Background and Objectives: Lipofilling is a commonly performed procedure worldwide for breast augmentation and correction of breast contour deformities. In breast reconstruction, fat grafting has been used as a single reconstructive technique, as well as in combination with other procedures. The aim of the present study is to systematically review available studies in the literature describing the combination of implant-based breast reconstruction and fat grafting, focusing on safety, complications rate, surgical sessions needed to reach a satisfying reconstruction, and patient-reported outcomes. Materials and Methods: We adhered to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) throughout the whole review protocol. A systematic review of the literature up to April 2022 was performed using Medline, Embase, and Cochrane Library databases. Only studies dealing with implant-based breast reconstruction combined with fat grafting were included. Results: We screened 292 articles by title and abstract. Only 48 articles were assessed for full-text eligibility, and among those, 12 studies were eventually selected. We included a total of 753 breast reconstructions in 585 patients undergoing mastectomy or demolitive breast surgeries other than mastectomy  due to breast cancer or genetic predisposition to breast cancer. Overall, the number of complications was 60 (7.9%). The mean volume of fat grafting per breast per session ranged from 59 to 313 mL. The mean number of lipofilling sessions per breast ranged from 1.3 to 3.2. Conclusions: Hybrid breast reconstruction shows similar short-term complications to standard implant-based reconstruction but with the potential to significantly decrease the risk of long-term complications. Moreover, patient satisfaction was achieved with a reasonably low number of lipofilling sessions (1.7 on average). Breast cancer is the most common malignancy in women, with a global incidence of 2,088,849 new cases and 626,679 related deaths reported in 2018 [The percentage of U.S. women who opted to undergo breast reconstruction after breast cancer was estimated at 43.3% based on data from NSQIP 2014 [Among different types of breast reconstruction, fat grafting has been used not only as a single reconstructive technique, but also in combination with other procedures. Lipofilling might not only be useful in improving breast contour after implant-based reconstruction, but also in increasing mastectomy flap thickness prior to or associated with immediate breast reconstruction. In some cases, adjuvant lipofilling is performed after tissue expansion or at the time of expander/implant substitution, but also in autologous breast reconstruction .Although initial skepticism has surrounded the oncological safety of fat grafting in breast reconstruction, studies found strong evidence demonstrating no increase in breast cancer recurrence or mortality ,5,6,7,8.Autologous fat transplant (AFT) has been associated with increased skin trophism and vascularization, reduced post-operative pain, and improved cosmetic results ,10,11. HLipofilling is a commonly performed procedure worldwide for breast augmentation and correction of breast contour deformities ; howeverThe aim of the present study is to systematically review available studies in the scientific literature describing the combination of implant-based breast reconstruction and fat grafting, focusing on safety, complications rate, number of sessions needed to reach a satisfying result (both from an aesthetic and volumetric point of view), and patient-reported outcomes.The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guideline was followed throughout the design, implementation, analysis, and reporting of this systematic review . Two revThe research produced 8924 articles, updated to April 2022. The flow chart of the study selection is outlined in Initially, 292 articles were screened by title and abstract. However, only 48 articles were assessed for full-text eligibility, and among those, 12 studies were eventually included ,22,23,24A total of 753 breast reconstructions in 585 patients undergoing mastectomy or demolitive breast surgeries other than mastectomy  due to breast cancer or genetic predisposition to breast cancer  were included. The mean volume of fat grafting per breast per session ranged from 59 to 313 mL. The mean number of lipofilling sessions per breast ranged from 1.3 to 3.2 . The mean follow-up was 17.8 months (range 6–48.5 months). Correlations between volume of fat grafted and complications, as well as correlations of type of complications with injected volume and with injection timing (with respect to radiotherapy) could not be statistically analyzed due to small sample sizes, nor could nonstandardized treatments with variable patient characteristics, timing, or reconstruction technique. Among the different techniques of manipulation of the harvested adipose tissue, Coleman’s technique was the most common, being used in 8 out of 12 studies; and, in all studies, adipose tissue injection was performed with 1 to 2.5 mL Luer-lock syringes and a blunt tip one-hole concave Cannula (Coleman Infiltration Cannula).n 14, 1.9%), followed by infection requiring implant removal , seroma , dehiscence , mastectomy skin necrosis , 0.8%), implant malposition , minor pneumothorax , and implant rupture . The capsular contracture rate was not clearly stated by some studies; for this reason, it was separately analyzed from other complications. It was mentioned in 6 out of 12 studies included for a total of 17 cases out of 337 reconstructed breasts (5%).Overall complications were 60 (7.9%), of which 19 (2.5%) were classified as major complications  and 41 (5.4%) were classified as minor complications (not requiring reintervention). Among total complications, the most common was cystic fat necrosis  ,19,20,21Although radiotherapy has been proposed as the most important factor associated with the number of lipofilling sessions needed to complete a breast reconstruction and with the rate of complications , on the Gronovich et al.  and HammIn the reviewed studies, high patient satisfaction was achieved with a reasonably low number of lipofilling sessions, averaging 1.7 sessions (range from 1.3 to 3.2 in different studies) with studies using the Likert scale . In a study by Cigna E et al. using the VAS scale (range 1–10), the average patient satisfaction went up from 5.2 to 7.9 and surgeon satisfaction went up from 4.9 to 7.7. Studies using BREAST-Q reconstruction questionnaire reported “high” to “very-high” levels of satisfaction with final outcome . These eA further role of fat grafting may be related to the ability to expand indications for prepectoral breast reconstruction. The prepectoral approach in breast reconstruction is often avoided due to the increased risk for mastectomy flap necrosis and contour deformities . HoweverNonetheless, the combined reconstructive approach has some relevant limitations to be considered. First, several surgical procedures are often necessary to achieve final results, increasing the costs of the reconstruction. Second, it delays the psychological acceptance of the reconstructed breast .Despite the promising results available in the literature and widespread use of hybrid reconstruction techniques in daily practice, our study is subjected to some limitations. First, we have to highlight the lack of comparable interstudy data  and heterogeneity in patient-reported outcomes (e.g., only two of the included studies ,22 reporTo the best of our knowledge, this is the first systematic review of the literature on the use of implant-based reconstruction associated with fat grafting. Despite this limitation, our study demonstrates efficacy of the use of fat grafting in addition to prosthetic breast reconstruction. However, we confirmed the previously reported lack of evidence in patient-reported quality of life, and we point to the need for a high-quality randomized prospective trial comparing hybrid and standard reconstruction ,56.Hybrid breast reconstruction appears to be a minimally invasive, safe, easy-to-perform, and effective choice in breast reconstruction. Fat grafting may represent a valuable tool in plastic surgeons’ hands in reducing the risk of long-term complications, such as capsular contracture. It showed the capability to restore the thickness and trophicity of the mastectomy flap, allowing implant-based reconstruction even in the presence of poor skin quality and previously irradiated breasts.However, the data we obtained from the included studies are tempered by the low quality of the available literature. Future larger series and randomized controlled trials are needed to confirm the results of our study and identify which patients would benefit the most from combined breast reconstruction.


