-up, nineteen patients  received antiseizure treatment. The median number of ASM was two. All of the patients who received ASM were treated with vigabatrin , ten with valproic acid , five with carbamazepine , five with levetiracetam , two with clobazam , and two with topiramate . Two children received adrenocorticotropic hormone (ACTH), and two patients were on the ketogenic diet.At least one adverse effect was reported in all patients  . The numTwelve individuals began the treatment between birth and six months of age. In this group, the most common AE was anemia in eleven , hyperlipidemia in ten , with the most frequent being hypertriglyceridemia in seven , and elevated PLT in seven . One patient had low PLT .Fourteen patients continued or started treatment with sirolimus between six and twelve months of age. The most common side effects were anemia in eight (8/14. 57.14%), hyperlipidemia in eleven , and elevated PLT in five .Fourteen patients were on treatment between one and two years of age. Nine of them had anemia , thirteen had hyperlipidemia , and eleven had elevated PLT . Two patients with hyperlipidemia were on the ketogenic diet at that time.The data was available for ten patients two years and older. The most common AE was elevated PLT in seven , anemia in five , and hyperlipidemia in five .D-dimers and fibrinogen were rarely tested despite high PLT.Throughout the follow-up, infections were reported in 16.67% up to 50% of the patients, most common in individuals two years and older. On the other hand, neutropenia was reported in 10% up to 41.67% of the patients, and it was most prevalent in patients under six months of age. One individual required hospitalization several times. One patient’s parents noticed a pattern between higher serum sirolimus levels and the frequency of infections.Three cases of mouth ulcerations were reported, two in patients younger than six months  and one in a patient older than two years .The majority of adverse effects were grade 1 and 2 according to the CTCAE. All patients with anemia reported hemoglobin levels between 8.5 g/dL and the LLN for the respective age group.Elevated PLT in most participants was between 450 and 600/µL. One child reported a PLT count of 745/µL.None of the participants in the study had to be hospitalized due to hyperlipidemia. In most patients with elevated LDL-C, the levels were between 130 mg/dL and 280 mg/dL. Hypertriglyceridemia was reported to be between 150 mg/dL and 300 mg/dL. One patient had a triglyceride level of 750 mg/dL. The individual was on the ketogenic diet at that time; a modification of proportions led to the normalization of lipid levels. The levels exceeded 1000 mg/dL in none of the participants.One patient was hospitalized two times due to infections: varicella and not identified bacterial infection treated with antibiotics. Both times the patient presented with fever and epileptic seizures. No AEs grade 4 or 5 were reported.The prevalence of anemia, thrombocytosis, and hyperlipidemia was first calculated for all patients who had the factor tested. Then the frequency was estimated among the patients with normal hemoglobin and RBC, PLT, or serum lipids prior to the sirolimus administration.Anemia was reported in 40–70% of individuals during the follow-up. This AE was observed for the first time in the first three months of pharmacotherapy in more than half of the patients.Thrombocytosis was reported for the first time in almost 40% of the patients in the first three months after starting the treatment. After six months of pharmacotherapy, in this group, the frequency of elevated PLT rose to 70%; it remained elevated in over two-thirds of the patients until the end of the follow-up.In the first three months of treatment, lipid blood levels were elevated in more than 60% of the patients. This number dropped to 40% in the following three months. Regardless of the group size, hyperlipidemia remained present in 40–60% of individuals during the follow-up.Randomized clinical trials, EXIST-1, and EXIST-3 proved everolimus as effective and safe in young children ,21,22. OThe AEs were observed in all participants during the follow-up, yet they tended not to be severe and did not lead to pharmacotherapy discontinuation. Clinical trials on sirolimus used in older patients with TSC also found it to cause low-grade AEs, grade 1 or 2 ,16,28. CIn research studies on sirolimus, the target serum levels range between 4–20 ng/mL, typically 5–15 ng/mL ,26,28,29TSC2 mutation was found, which is known to correlate with more severe TSC symptoms [All the included participants started the treatment with mTOR inhibitors due to SEGA, CRs, or refractory seizures, which are the most common causes according to Krueger et al. and Saffari et al. ,17,28. Isymptoms . As a reHyperlipidemia, anemia, and thrombocytosis were the most frequently reported