ClinicalTrials.gov” web platform in the last 10 years, to list and describe their characteristics, and to identify possible gaps to be filled in the future research.Clinical trial registration has become a valuable tool that can be used to track the status and nature of trials conducted on a specific topic. This approach has been applied to many areas of research, but less is known about the characteristics and trends over time of clinical trials focused on diet and health. The aim of this study was to analyze diet-related clinical trials registered on the National Institute of Health “ClinicalTrials.gov database. Intervention studies registered from January 2010 to December 2020, conducted on adults, with a follow-up of ≥2 weeks, evaluating the impact of different diets on all outcomes except those assessed with scales or questionnaires were considered.A search was performed on the n = 381 trials), followed by those based on a modification of macronutrients (n = 288) and time-restricted feeding and intermittent fasting diets (n = 140). The main measured outcomes included anthropometric parameters and body composition (57.8%), glycemic control parameters (49.7%), lipid parameters (40.1%), inflammatory markers (29.1%), and blood pressure and/or heart rate (24.5%). A growing body of studies also focused on microbiota and host metabolism (17.8%). Most studies had a duration of less than 12 weeks (~60%), and more than 90% of studies enrolled volunteers with overweight/obesity or other diseases. Regarding aging, only 21 studies focused only on older adults.At the end of the selection process, a total of 1,016 registered clinical trials were identified and included in the analysis. The most investigated dietary approaches were balanced diets (The number of studies investigating the relationship between diet and health has increased over the years. Despite the growing interest in the topic, there are some gaps, such as the limited duration of most trials, the underrepresentation of some population groups, and the limited number of studies for some diets that, although popular in the population, lack robust scientific evidence. The paramount importance of diet on human health is supported by a large body of scientific evidence. The Global Burden of Disease highlighted that, in 2017, 11 million deaths and 255 million disability-adjusted life years (DALYs) were attributable to dietary risk factors  577,014. A large number of epidemiological and clinical evidence for health promotion is available for “traditional” diets, such as the Mediterranean diet or for low-carbohydrate diets  at the National Institutes of Health (NIH) of the United States  Population: adults (aged 18–64 years) and/or older adults (>65 years); 2) study type: interventional studies with a follow-up of ≥2 weeks; 3) type of intervention: diets as a whole ; 4) outcome: all outcomes except those assessed with scales or questionnaires; 5) status: all except “withdrawn”; and 6) registration date: all the trials registered from 1 January 2010 to 31 December 2020. The search was performed on 13 April 2021.To identify studies focused on diet, a search was performed on the The study selection and the evaluation of the eligibility of the clinical trials were performed by two independent reviewers (D.M. and M.D.). Any discrepancy between reviewers was solved through consultation with a third independent author (G.P.) to achieve a consensus.ClinicalTrial.gov” in “All Available Columns” and “Tab-separated values”: URL of the registered trial, title, year of first publication in the “ClinicalTrials.gov” register, study location, gender, age group, health status of study participants, number of enrolled subjects, type of diet, outcomes of interest, duration of the intervention, and funding sources. Data extraction from the registry was performed by two reviewers (MD and GP). A third author (DA) checked the extracted information to ensure the accuracy of the retrieved data.The following data were extracted from an XML dataset downloaded from the US National Library database “ClinicalTrials.gov” was extracted to explore temporal trends. The health status of enrolled participants was assessed by considering the field Conditions. Trials stating that included participants were “healthy” were categorized as studies conducted on a healthy population. The other clinical conditions were analyzed by performing frequency statistics and were manually classified as follows: 1) overweight/obesity, 2) diabetes, 3) metabolic syndrome, 4) cardiovascular disease/risk, 5) cancer , 6) gastrointestinal condition, 7) liver disease, 8) kidney disease, 9) neurodegenerative disease, 10) polycystic ovary syndrome, and 11) other .The year of first publication on “Intervention field. As a first step, the 10 most used diets in clinical trials, defined as “top 10 diets,” were analyzed. The number of studies  in which these diets were evaluated, the relative value per year, and the number and type of outcomes considered were reported. Then, to facilitate the organization and presentation of data, the diets were manually grouped into eight categories, termed “dietary approaches,” that shared common characteristics: 1) balanced diets, 2) diets based on the modification of macronutrients, 3) diets based on food timing, 4) diets that excluded specific foods or food groups, 5) diets based on the modification of specific nutrients/nonnutrients, 6) diets with meal replacement, 7) diets fortified with specific foods, and 8) other. The type of diet being studied was extracted from the Primary and Secondary outcomes fields. Also in this case, the following categorization of outcomes was manually performed: 1) anthropometric parameters and body composition, 2) hemochrome parameters, 3) lipid parameters, 4) glycemic control parameters, 5) markers of liver function, 6) markers of kidney function, 7) markers of thyroid function, 8) minerals and vitamins, 9) hormonal parameters, 10) blood pressure and/or heart rate, 11) inflammatory markers, 12) oxidative stress parameters, 13) cancer biomarkers, 14) microbiota and metabolites, 15) genetic markers, and 16) other . Also in this case, trials assessing multiple outcomes were labeled with as many categories as were relevant, and consequently, the percentages of trials by outcome sum to greater than 100%.Regarding outcomes, data were extracted from Age field and classified following the categorization in “ClinicalTrials.gov”: adults only (18–64 years), older adults only (≥65 years), and both adults and older adults (>18 years). As to the number of enrolled volunteers, data were extracted from the Enrollment field and categorized into 4 groups, namely, 1–50 participants, 51–100 participants, 101–500 participants, and >500 participants. The duration of the intervention was extracted from the Duration field and was categorized into ≤ 12 weeks, 13–24 weeks, 25–52 weeks, and >52 weeks. The funding source was categorized into Academic Medical Centers/Hospitals/United States Government/Others (AMC/Hosp/US Govt/Others), industry, and both. The category “US Govt” was generated from the “ClinicalTrials.gov” database categories NIH and US Fed, while “Other” was composed of charities and foundations. The category “Industry” was taken directly from the categorization in ClinicalTrials.gov.The age of the participants was taken from the Descriptive analyses were performed using the statistical package IBM Statistical Package for Social Science for Macintosh version 27.0 . In particular, frequency statistics were performed, and data were expressed as numbers and percentages of the total. Given the lack of specific reporting guidelines developed for this type of study registry analysis, we adhered to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guidelines for cross-sectional studies .ClinicalTrials.gov database by using the search strategy  were conducted on women, 5.1% (n = 52) on men, and 79.5% (n = 808) on both sexes. In terms of age groups, 70.2% of the trials (n = 713) included both adults (18–65 years) and older adults (>65 years), while almost one-third were conducted in adults only , and 2% (n=29) were conducted in older adults only. Almost half of the trials enrolled ≤ 50 participants , while 27.6% (n = 280) and 24.8% (n = 252) of the studies included 51–100 and 101–500 participants, respectively. Only 2.4% (n = 24) of the trials enrolled >500 participants. Regarding the duration of the intervention, 54.8% of the trials (n = 557) had a follow-up of ≤ 12 weeks, in 23.8% (n = 242) ranged from 13 to 24 weeks, and in 13.2% (n = 134) ranged from 25 to 52 weeks, while 8.1% (n = 82) had a duration of more than 1 year. The study duration was not specified in 1 trial (0.1%).Overall, 15.4% of the trials (n = 69), while 91.5% of the trials enrolled volunteers with diseases , diabetes (n = 161), cardiovascular disease/risk (n = 100), cancer (n = 70), metabolic syndrome (n = 53), gastrointestinal conditions (n = 50), liver disease (n = 44), kidney disease (n = 32), neurodegenerative disease (n = 24), polycystic ovary syndrome (n = 15), and other (n = 104). In 17 trials (1.7%), participants' health condition was not specified. The analysis of funding sources showed that 91.3% of trials (n = 928) were funded by academic medical centers, hospitals, government agencies, or others, 1.2% (n = 12) by industry, and 7.5% (n = 76) by both.With regard to the health status of the enrolled participants, they were defined as “healthy” in the 6.8% of the trials  in 2019 and a subsequent slight decrease in 2020 , followed by Italy , Canada , the United Kingdom , China , Denmark , Germany , Brazil , Spain , Sweden , Israel , Mexico , Iran , Norway , and the Netherlands . The other countries shown in the map reported less than 10 eligible trials registered on “ClinicalTrials.gov” over the past decade.By considering the year of first publication in the “ in 2020 . Conside in 2020 , more thAs to the examined outcomes , anthropClinicalTrials.gov” between 2010 and 2020 are reported in The top 10 diets used in clinical trials registered on “By considering the dietary approach used in the registered trials, balanced diets accounted for the largest proportion of trials (37.5%), followed by diets based on the modification of macronutrients (28.3%), diets based on food timing (13.8%), diets that excluded specific foods or food groups (10.9%), diets based on the modification of specific nutrients/non-nutrient intake (7.1%), diets with meal replacement (4.4%), diets added with specific foods (4.2%), and other diets (2.9%) .Comparing the different dietary approaches , studiesRegistration of trials over time according to dietary approaches is shown in ad-hoc clinical trials focused on different tasks of health outcomes and to the fact that in the last years, registration has been recommended also for dietary intervention studies. A major focus was on anthropometric measurements, which have been the most considered for all the diets, except the DASH one. This is likely due to the well-recognized role that an adequate dietary regimen has on the maintenance of an adequate body weight and, in turn, on the modulation of the glycemic and lipid profiles, as well as in the prevention of inflammatory status. Other than these “classic” clinical chemistry measurements, there is a growing interest in the study of the diet-derived gut microbiota modulation, both general and species-specific, which is more considered due to the increased awareness of its potential association with many health factors  . Among tFew studies  have been conducted on diets that excluded specific foods or food groups, such as gluten-free or vegetarian/vegan diets. The former ones are specifically designed for people with coeliac disease or gluten-sensitivity conditions, although they are often used with the aim of losing weight in a very tight time, likely due to the misperception that gluten may be responsible for weight gain and fastidious gut discomforts, such as bloating or soreness . RegardiBesides focusing on the types of diets considered in the registered trials, it is noteworthy to analyze the main characteristics of such studies. Among them, duration is surely one of the most complicated aspects of the study design to be considered. From one side, the length of the study should consider the necessary period of time to expect a change in the considered outcomes, the fluctuation of such values along the time, etc. . HoweverRegarding health status, on average only ~7% of the total registered trials enrolled apparently healthy individuals while, as expected, most of the studies enrolled overweight/obese subjects, followed by the ones affected by diabetes or other cardiovascular diseases. This is not surprising considering that it is difficult and, in many cases, not clinically relevant to the modulation of health outcomes in healthy people who have already health-related markers in physiological ranges. On the contrary, altered values of health or disease outcomes, i.e., body weight, glucose-, lipid- or inflammation-related markers in population with or at risk of chronic diseases, may be more sensitive to a dietary recommendation or to change of dietary regimens , such asMost of the registered trials have been performed by enrolling both men and women, which reflects the gold standard way to evaluate the health effects of foods/diet without gender/sex bias. However, it also emerged that there were a higher number of trials, up to 20% in the balanced diet group, conducted on women than those on men. These data may be discussed in different ways, mainly taking into account the main outcomes and the complexity of the two-body systems. In fact, an interesting US report pointed out that only some CVD trials focusing on hypertension, diabetes, and stroke, but not heart failure, coronary artery disease, and hyperlipidemia, showed a higher enrollment of women than men . In thisRegarding age, it is intriguing to notice that most of the studies were performed on adults or in both adults and older adults, while only 21 studies were focusing only on older adults (age > 65 years). Due to the well-recognized importance of dietary habits and lifestyle in healthy aging , 25, thiClinicalTrials.gov, which allows to have a real-time picture of efforts carried out in these years, including those that have not led to publications, yet. Additionally, this approach allows to retrieve information that is often missing in publications, including details on the characteristics of the study population, the study design, and all outcome measures. Regarding limitations, it is noteworthy that the search on databases of registered clinical trials does not allow to retrieve information related to the results of the studies. Moreover, it cannot be excluded that the presence of other studies focused on diets and health that could have been found by using other specific terms, such as “eating” or “meal(s),” or that have not been registered or are registered in other databases. In fact, recent findings regarding the ICTRP revealed that less than 50% of the total clinical trial registrations belong to ClinicalTrial.gov database, with a decreasing trend due to a non-US clinical research rise of registrations (This work has several strengths and limitations worth to be noted. The first strength is related to the approach of using clinical trials registered in trations . FurtherIn conclusion, results from the present descriptive analysis underline the continuous interest not only in the study of traditional and balanced diets, such as the Mediterranean diet, but also for diets focused on modifications of macronutrients and, more recently, on food timing, with large variability in terms of duration of the study, selected outcomes, and characteristics of participants. These findings contribute to describe past efforts on this topic and to identify possible gaps and fields of research to cover in the next future to provide further evidence on the relation between diet and human health. Attributing specific health effects to diets is one of the greatest challenges in nutritional science. When clinical studies are underrepresented, as in some cases we have highlighted, a good strategy is to consider all available evidence and not just individual studies. Different studies, settings, and methodologies that produce similar results on the same question give a reasonably good indication of the existing relation between adherence to a particular diet and a specific health outcome.https://clinicaltrials.gov/.Publicly available datasets were analyzed in this study. This data can be found here: MD, DM, and DA conceived the study and were involved in the protocol design, data collection, analyses, and in the interpretation of the results. GP was involved in the protocol design and data collection. MD, CDB, DM, and DA drafted the manuscript. MP, PR, MS, and FS were involved in the interpretation of the results and reviewed the final draft. MD, DM, and DA had primary responsibility for the final content. All authors contributed to the article and approved the submitted version.DM is grateful for the support granted by Piano di sostegno alla ricerca-Linea 2, azione A, grant no. PSR2020-DMART.