AEs during the follow-up, regardless of age or the duration of the treatment.The majority of participants reported mild or moderate hyperlipidemia, especially hypertriglyceridemia, during the follow-up. It is consistent with previous clinical trials, which found mTOR inhibitors lead to elevated, yet not life-threatening, serum lipid levels ,12,13,16Two patients with the highest blood lipid levels were on a ketogenic diet at that time, also known to cause hyperlipidemia . In thosAnemia is one of the most often reported sirolimus-induced AEs ,25,26. IAccording to the literature, mTOR inhibitor discontinuation due to anemia is rare ,26. DuriSirolimus use in human trials often leads to thrombocytopenia ,36,37,38Elevated PLT was frequent in all age groups, yet in none of the individuals did it lead to any further clinical consequences, nor did it require intervention. No literature on the sirolimus impact on PLT elevation was found. Interestingly, due to its immunosuppressive effect, sirolimus can be applied as rescue therapy for thrombocytopenia in primary antiphospholipid syndrome .Sirolimus belongs to a class of immunosuppressants and has proven its efficacy in solid organ transplantation. Its immunosuppressive effect may increase the susceptibility to infections, in particular pneumonitis or stomatitis ,42,43,44Stomatitis or mouth ulcerations are likely dose-dependent and often reported in older children, requiring short-term or complete treatment withdrawal ,16,18. DFew interventions were needed to minimalize the AEs caused by sirolimus use. In most cases, a dose adjustment or temporary discontinuation had to be undertaken. Sirolimus dosing alternations may cause imprecise serum level calculations and require further modifications to the dosing scheme.The typical drop-out rate in the studies on mTOR inhibitors in patients with TSC is 0–5% ,45,46. IThe study was a retrospective data collection, which may be subject to patient, parent, and physician recall bias and differences in the history-taking methodology. The relatively small number of participants assessed in this analysis can lead to conclusions that should not be applied to all patients.The analysis is the first to assess the safety of sirolimus in infants and young children under two years of age. In the EU and the US, mTOR inhibitors are currently recommended as a treatment for distinct TSC clinical manifestations. Clinical trials have proved everolimus’ efficacy and safety, yet the research on sirolimus is limited. The innovative character of this study is emphasized by the size of the cohort, which included all patients from two clinical centers specializing in child neurology in Poland.Adverse effects associated with sirolimus use in infants and young children with TSC are common yet not life- or health-threatening. The most frequent AEs in this group of patients are anemia, hyperlipidemia, and thrombocytosis. Mouth ulcerations or stomatitis are not common, and infections tend to be mild. Sirolimus appears to be safe and well tolerated in young patients with TSC.Further prospective studies are recommended to support the findings.


Gastric cancer (GC) is one of the most common malignant cancers worldwide. The development of potential antitumor agents is being investigated and stimulates more clinical trials. Overall survival (OS) is consistently considered the primary endpoint for clinical trials on treatment effect assessment. However, finding an appropriate endpoint more sensitive and easy for trials is vital. For adjuvant chemotherapy, current evidence has shown that disease-free survival (DFS) could be a surrogate endpoint for randomized controlled trials (RCTs) with GC, but evidence for neoadjuvant chemotherapy (NCT) or chemoradiotherapy (NCRT) is inadequate. This study was designed to evaluate the possibility that event-free survival (EFS) surrogates OS in RCTs of NCT/NCRT of gastric orss gastroesophageal (GC or GEJ) adenocarcinoma patients (ADK).2. We used Review Manage pooling HRs of OS and EFS at the trial level. I2 was used to demonstrate the heterogeneity of inclusions. Publication bias was summarized and illustrated through funnel plots. All analyses were on two sides with a setting statistical significance as p < 0.05.A literature search was conducted through databases including PubMed, the Cochrane Library, and Embase. References and articles from other sources were also included. A total of 8 RCTs with 2,837 patients were eventually analyzed. Hazard ratios (HRs) of OS and EFS were directly approached. The surrogacy of EFS was assessed through the correlation of determination R2 for OS was 21% and 51% for EFS, and a fixed-effect model was used. The pooled HR of OS was 0.83 , and that of EFS was 0.78 . The regression correlation coefficient between EFS and OS was 0.76 , and the coefficient of determination R2 = 0.826.Eight