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: Suboptimal health status (SHS) measurement has now been recognized as an essential construct in predictive, preventive, and personalized medicine. Currently, there are limited tools, and an ongoing debate about appropriate tools. Therefore, it is crucial to evaluate and generate conclusive evidence about the psychometric properties of available SHS tools. Objective: This research aimed to identify and critically assess the psychometric properties of available SHS instruments and provide recommendations for their future use. Methods: Articles were retrieved by following the guidelines of the PRISMA checklist, and the robustness of methods and evidence about the measurement properties was assessed using the adapted COSMIN checklist. The review was registered in PROSPERO. Results: The systematic review identified 14 publications describing four subjective SHS measures with established psychometric properties; these included the Suboptimal Health Status Questionnaire-25 (SHSQ-25), Sub-health Measurement Scale Version 1.0 (SHMS V1.0), Multidimensional Sub-health Questionnaire of Adolescents (MSQA), and the Sub-Health Self-Rating Scale (SSS). Most studies were conducted in China and reported three reliability indices: (1) the internal consistency measured by Cronbach’s α value ranged between 0.70 and 0.96; (2) the test–retest reliability; and (3) the split-half reliability coefficient values ranged between 0.64 and 0.98, and between 0.83 and 0.96, respectively. For the values of validity coefficients in the case of SHSQ-25 > 0.71, the SHMS-1.0 ranged from 0.64 to 0.87, and the SSS ranged from 0.74 to 0.96. Using these existing and well-characterized tools rather than constructing original tools is beneficial, given that the existing choice demonstrated sound psychometric properties and established norms. Conclusions: The SHSQ-25 stood out as being more suitable for the general population and routine health surveys, because it is short and easy to complete. Therefore, there is a need to adapt this tool by translating it into other languages, including Arabic, and establishing norms based on populations from other regions of the world. Health has been traditionally conceptualized as a biological function at typical levels of efficiency . HoweverSHS has been conceptualized by Feng  under thMany developed countries, including Saudi Arabia, are undergoing vivid shifts due to fast-paced economic growth, which impacts lifestyles and social systems. The literature from other high-income countries has pointed out that unhealthy lifestyles significantly impact health, and mainly increase the vulnerability for sub-optimal health status . Among tSHS is a heightened concern for medical professionals and public health experts because it is a significant risk marker for chronic illnesses. SHS is differentiated from a sub-clinical disease state because it is a low-quality health state that cannot be classified as a disease state . The sigDifferent quantitative, qualitative, and mixed approaches have been used to measure SHS ,15. AmonInterestingly, there are also controversies about whether the measurement of SHS aligns with the SHS theoretical framework, which assumes that environmental factors and psychological states determine SHS, and betterment in these factors should alleviate SHS . TherefoSHS has now been recognized as an essential construct in personalized medicine to decrease the risk of developing disease and enhance general health. Moreover, the idea of SHS reflects the belief that chronic diseases can be effectively predicted and prevented before a clinical manifestation of severe pathologies from the view of predictive, preventive, and personalized medicine ,5. It isWhat instruments are available to measure SHS for different segments of populations?What are the key strengths and weaknesses of measures used to assess SHS in target populations?To what extent have the psychometric properties of these SHS instruments been evaluated for use in target populations?What are the current gaps in generating conclusive evidence about the psychometric properties of these tools in different populations?Appropriate measurement of SHS is vital to designing effective community health interventions. Therefore, the purpose of this article is to answer the following research questions:Given that self-report measures are widely used to assess SHS, practitioners and researchers need validated and reliable self-report instruments that can easily be administered to assess SHS. Therefore, it is essential to conduct a systematic review of the psychometric properties and utility of subjective SHS measures to guide in selecting appropriate SHS instruments for health research and clinical assessment. However, the currently available systematic reviews focused on the construct and conceptual framework of SHS ,5 to datA systematic review of global studies was conducted to assess the measurement properties and robustness of scales used to assess SHS. In this study, we followed the guidelines of the Centre for Reviews and Dissemination  and prefThe term suboptimal health status was first coined in 2002 by a Chinese researcher, as cited in , and theBy the Consensus-based Standard for the Selection of Health Measurement Instruments (COSMIN) , we searThis search strategy and filter were applied in an original article search on each database. The article search was completed by three  out of six investigators on this project, from January 2022 to February 2022. It was limited to articles published between Jan 2003 and Jan 2022 in the English language only. The inclusion and exclusion criteria for determining the eligibility of articles were as follows: (1) original studies on the measurement of SHS; (2) conducted with adult and adolescent populations; (3) studies undertaken in the last 20 years (Jan 2002 to Jan 2022); (4) studies complying with ethical standards such as the Declaration of Helsinki codes. Studies exclusively focusing on evaluating clinical interventions, systematic reviews, and meta-analyses were not included. This search yielded over 4500 research articles from each database, and the Microsoft Excel and CVS files from the databases were downloaded for the initial inspection of datasets. This MS Excel file contained basic identifying information about the articles, such as authors’ names, titles, publication dates, journal names, Doi numbers; then, an identification number was allotted by the database to each article. This basic information was used to remove the duplicated records, and a list of final articles was finalized for initial review by the researchers against the eligibility criteria. The article search, retrieval, and selection steps are presented in the flowsheet diagram .In the first stage, two researchers (MAA and COA) carefully read the article titles and abstracts to filter the articles that match the eligibility criteria. Where doubts arose regarding inclusion, the method section of the article was read to access details. This search revealed that 54 articles met the inclusion/exclusion criterion. In the second stage, about 12 to 14 articles were assigned to four investigators on the project , who read complete articles and filled in a data extraction form prepared by the research team containing questions based on The Consensus-based Standards for the Selection of Health Measurement Instruments (COSMIN) checklist to assess the procedural diligence and main findings related to measurement properties of the instruments ,27. In tThe study’s methodological quality was assessed using the adapted version of the COSMIN Risk of Bias checklist manual for systematic reviews of PROMs . We asseThis systematic search retrieved four subjective measures for assessing SHS in adults, adolescents, and university students. These included the following: (1) Suboptimal Health Status Questionnaire- 25 (SHSQ-25), (2) Sub-health Measurement Scale Version 1.0 (SHMS V1.0), (3) Multidimensional Sub-health Questionnaire of Adolescents (MSQA), and (4) Sub-Health Self-Rating Scale (SSS) .The psychometric properties of these tools were assessed through some reliability and validity indicators, as shown in The SHSQ-25 is the most commonly used SHS screening tool. China’s Capital University of Medical’s Wei Wang group invented it in 2007 ,4, and rThe SHSQ-25 is rapid and easy to complete; therefore, it is appropriate for the general population and healthcare settings ,13,15. ISHSQ-25 items are scored on a 5-point Likert scale, from never to always ,31,32. TWe found four studies assessing the instrument’s internal consistency and test–retest reliability. Yan et al. (2009) examined 3000 Chinese individuals, and found item-sub-scale correlations ranging from 0.51 to 0.72, and a Cronbach’s α 0.93 for all sub-scales . FurtherThe second instrument was the Sub-Health Measurement Scale V1.0. It is a self-reported multidimensional inventory designed to assess physiological, psychological, and social symptoms to determine SHS. This inventory was devised by researchers in China . The invThe SHMS V 1.0 has been widely used to determine the SHS of participants, especially among nurses, urban residents, college students, and midwives ,35,36,37The SHMS V1.0 has a straightforward scoring system. On a five-point Likert scale, from 1 (never) to 5 (often), respondents are asked to rate how often they experienced various types of discomfort over the past six months . A set op < 0.01) [We found five studies assessing SHMS V 1.0 psychometric properties; the structural validity showed a high correlation between an item and dimensional