RCTs of 2,837 patients were analyzed at the trial level. The IA strong correlation was observed between OS and EFS at the trial level. EFS could be a surrogate endpoint for neoadjuvant RCTs of GC and GEJ adenocarcinoma. Further studies and evidence from individual data are expected. With more than a million new diagnoses per year, gastric cancer (GC) ranks as one of the most common malignant cancers worldwide. It remains the fourth main cause of cancer-related deaths . Among aSurgery is the main and curative strategy for resectable GC or GEJ ADK, but the effect of surgery alone is inadequate, especially for locally advanced GC . For latOverall survival (OS) is commonly accepted as the gold standard endpoint in these trials. This means that the time from randomization to death of any reason or the last follow-up data is censored , 7. It iStudies were searched through PubMed, the Cochrane Library, and Embase, and the references cited by the included studies, relative reviews, and meta-analyses were also searched. The whole search procedure lasted from January 10, 2021, to February 10, 2021. The literature search strategy in PubMed was (((((gastric cancer) OR (gastric adenocarcinoma)) OR (gastric neoplasms)) OR ) AND ((((neoadjuvant chemotherapy) OR (neoadjuvant therapy)) OR (neoadjuvant chemoradiotherapy))) OR (neoadjuvant drugs))) AND  OR ) OR ) OR ). The keywords were consistent in other databases. Unpublished studies were not searched, and a recently published study was taken into the final analysis. Articles meeting the following criteria were included: 1) enrolling patients with GC or GEJ ADK; 2) comparing the effect of NCT and surgery alone, or NCRT and NCT between two arms; 3) RCT; 4) OS and EFS or DFS or PFS being used as endpoints for study; and 5) hazard ratios (HRs) of endpoints for two comparative arms could be reached. Exclusion criteria consist of the following: 1) patients with other tumor histology or location except for GC or GEJ ADK; 2) no neoadjuvant therapy related or other topics not comparing treatment effect of neoadjuvant therapy; 3) non-English publication; 4) articles of systematic reviews, case reports, replies, and meta-analyses; 5) studies of non-RCTs; 6) no OS or EFS or DFS or PFS data available; 7) HR could not be accessed directly from the study; 8) abstracts, articles of trial design, or conference summary; or 9) no full-text available studies. All titles and abstracts of the searched results were first scanned, and then the full text was carefully browsed. The study selection was conducted separately by two researchers on our study team. When there were different opinions about study selection, the discussion was conducted, and agreement eventually was reached.Countries, number of patients, tumor location, tumor histology, therapy regimen, and other basic characteristics of the experimental and control arms were extracted. HRs published were directly reached and used at the trial level. The Cochrane risk-of-bias tool for RCTs was used to assess the quality of studies. The low, high, or unclear risk was determined for each included trial.2 was used to demonstrate the heterogeneity of studies. Funnel plots were used to evaluate publication bias, and no publication bias was set as symmetric funnel plots within the 95% region. The strength of the correlation between the HRs of OS and EFS was evaluated by linear regression using SPSS . All analyses were on two sides with a setting statistical significance as p < 0.05.We used Review Manager  to pool trial-level HRs of OS and EFS, and a 95% CI was applied. IIn total, 3,789 studies were initially identified, and 8 RCTs of 2,837 patients were eventually included for final analysis after screening titles, abstracts, and then full texts  12–14) –14 12–14The basic characteristics of the trials are shown in via the 5th Union for International Cancer Control (UICC), IB-IVA in one study via the 6th American Joint Committee on Cancer (AJCC), IIB-IV in one study via the 7th AJCC, and IB-IV in one study through the Japanese Classification of Gastric Carcinoma, 2nd English version. The clinical stage in the other three articles was unavailable  in a fixed-effect model. The I2 of EFS was 51%, and the pooled HR was 0.78 . Similar pooled HRs were reached for OS and EFS, with low–middle heterogeneity shown among the included studies . Extra validation of studies revealed that the predicted HROS from the observed HRDFS was near the observed HROS and within the 95% prediction CI. Therefore, the therapeutic effect on DFS could be a reasonable prediction of that on OS, and DFS can be used as the primary endpoint in future clinical trials of adjuvant chemotherapies. However, the exploration of the relative possibility for trials of NCT or NCRT was unfavorable. Two analyses evaluated whether DFS could do so on GC; however, totally opposite