scores (0.656 to 0.878). The correlation between each dimension and sub-scale scores was strong (0.586 to 0.868) . We foun < 0.01) , as indiOur systematic search found the MSQA, an adolescent assessment tool, as the third SHS instrument. Chinese researchers developed a self-reported questionnaire to assess teenage psychological problems . The MSQThe MSQA measures emotional, behavioral, and social symptoms . SummingWe found four studies assessing the psychometric properties of the MSQA. The test–retest reliability was around 0.87 in three studies. Moreover, the Cronbach alpha coefficient and split-half reliability were around 0.96 and 0.94, respectively ,44,45,47The search retrieved the Sub-Health Self-Rating Scale (SSS), which Chinese researchers developed to assess the SHS of university students . It deteThe SSS is scored by adding the raw scores on items or sub-scales . Each itp = 0.000), and the Kaiser–Meyer–Olkin (KMO) score of sample adequacy was 0.94, as shown in The SSS’s psychometric qualities were assessed using the Cronbach α coefficient of 0.942 [To sum up, the systematic review of articles on SHS measurement tools demonstrated some evidence about the psychometric properties of instruments; most of them were based upon studies from China. Among the reliability indicators, three parameters were commonly reported: (1) the internal consistency measured by Cronbach’s α value ranged between 0.71 and 0.96; (2) the test–retest reliability ranged from 0.64 to 0.98; and (3) the split-half reliability coefficient values ranged between 0.64 and 0.98, and between 0.83 and 0.96, respectively. All three indicators of reliability revealed acceptable levels of evidence about the reliability of these measures.The indicators of the validity of tools were determined through (1) construct validity, (2) convergent validity, and (3) divergent validity, and it was accomplished for three subjective measures, namely the SHSQ-25, MSQA, and SSS. For the validity coefficient values in the case of SHSQ-25 > 0.71, SHMS-1.0 ranged from 0.64 to 0.87, and SSS ranged from 0.74 to 0.96, and can be considered as acceptable.We believe this is the first systematic review of the validity and reliability of SHS instruments. We found four instruments measuring SHS, and there have been few studies conducted on the psychometric properties of these SHS instruments. Encouragingly, all instruments showed some psychometric testing, with comparatively higher coefficient values (0.70–0.98) on the reliability tests and correlation coefficient values (0.70–0.92) for the validity test.Utilizing standardized measures to assess the SHS has many advantages. When evaluating an intervention, there is no rationale for creating new  instruments when excellent standardized tools already can be used for free or at a minimal cost, given that these instruments were relatively good in discriminating SHS. It was imperative to scrutinize items or elements used in measuring SHS by each instrument. For example, the SHMS V1.0, which has a total of 39 items, has 4 separate items used to make general health self-evaluations, with the remaining 35 divided among three symptom dimensions. The first dimension comprises 14 items that assess physiological symptoms; the second dimension includes 12 items and assesses psychological symptoms; and the third dimension consists of 9 items that assess social symptoms ,40,41,46On the other hand, the SSS instrument had a good correlation coefficient on the total scale and the sub-scales. Each question had a correlation of 0.52–0.89 for physiological, psychological, and social dimensions . The queInterestingly, the SHSQ-25, which has 25 items in five domains, has been validated in Chinese, African, and European populations ,29. The Additionally, the MSQA, designed to assess uncomfortable symptoms experienced by respondents, had 71 questions divided into six symptom dimensions . The MSQIn short, comments on the validity of individual health and well-being tools were based on articles that evaluated or reported on the validity of the measurement tools. These articles’ findings were taken at face value. However, many of the articles did not use standard terms for validity, and did not evaluate validity in the same way. Even though some tools have limitations, the full table was provided for information. The scoring has some subjective parts, and different criteria for scoring could be used to come up with different total scores and rankings. However, we used a logical framework to separate the tools that could be used to evaluate SHS in community interventions. With the information in this paper, researchers and clinicians can find and use instruments that have real proof in possessing appropriate psychometric properties to measure concepts that are relevant to their goals.In conclusion, four instruments for SHS were found through our systematic analyses, and their psychometric properties were found to be adequate. Since they have been proven to be reliable and valid, are easy to use, and often have established population norms, and they are good choices. However, the SHSQ-25 was found to be better for the general population than the SHMS V1.0, MSQA, and SSS as a result of the length and population-specific focus of the SHMS V1.0, MSQA, and SSS. In contrast, the SHSQ-25 is brief and simple to complete, and its psychometric qualities have been examined in numerous populations around the world. Therefore, it is important to adapt the SHSQ-25 and translate it into various languages, such as Arabic, in order to evaluate out how they perform in the general populations of other countries.


Direct oral anticoagulants (DOACs) have been used more frequently for the prevention and management of thromboembolic disease in comparison to their predecessors. DOACs provide greater ease of administration, shorter half-lives, less monitoring, and fewer drug-drug interactions. With the rise of DOACs such as rivaroxaban, the opportunity for abuse also increases. Therefore, standardization of care based on rivaroxaban misuse must also be explored, an area in which there is not ample information. We present a case where a patient consumed a stockpile of her home medications in hopes to commit suicide. A 64-year-old female presented to the emergency department due to the ingestion of rivaroxaban 5,000 mg along with ingestion of acetaminophen 30,000 mg and isosorbide mononitrate 1000 mg in the setting of intentional self-harm with multiple declarations of being classified as Do Not Resuscitate. There have been documented cases of rivaroxaban overdose, however, there are no documented cases with levels of ingestion reaching 5,000 mg along with signs of severe bleeding. Our case study reviews the previously documented management of rivaroxaban abuse and the treatment that was given to our patient in the setting of extreme anticoagulant ingestion. Rivaroxaban, also known as Xarelto, is a factor Xa inhibitor used in the management of non-valvular atrial fibrillation, deep vein thrombosis, and pulmonary embolism . Its metThroughout the literature, there are a few case reports associated with rivaroxaban overdose and the management strategies performed. In the case studies that have been published, the maximum known dosage of rivaroxaban has not been more than the dose that was ingested by our patient. Furthermore, although there are documented cases of severe bleeding with the ingestion of rivaroxaban, the amount ingested is not known, and therefore management is unclear. Here, we present a case of a severe Xarelto overdose that had significant bleeding.A 64-year-old 93 kg female with a past medical history including hypertension, history of multiple pulmonary embolisms, paroxysmal non-valvular atrial fibrillation, heart failure with preserved ejection fraction, coronary artery disease (CAD) status post percutaneous coronary intervention and drug-eluting stent (DES) most recently for angioplasty to the left circumflex artery, cerebral vascular accident (CVA) with residual left-sided weakness, a major depressive disorder with post-traumatic stress disorder (PTSD), and previous suicide attempts, presents to the emergency department (ED) for intentional self-harm and a suicide attempt. The patient had been discharged the previous day with home health due to the unavailability of an open bed for inpatient rehabilitation. Upon discharge, the patients’ medications were filled for a 90-day supply, including rivaroxaban and isosorbide mononitrate. The following day, the home health nurse arrived for their assessment. The home health nurse reported that the patient had multiple old prescription bottles beside her, including rivaroxaban, isosorbide mononitrate, and an over-the-counter acetaminophen bottle.Upon arrival in the ED, it was found the patient had ingested rivaroxaban 5000 mg, acetaminophen 30,000 mg, as well as isosorbide mononitrate 1000 mg. The patient's review of systems was positive for nausea, lightheadedness, headache, and suicidal ideation. A physical exam revealed oozing epistaxis of the bilateral nares, bilateral lower extremity non-pitting edema, a right forearm oozing hematoma, and chronic residual left-side weakness. The patients’ initial vital signs were within normal limits. The patient’s labs of significance included a white blood count of 15.7 × 10*3/µL , hemoglobin of 11 g/dl , hematocrit of 34.7% , PT of 38.5 seconds , INR of 3.9 , PTT of 77.3 seconds , salicylate level of less than 2.8 mg/dl , and acetaminophen level of 263.4 µg/ml . The patient’s ethanol and urine drug screens were both negative. A head computed tomography (CT) scan and chest X-ray (CXR) revealed no acute pathology.The patient arrived at the ED within 30 minutes of ingestion and was given activated charcoal. Activated charcoal-sorbitol 50 g/240 mL suspension for a total of 100 g was given due to the ingestion of toxic amounts of medication. Due to the excessive amount of Tylenol ingestion, N-acetylcysteine 150 mg/kg bolus followed by a 15 mg/kg/hour infusion was given for acetaminophen toxicity. Four-factor human prothrombin complex concentrate (Kcentra) 4770 units was given, as well as Phytonadione (Vitamin K) 10 mg. While still in the ED, the patient was coughing, which resulted in hemoptysis that was controlled with nebulized tranexamic acid (TXA). The management of this patient was complicated by continuous bleeding for approximately four hours after the patient’s initial presentation and after the perfusion of Kcentra. The decision to begin an Andexanet alfa bolus of 13,950 mg (150 mg/kg) was made and followed by a 15 mg/kg infusion. The patient was then admitted to the intensive care unit for monitoring.Throughout treatment and during the patient’s hospital stay, repeat laboratory data 24 hours post-treatment showed an acetaminophen level of less than 2 µg/ml. PT and INR eventually returned to baseline levels around 36 hours after the administration of reversal agents. In Table The first orally dosed direct factor Xa inhibitor was rivaroxaban . It bindIsosorbide is a nitrate that exerts its pharmacologic effect by releasing nitric oxide (NO), an endothelium-derived relaxing factor (EDRF) . NO is eAcetaminophen toxicity is the second most common cause of liver transplantation worldwide and the most common cause of liver transplantation in the US . AcetamiThree cases of massive rivaroxaban overdose were discovered in the literature review: 1400 mg, 1800 mg, and 1960 mg -22. NoneThis case report allows us to explore the treatment and management of an unprecedented presentation of rivaroxaban, isosorbide mononitrate, and acetaminophen overdose resulting in major hemorrhage. Although treatment and management strategies for acetaminophen overdose are well described, the same cannot be said for the treatment and management of an isosorbide overdose and massive rivaroxaban overdose. The strategies used in the case were based upon limited evidence, and the decisions made throughout this case will hopefully allow for a standardization of care in the setting of any level of anticoagulant use or misuse.


Plastic pollution has emerged as a growing concern worldwide. In particular, the most abundant plastic debris, microplastics, has necessitated the development of rapid and effective identification methods to track down the stages and evidence of the pollution. In this paper, we combine low-cost plastic staining technologies using Nile Red with the continuous feature offered by microfluidics to propose a low-cost 3D printed device for the identification of microplastics. It is observed that the microfluidic devices indicate comparable staining and identification performance compared to conventional Nile Red staining processes while offering the advantages of continuous recognition for long-term environmental monitoring. The results also show that concentration, temperature, and residency time possess strong effects on the identification performance. Finally, various microplastics have been applied to further demonstrate the effectiveness of the proposed devices. It is found that, among different types of microplastics, non-spherical microplastics show the maximal fluorescence level. Meanwhile, natural fibers indicate better staining quality when compared to synthetic ones. It has been estimated that the ongoing COVID-19 pandemic has exceedingly increased the demand for the use of plastics . Among tNevertheless, the current understanding of plastic pollution in terms of the quantity, type, lifetime, and associated health effects largely remains unknown. As a result, microplastic separation and identification serve as an important approach to providing evidence and metrics of the pressing environmental issues caused by plastic pollution . For exaAs a result, visual inspection, though not as effective as other sophisticated counterparts, is still widely applied for faster recognition ,23. CurrNevertheless, the focus of current studies on microplastic staining and identification has been largely given to batch-by-batch or case-by-case analysis ,34,35,36Herein, we further explored the staining capabilities of Nile Red through a microfluidic device capable of continuously staining microplastics for rapid identification. The proposed device has two inlets for respective Nile Red and sample injection A, and a In this paper, the process of static microplastic identification using Nile Red was carried out without a filter. Specifically, the staining Nile Red solution was added directly into an Eppendorf tube containing the microplastics sample and placed inside an oven B. On theThe staining solution was prepared by dissolving Nile Red  in methanol to different concentrations. We have considered the limit of solubility of Nile Red in methanol (1 mg/mL) to be the stock solution for further dilution, from which the solution was diluted into 50X, 100X, 250X, 500X, and 1000X samples.In this paper, lab-prepared and commercially available microplastics were adopted in lieu of naturally formed microplastics. More specifically, microspheres made of polyethylene (PE), ranging from 10–45 µm  were applied to determine the optimal parameters for staining. Other plastics including the microspheres made of polystyrene (PS) with sizes from 9.5–11.5 µm , cotton and acrylic fabric acquired from clothing, polypropylene (PP) and non-spherical PE prepared from plastic storage containers were also applied to test the versability of the proposed method. All the samples were mixed with deionized (DI) water prior to staining. Note that commercial microspheres were diluted in a concentration of 10 mg/mL, while the other samples were diluted to 1 mg/mL, which is because the commercial particles were more available than the ones obtained from other sources.To prepare the samples for static experiments, 100 µL of Nile Red solution was thoroughly mixed with 100 µL of PE microplastic solution inside an Eppendorf tube, followed by baking inside an oven . On the other hand, all static experiments were performed using PE microspheres. To investigate the effect of Nile Red concentration on staining performance, different concentrations were tested: 100X, 250X, 500X, and 1000X; in addition, different temperatures  were applied to study the effects of temperature. All the samples were placed inside the oven for 10 min, and analysis was conducted immediately after baking.To create the microfluidic devices, soft lithography, a commonly used method in microfluidics, was applied. Specifically, a 3D printer  was used to create the molds for casting polydimethylsiloxane (PDMS) to obtain the final devices. After curing the PDMS mixture in an oven overnight at 65 °C, a corona treater  was used to permanently bond the device onto a glass slide. Finally, the device was placed inside the oven and a syringe pump  was used to run the samples as well as the staining agents inside the device.https://imagej.nih.gov/ij/, accessed on 10 November 2021) was used to analyze and quantify the results. Each experiment was performed four times for statistical analysis. We have not filtered the particles prior to observation, instead, we have directly placed a droplet of the diluted sample on top of a glass slide.For both static and microfluidic staining, an inverted microscope (Zeiss Axio Vert.A1) was used. To visualize the fluorescent signal from the samples, an illumination system  with a wavelength of 365 nm was used. All the images were recorded using a camera attached to the microscope . ImageJ . In this paper, the total microchannel length was 400 mm, and its cross-sectional area was 2 × 2 mm. Using this design, we could achieve 5, 6, 7, 8, 9, 10, 11, and 12 min of residency time by applying corresponding flow rates of 7.82, 6.52, 5.58, 4.89, 4.34, 3.91, 3.55, and 3.26 µL/min, respectively. Note that the microfluidic device was placed inside the oven while the syringe pump was kept outside. The input and output hoses were