outcomes were reached. Petrelli et al. (2 = 0.89), while log(HROS) and log(HRDFS) at the trial level had poor links from the above 16 studies (R2 = 0.27). It concluded that OS could not be surrogated in terms of NCT or NCRT effect on GC, GEJ, and esophageal cancer, although the association between two endpoints of GC could be better than esophageal cancer (R2 = 0.78 vs. 0.2). Ronellenfitsch et al. . A strong association between two endpoints was observed at both the individual level (rs = 0.8943) and trial level . The provided surrogate threshold effect of DFS for OS was 0.79. Based on the above statistical results, researchers concluded that DFS could be a suitable surrogate endpoint for OS in NCT or NCRT trials. However, the tumor histology and location of the study population enrolled were not limited to GC or GEJ ADK. Squamous cell carcinoma, mixed adenosquamous carcinoma, or ADK from the thoracic esophagus was not excluded. The selection process of the endpoint should be careful about hypotheses to be proven . Correlation analysis revealed a good association between the two endpoints (R2 = 0.826). The results and conclusions were consistent with those of Ronellenfitsch et al. (In terms of our exploration, although the study by Shapiro et al. included enrolled ADK and squamous cell carcinoma patients, HRs of the ADK subgroup were available and included in our final analysis. Two studies with a small part of the population diagnosed with ADK in the lower third esophagus were included considering lower esophageal ADK close to GC ADK in histology. Most esophageal cancers are tumors of squamous cell carcinoma. ADK of the lower esophagus is quite similar to even distal GC, showing that esophageal ADK relies more on GC than on esophageal cancers. Similarly, robust molecules also strongly demonstrated that GEJ cancers are closer to GC than esophageal cancers, regardless of heterogeneity . Our stuh et al. . Accordih et al. . Thus, dOur study has limitations. First, two included studies enrolled patients with lower third esophageal ADK, which may introduce bias. Then, most included trials were conducted in European countries and parts of Korea and Japan. Evidence is deficient for other countries and regions. The above limitations restrict the wide application of conclusions. Additionally, due to potential differences in the definition of EES/PFS/DFS and assessment criteria for progression and recurrence in clinical trials, the application of conclusions in actual trials should be more cautious. Our study is the first to offer proof of the probability of EFS as a surrogate of OS in NCT or NCRT clinical trials of GC or GEJ ADK. Further analyses of individual data and trials from other regions are expected to provide more evidence.In summary, a strong correlation was observed between OS and EFS at the trial level. EFS could be a surrogate endpoint for neoadjuvant RCTs of GC and GEJ adenocarcinoma. Further studies and evidence from individual data are expected.The original contributions presented in the study are included in the article. Further inquiries can be directed to the corresponding author.HL: conceptualization, investigation, methodology, data curation, formal analysis, and writing—original draft. YW, CQ, and TX: data curation, formal analysis, and supervision. ZP, JL, and LS: conceptualization, investigation, methodology, and supervision. XZ: conceptualization, investigation, methodology, data curation, supervision, and project administration. All authors contributed to and approved the final revision submitted.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The reviewer YW declared a shared affiliation with the authors to the handling editor at the time of the review.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Rheumatoid arthritis (RA) and Parkinson's disease (PD) are two common chronic diseases worldwide, and any potential link between the two would significantly impact public health practice. Considering the current inconsistent evidence, we conducted a meta-analysis and systematic review to examine the risk of PD in patients with RA.Two investigators (DL and XH) conducted a comprehensive search of PubMed, Embase, and Web of Science using medical subject headings terms combined with free words to identify relevant papers published from inception through December 31, 2021. All studies that explored the relationship between RA and PD were included for quantitative analysis and qualitative review. Random- and fixed-effects models were used to pool the risk ratios (RRs) of PD in patients with RA. The Newcastle-Ottawa scale was used to assess the quality of included studies. This study followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA 2020) guideline.P = 0.034). No apparent effects of gender, age, region, follow-up time, or study design on PD risk were observed. Sensitivity