long enough to enable sample collection and syringe manipulation outside the oven. From the information acquired during the static experiments, we have performed the microfluidic experiments with the most promising configurations with respect to concentration and temperature . Different flow rates were tested to compare the performance of static and microfluidic staining regarding the residency time. As expected, lower flow rates provided better results, which is in accordance with the static experiments ,49. NoneBesides PE microspheres, we further demonstrated the capabilities of our device for identifying other types of plastic. In this regard, multiple types of microplastics were applied, including microspheres (PS), fibers (cotton and acrylic), plastic parts scratched from storage containers (PP and PE). Moreover, yeast was adopted as a model of potential organic particles in seawater. In this paper, we have presented a novel microfluidic identification method for the continuous recognition of microplastics in water. Our method combines the Nile Red staining protocols with the high-throughput advantages imposed by microfluidics ,47,51. WIn addition, though microfluidic results are still not as good as the static ones, future improvements can be carried out by adopting a better mixing strategy for Nile Red and samples ,51,52. CThe device can be further improved by coupling an on-chip heater, eliminating the need for an oven , thus reNote that the concentration of microplastics in seawater samples varies widely, being less concentrated off-shore (down to 8 particles/m³) . In addiOverall, we have suggested the adoption of a microfluidic device for the continuous analysis and further detection of microplastics. Nile Red has proven to be effective for the identification of microplastics. Static experiments were performed to systematically assess the influence of staining agent concentration, temperature, and residency time. Based on the results, the microfluidic configuration for continuous staining was optimized, leading to the best fluorescence results among the tested configurations. Our method demonstrated to be feasible for the identification of different types of microplastics with the advantage of continuous staining and with the possibility of future integration for in situ identification along with higher throughputs. This platform demonstrated to successfully identify microplastics in a continuous manner, representing a valuable option for environmental management.


This study examines how different ways of presenting information about the ecological threats stemming from the use of disposable plastics may affect people’s willingness to reduce their use. To test our hypotheses, we used a 2 × 3 × 2 between-subjects experimental design, utilizing a sample of 1001 Hong Kong residents. The independent variables tested included: (a) message frame (gain vs. loss), (b) modality (text vs. image vs. infographic), and (c) information source . The findings demonstrate that the loss frame was more effective than the gain frame in persuading participants to reduce the use of disposable plastics. Furthermore, compared to image-based messages, text-based and infographic-based messages were more effective in promoting the reduction in the use of disposable plastics. For information sources, however, we found no main effect on behavioral intentions. However, this study still suggests an interaction effect of the loss frame and NGO source, as well as the interaction between text-based modality and government source, both leading to more positive outcomes. Furthermore, the study reveals that negative emotional responses mediate the effect of media frames on behavioral intentions. The findings offer useful insights for designing more effective communication campaigns aimed at curbing the use of disposable plastics. Single-use plastic waste poses a serious threat to the natural environment. Globally, it is estimated that over 380 million tons of plastics are produced every year, and up to 50% of that is for single-use purposes. Data show that more than 10 million tons of plastic are dumped into oceans annually, and more than 1 million marine animals are killed by it every year . In HongDrawing on prospect theory , messageLastly, we also examined the potential mediating role of emotional arousal (positive vs. negative), which is one of the key determinants of pro-environmental behaviors . AlthougPrevious research has examined the drivers of environmentally sustainable behaviors in various social and cultural contexts. For instance, Heidbreder, Steinhorst, and Schmitt conducted an experimental study to investigate whether the “Plastic Free July” campaign could reduce plastic consumption in German, with the result that participants exposed to information about this campaign were significantly less likely to use disposable plastics than the control group who did not receive this information . Walker Goal framing is a type of framing effect that is frequently employed in environmental communication studies addressing environmental sustainability ,10,27,28Previous studies demonstrated the effectiveness of gain- and loss-frame messages in changing people’s behaviors. For instance, Cho and Bolster demonstrated the persuasiveness of loss-frame messages among smokers , and MitRQ1: Which frame (gain vs. loss) is more effective in promoting the reduction in the use of disposable plastics?Previous studies demonstrated that multimodality is an important factor affecting information processing. According to the cue summation theory , multichH1. Media modality  has an impact on the reduction in the use of disposable plastics. More specifically, infographics will be more effective than (a) text- and (b) image-based messages in promoting the reduction in the use of disposable plastics.In addition, previous studies also demonstrated that the sender-related factors might influence information processing, especially when the audience lacks the motivation or knowledge to scrutinize messages about environmental issues ,6. Carl H2. Information source  has an impact on the reduction in the use of disposable plastics. More specifically, the messages from the government will be more effective than those from NGOs in promoting the reduction in the use of disposable plastics.In addition, there is still a lack of evidence on how the combination of media frame, media modality, and information source may exert influence on behavioral intentions, so we also posed the following research question:RQ2: How will the three-way interactions of media frame (gain vs. loss), media modality (text vs. image vs. infographic), and information source (government vs. NGO) impact the reduction in the use of disposable plastics?Previous studies found that emotions have a large influence on human behavior, in which appealing to emotions is an effective approach to persuasion ,54. In eH3. (a) Positive and (b) negative emotions will mediate the effect of media frames on the reduction in the use of disposable plastics.M = 40.3, SD = 12.6). A total of 95.7% of participants have received secondary school education or above, and 74% had a monthly income of more than HKD 10,000. Compared with the population in Hong Kong, the sample in our study was younger and better educated.This study used data from an online panel survey conducted in 2020 by a reputable market research company in Hong Kong. A total of 1001 Hong Kong residents were recruited from a proprietary consumer panel using gender and age quotas. The population in Hong Kong is estimated to be 7.41 million, of which 45.5% are male, with a median age of 46.3. Additionally, 82% received secondary education or above . In our The study was conducted using an embedded experimental design that tested the effectiveness of different types of message appeals, modalities, and sources. The experimental setting was set up in the Qualtrics system, and the random assignment was conducted through Qualtrics using a randomizer tool. After providing consent, participants answered questions about their demographic information, environmental knowledge and concern, media use habit, and single-use plastics habit. Next, they were randomly assigned to receive one of our experimental stimuli.n = 74), group 2 , group 3 , group 4 , group 5 , group 6 , group 7 , group 8 , group 9 , group 10 , group 11 , group 12 , and group 13 . Twelve versions of an ad advocating for the reduction in the use of disposable plastics were created (see A 2 (frame: gain vs. loss), * 3 , and * 2 (source: government vs. NGO) between-subjects experimental design was employed in this study. Participants were randomly assigned to one of the 13 experimental conditions: group 1 , 2 , and 3 (Others), and education was assessed according to the level achieved by the respondent up to graduate degree . Household income was measured using 14 categories .In order to control for the factors that could affect single-use