analysis showed that pooled results were relatively stable, and no publication bias was detected. The Mendelian randomization study indicated a significant inverse association between RA and PD  and that each one standard deviation increase in the risk of RA was significantly associated with a lower risk of PD. Of note, the current study is limited by the relatively small number of included studies and unmeasured confounding factors, especially for RA-related anti-inflammatory agents.Four population-based studies involving 353,246 patients and one Mendelian randomized study were included in our study. The pooled result showed a significantly reduced risk of PD in patients with RA than in the general population (RR = 0.74, 95% CI: 0.56-0.98, This study supports that people with RA had a lower PD risk than those without RA. Further studies are needed to explore the underlying molecular mechanisms of the interaction between the two diseases. Parkinson's disease (PD) is a chronic progressive motor dysfunction characterized by static tremor, rigidity, and bradykinesia –4. As thRheumatoid arthritis (RA) is an inflammatory autoimmune disease. Similar to PD, RA is a common chronic disease that affects approximately 1% of people worldwide . RA can Both RA and PD are highly prevalent diseases worldwide, and any correlation between the two would have important implications for public health practice and potentially provide new targets and ideas for the management of PD. Considering the current conflicting evidence and the lack of a study comprehensively summarizing current data, we conducted this meta-analysis and systematic review to elucidate the impact of RA on PD risk and provide higher quality evidence.This study was reported following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA 2020) guidelines . The proTwo investigators (DL and XH) independently searched PubMed, Embase, and Web of Science databases for relevant literature published from inception through December 31, 2021. The search strategy consisted of subject terms  and synonyms  for RA and PD; the medical subject headings terms were linked by “AND” and the synonyms by “OR.” No language restrictions or filters were used. The specific search strategy for each database is available in the Studies that fulfilled all of the following criteria would be considered for inclusion in meta-analysis: (1) RA and PD were clearly defined or met diagnostic criteria, such as the readable codes or standard clinical diagnostic criteria; (2) correlations between RA and risk of PD were explored; (3) studies reported relative risk estimates and corresponding 95% confidence intervals (CIs), such as risk ratio (RR), hazard ratio (HR), odds ratio (OR), and standardized incidence ratio (SIR), or provided sufficient data to calculate these ratios; and (4) the study design was a longitudinal observational study, including case-control and cohort studies, whether prospective or retrospective.Studies meeting any of the following criteria would be excluded: (1) studies that did not generate original data, such as reviews, meta-analyses, commentaries; (2) non-human studies, such as cellular or animal model experiments; (3) case reports or case series of fewer than 10 patients; (4) overlapping study participants; for multiple studies from the same population, the study with the largest sample size and the most complete outcomes would be included.Studies that did not fully meet the above criteria but explored the association between RA and risk of PD would be qualitatively analyzed as part of the systematic review.Two investigators (DL and XH) independently screened the titles and abstracts of the initially retrieved records according to the eligibility criteria. Potentially eligible papers were read in full, and reasons for exclusion would be recorded.For included studies, two researchers independently (DL and TC) extracted the following information: first author, publication year, study period, age, gender, population source, sample size, study design, effect size, methods of PD and RA identification, adjusted/matched confounders, and follow-up time.The Newcastle-Ottawa scale (NOS) was used to assess the quality of included studies, and three main aspects were involved:  selectioQ-test and Higgins' I2 statistics; pooled results of the fixed-effects model were reported when I2 <50% and P > 0.1, otherwise, heterogeneity between the included studies was considered significant and results of the random-effects model were reported. We performed sensitivity analyses by excluding one study at a time and comparing the results of the fixed- and random-effects models. Publication bias was assessed by observing the symmetry of the funnel plot and performing Egger's and Begg's tests. We performed subgroup analyses according to age, sex, follow-up, region, and study design to explore the effects of these variables on the pooled results. Data for this study were analyzed using STATA/MP 16.0 . All P-values were two-tailed, and the statistical significance threshold was 0.05.We used fixed- and random-effects models to calculate pooled risk estimates of PD in RA patients compared to the non-RA population . For stuThe predeveloped database search strategies and browsing the reference lists of relevant papers yielded 2,295 records. After removing 875 duplicates and excluding 1,382 apparently irrelevant papers, 38 papers were read in full. Among them, 15 did not report results of interest, 9 were case reports or case series, 5 were cellular or animal model studies, and 4 had duplicate data. A total of five studies were finally included in this review .Of the eligible studies, four were population-based studies, and one was the Mendelian randomization study , 25, 26.The NOS scores of the included studies ranged from 7 to 9, indicating a high level of overall quality. These studies had clear exposure and outcome definitions, appropriate adjustment for confounders, and sufficiently long follow-up .2 = 87.9%, P < 0.001), so the random-effects model was used . Mendelian randomization analysis was performed in two samples and showed that each one standard deviation increase in the risk of RA was significantly associated with a lower risk of PD  .We performed stratified analyses according to sex, age (<65 vs. ≥65 years old), duration of follow-up (<5 vs. ≥5 years), region, and study design.No significant differences in effect sizes were observed between the above subgroups, and RA was associated with a reduced risk of PD in all conditions. However, the pooled results for age subgroups , European region , and cohort studies  were not statistically significant; this may be due to the small number and substantial heterogeneity of the included studies resulting in conservative confidence intervals .We performed sensitivity analyses to test the robustness of the combined results by two methods. First, the impact of individual studies on the overall pooled result was assessed by excluding one study at a time. Although the statistical significance of the pooled result disappeared after excluding certain studies, patients with RA still tended to have a lower risk of PD . In addiP-values for both Begg's and Egger's tests were >0.05 , indicating a low likelihood of potential publication bias.The funnel plot was not performed because of the small number of included studies at the end. The To our knowledge, this is the first study to systematically assess the association between RA and PD risk. The pooled result from four population-based studies showed a 26% reduction in the risk of PD among patients with RA compared to the general population. Despite the small number of included studies, the results from high-quality studies and more than 300,000 participants can be considered a stringent estimate. A Mendelian randomization study provided further strong evidence for the inverse association between RA and risk of PD. Overall, the inverse association between RA and PD risk is relatively stable in populations with different characteristics.I2 = 0%, P = 0.737), and the pooled results remained statistically significant . The cohort study by Li et al. used a database covering the Swedish Hospital Discharge Register from 1964 to 2007, and standardized incidence ratios were calculated by the ratio of observed to expected number of cases; they found no correlation between RA and risk of PD   . Interes . Intere89-1.26) .RA often requires treatment with anti-inflammatory drugs that may affect the correlation between RA and risk of PD. Drugs commonly used in RA patients are non-steroidal anti-inflammatory drugs (NSAIDs) and immunosuppressive drugs including traditional disease-modifying anti-rheumatic drugs (DMARDs)  and biologic DMARDs  , 28. SomAlthough our meta-analysis and Mendelian randomization study demonstrated that RA patients have a lower risk of PD, the exact mechanism of the reduced risk is unclear. In fact, the current results contradict the hypothesis on the pathological mechanisms of RA and PD. Inflammation is thought to play a critical role in RA and PD, and long-term chronic inflammation and excessive production of inflammatory mediators such as interleukin (IL)-1, IL-6, TNF-α, and chemokines may trigger inflammatory processes and activate microglia in the brain, leading to degeneration of dopaminergic neurons and an increased risk of PD , 44–46. First, the association between RA and PD may be influenced by lifestyle factors. A classic example is smoking; smoking is an important risk factor for RA but is associated with a reduced risk of PD , 47, 48.The strength of our study is that the pooled results from large sample size studies could provide a stringent estimate. Although there may be multiple theoretical mechanisms for the reduced risk of PD, the implications of anti-inflammatory drugs associated with RA are of interest. Due to limited data, subgroup analyses stratified by medications were not performed, but the study by Sung et al. provided evidence that biologic DMARDs use may reduce the risk of PD . PreviouThere are several limitations to this study that should be noted. The main limitation is the relatively small number of included studies, but the large sample and high-quality characteristics of these studies partially compensate for this and make the conclusions relatively stable. All included studies were derived from medical record databases and used readable codes to identify cases, whereas diagnostic criteria for PD and IBS may be inconsistent and subject to misdiagnosis and underdiagnosis. In addition, as discussed above, the results of observational studies may be biased by unmeasured and unconsidered variables.According to the meta-analysis of population-based studies and the Mendelian randomization study, patients with RA had a lower risk of developing PD than those without RA. The current results may be partially explained by anti-inflammatory drugs related to RA. Further studies are needed to explore the underlying molecular mechanisms of the interaction between the two diseases, which may help provide new insights into prevention and therapeutic targets for PD.The original contributions presented in the study are included in the article/DL contributed to the conception and design of the study. DL and XH performed the database search and study selection. DL and TC extracted data, conducted data analysis, and wrote the manuscript. XH checked and verified all data. All authors contributed to manuscript revision, read, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Mean platelet volume (MPV) is an indicator of platelet activation and has been proposed as a diagnostic marker for several kinds of cancers. We investigated the value of MPV as a diagnostic marker for prostate cancer (PCa) and examined whether MPV in combination with prostate-specific antigen (PSA) could increase the sensitivity or specificity of PSA for PCa diagnosis. For this study, 107 pathologically confirmed PCa and 177 non-PCa patients who underwent prostate biopsy were retrospectively studied. Clinical data and pre-biopsy hematological parameters were collected. The above parameters were compared between PCa and non-PCa patients. The correlation between MPV and clinical characteristics was analyzed. Receiver operating characteristic (ROC) analysis was used to explore the diagnostic value of MPV for PCa. Among all parameters analyzed, the difference was only found in MPV, platelet distribution width (PDW), and PSA between PCa and non-PCa patients. MPV was significantly decreased and PDW increased in PCa than that of non-PCa among men. ROC analysis identified MPV ≤ 9.05 fl as a cut-off value for potential PCa with area under the ROC curve (AUC) = 0.783, 95% CI = 0.733–0.833, sensitivity = 0.746, and specificity = 0.708. AUC and the sensitivity of MPV were comparable with total PSA (TPSA) or free PSA (FPSA). However, the specificity of MPV was larger than that of TPSA (0.461) or FPSA (0.561). Furthermore, MPV combined with TPSA or FPSA further enhanced the specificity of TPSA (0.844) or FPSA (0.927), but PDW did not. These findings suggested that MPV could have a predictive value for the diagnosis of PCa. MPV in combination with TPSA or FPSA could enhance the specificity of PSA and may reduce the rate of unnecessary biopsy for patients with high levels of PSA. Prostate cancer (PCa) is the most diagnosed cancer and the second leading cause of cancer-related death for men in the United States , 2. The Recent investigations showed that platelets play critical roles in mediating the growth, dissemination, and angiogenesis of cancer cells . ActivatSeveral studies also revealed the association of MPV and PDW with prostate cancer –17. It in = 160) or normal (n = 10) by histology. For the 160 patients with BPH, their lower urinary tract symptom (LUTS) is not severe enough for surgical treatment, hence included in this study. Individuals with long-term administration of drugs that could affect blood examination  were excluded. Patients with hematological disorders, hypertension, or diabetes mellitus were also excluded.This study was approved by the Ethics Committee of the Second Hospital of Shandong University. We retrospectively investigated 284 patients admitted to the Second Hospital of Shandong University between July 2015 and 2020 for prostate biopsy because of high PSA levels (>4 ng/ml). Among them, 107 had pathological confirmed PCa, and 177 had pathological non-PCa who was proved to be benign prostatic hyperplasia , medical history, and physical examination. Laboratory data included total PSA (TPSA), free PSA (FPSA), ratio of TPSA to FPSA, and complete blood count analysis, which examined the number of platelets, white blood cell (WBC), neutrophil, lymphocyte, monocyte, eosinophils, basophils, platelet, MPV, and PDW. For pathologically confirmed patients with PCa, the information of Gleason scores and tumor, nodes, and metastases (TNM) stage were also recorded. Laboratory examinations were done in the Second Hospital of Shandong University with a biochemical analyzer which is hisemikon blood cell instrument XN9000 . The serum PSA was measured using an automatic electrochemistry luminescence immunoassay system (CLEIA)  . CompletSD. The basic characteristics of PCa and non-PCa patients were compared by t-test. The correlation between MPV and the clinical characteristics was evaluated using the Spearman correlation test. Receiver operating characteristic (ROC) analysis was used to explore the diagnostic values of MPV, PSA, and their combination. All analysis was conducted by SPSS . The P-value < 0.05 was regarded as a statistical difference.Data were calculated as mean ± P = 0.001). On the other hand, PDW was significantly increased in PCa (15.43 ± 1.87%) than non-PCa among men . The platelet count among men with PCa (232.22 ± 67.59 × 109/L) tended to be more than that of the non-PCa study group (217.97 ± 69.81 × 109/L), but there was no statistical difference (P = 0.09). Both TPSA and FPSA were larger in PCa than non-PCa among men  or FPSA  . Compared with each alone, the AUC did not increase among MPV*TPSA, PDW*TPSA, MPV*FPSA, PDW*FPSA, or the three combinations. However, compared to each alone, the three parameters combination significantly increased the sensitivity but reduced the specificity  and 0.561  to 0.844 (MPV*TPSA) and 0.927 (MPV*FPSA).The combination of several biomarkers might lead to enhanced sensitivities and specificities. Next, we examined the ROCs of MPV or PDW in combination with TPSA (MPVDW*FPSA)  or the tcificity . To noteThe level of preoperative MPV has been found to be associated with many kinds of cancers. Our study showed that MPV in patients with PCa was significantly less than that of non-PCa among men. ROC analysis showed that MPV has the predictive value for PCa occurrence. No relationship was found between MPV level and TNM stage, Gleason score, and age, which indicated that MPV could be used in the diagnosis of patients with PCa independently of their cancer stage. ROC analysis further showed that MPV alone or in combination with PSA could enhance the specificity of PSA for PCa diagnosis. Thus, MPV should be considered as a valuable new biomarker for the correct identification of PCa among patients diagnosed with this disease.Mean platelet volume is correlated with platelet function and may be a more sensitive index than platelet number as a marker of clinical interest in various disorders . Our stuWe did not find the correlation of MPV or PDW level with PSA level in PCa patients. A negative correlation of MPV with PSA was identified in one study  showing Prostate-specific antigen was extensively used as a marker for screening and diagnosis of PCa, however, PSA may be elevated because of several reasons, including prostatitis or BPH in addition to prostate cancer. Thus, as the diagnostic marker of PCa, the sensitivity and specificity of PSA are low, and over-diagnosis and unnecessary biopsy often occur . ConsistOur study is retrospectively conducted, and the accuracy of our results requires validation by future prospective studies. The second limitation is that the subject population from a single medical center is relatively small, so a larger population of patients from multi-medical centers is needed. Another limitation of our study is that all our patients are hospitalized patients for prostate biopsy who have a PSA level larger than 4 ng/ml. Patients with PSA less than 4 ng/ml were missed for analysis, thus, MPV may not be used as a screening test for PCa.In summary, the present study revealed that MPV in combination with PSA could increase the specificity for differentiating patients with PCa from non-Pca ones. Because the measurement of MPV is easy, routine, and inexpensive, clinicians should check MPV in suspected patients with PCa. Men with a low MPV level and an increase of serum PSA should strongly be considered for biopsy. However, our results need to be validated with prospective and randomized investigations.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Second Hospital of Shandong University. The patients/participants provided their written informed consent to participate in this study.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.This work was supported by the National Nature Science Foundation of China (No. 82070783), and the National Science Fund of Shandong Province (ZR2021MH283).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.