plastics reduction behaviors, participants were asked a series of demographic questions, including age, gender, education, and income level. past behaviors, the following 16 items were adopted to assess single-use plastics consumption frequency .For ency see . All theEnvironmental knowledge was measured by 5 items on a 7-point scale, including (1) I am very knowledgeable about environmental issues; (2) I understand the environmental phrases and symbols on product packaging; (3) I know how to buy products that are environmentally safe; (4) I know more about recycling than the average person; and (5) I know how to select products and packaging that reduce the amount of waste ending up in landfills [M = 4.54, SD = 1.02, Cronbach’s α = 0.898). Environmental concerns measure individuals’ concern about the natural environment and the use of single-use plastics, including 10 items: (1) I worry about the endangered species in Hong Kong; (2) Improper disposal or dumping of industrial or domestic waste is a common phenomenon in Hong Kong; (3) The water quality in lakes, rivers, and streams is declining in Hong Kong; (4) Air pollution is a serious issue in Hong Kong; (5) It is essential to promote green living in HK; (6) I believe more environmental protection works are needed in HK; (7) Environmental protection issues are none of my business; (8) It is very important to raise environmental concerns among HK people; (9) Single-use plastics have severe negative consequences for humans and nature; (10) Recycling single-use plastics is important for the Hong Kong’s future. The items loaded onto a single factor using a principal components analysis with 49.6% of total variance explained. An additive index was created by taking the average of the responses . Environmental efficacy was measured by 4 items, including (1) As long as actions are taken to reduce plastic consumption, plastic pollution can be effectively controlled, (2) I believe that I have the ability to take action to mitigate single-use plastic pollution, (3) Although it may cause inconvenience, I can still change my behavior to reduce single-use plastic consumption, and (4) I can try my best in every way to reduce plastic pollution .Because single-use plastics use behavior may depend on a variety of psychological variables, we measured participants’ environmental knowledge, environmental concerns, and environmental efficacy. andfills . The restraditional media use was measured by three items, including watching local free-to-air TV, watching cable TV, listening to radio news, and reading traditional forms of media  . Digital media use was measured by four items, including the use of instant messaging apps , YouTube, and Facebook, and reading digitized forms of media  . Soft news consumption was measured by seven items, including cooking and home renovation shows, health and fitness information, movie and drama, fashion news, travel shows, celebrity news, and music videos . Hard news consumption was measured by five items, including financial news, political news, environmental news, science and technology news, and nature documentaries .This study not only measured traditional media use and digital media use behaviors but also assessed media content consumption. More specifically, The positive emotions were feeling happy, enthusiastic, optimistic, proud, content, and excited . The negative emotions include anger, fear, guilt, sadness, disappointed, nervous, worried, and frustrated .Emotional responses were measured by assessing 14 types of emotion with a 5-point scale, in which six were positive and eight were negative . The posthe intention of refraining from using single-use plastics, we asked respondents their willingness to engage in the following 19 behaviors, including “Bring a reusable glass or stainless steel bottle/travel mug when going out”, “Carry reusable shopping bags”, and “Avoid using plastic straws.” Answers were ranging from 1 to 7 and the responses were averaged to form an additive index .In terms of M = 5.53, SD = 1.24) was perceived as emphasizing the benefits of not using single-use plastics compared with the loss-frame message  = 4.18, p < 0.001). Meanwhile, a t-test indicated that respondents who were in the loss-frame message condition had significantly higher perception of loss  than those who were in the gain-frame condition  = 6.39, p < 0.001). Thus, both message framing conditions successfully created differential perceptions of gain and loss in participants.A manipulation check was performed to ensure that the appeals were perceived as intended. The results revealed that the gain-frame message  was perceived to be significantly less visual than graphic condition  and infographic condition .In order to ensure that the stimuli were perceived as text only, graphic only, or as an infographic, participants were asked to rate their opinion of the message on a semantic differential scale that was anchored by “text-based” (1) and “visual-based” (11). As expected, the text-based message  and those exposed to gain frames  reported higher behavioral intentions than control group  = 3.91, p < 0.05). However, the results of post hoc show that only the effect of the loss frame was statistically significant while the gain frame was not (p = 0.248). Thus, only the loss frame was an effective predictor of future behavioral intention, and RQ1 was answered.In order to examine the effectiveness of the media frame and answer RQ1, ANOVA results are displayed with means and standard deviations for all experimental groups in M = 4.96, SD = 0.895), text-based messages  and infographic-based messages  were more effective in increasing people’s intention to reduce single-use plastics use  = 3.91, p < 0.05, see Meanwhile, compared with image-based messages  refraining from using single-use plastics may affect the willingness to change behaviors, as well as the mediating role of affective arousal. The findings demonstrated that the loss frame was more effective than a gain frame in persuading individuals to reduce using disposable plastics. In addition, text and infographics were more effective than images. In particular, the study suggests a congruency between text-based messages from the government and the loss frame from NGOs conveyed by all three modalities. Moreover, the study also suggested the mediating role of negative emotion in the relationship between media frames and behavioral intentions. The study thus provided insights for both citizens and policymakers about the most effective strategies for designing messages to promote environmentally sustainable behaviors.As Tversky and Kahneman’s study suggests, people are likely to be more accepting of risks in a loss-frame condition, whereas they are more risk-averse in a gain-frame condition ; later sInterestingly, compared with images, text and infographics are more effective in persuading people to reduce using disposable plastics. One possible reason is that text and infographics provide more detailed information and require more time to process the information, which may stimulate cognitive activity and motivate the central route of information processing . GraphicThe government source, in general, does not show a significant difference from the NGO source in affecting participants’ behavioral intentions. As pointed out by some scholars, the environmental governance in Hong Kong relies on both the governmental authorities and social actors, and in particular, social actors are often the first movers in environment-related policies . This fiThis study also demonstrates the mediating role of negative emotions. Compared with positive emotions, negative emotions such as fear, anxiety, and guilt are often linked to higher arousal, which may lead to motivated avoidance behavior . For insThere are several limitations of this study that should be considered when interpreting the results. First, this study focused on the gain vs. loss framing based on the prospect theory, while there are other ways of framing that may also play an important role in curbing plastic consumption. For instance, Septianto and Lee demonstrated different effects of a “why” message  vs. a “how” message  . Future Finally, since this study was conducted among Hong Kong residents only, future research should aim to compare the effectiveness of the above campaign strategies in curbing the use of disposable plastics in other political and cultural contexts. Future studies should also explore different ways of segmenting target audiences so that effective intervention strategies can be designed with specific groups in mind.In this study we examined how the effectiveness of public engagement campaigns aimed at reducing the use of disposable plastics can be optimized by experimentally testing the effects of message frame, modality, and information source on behavioral intentions. The findings show that the loss frame was more effective than the gain frame in reducing the future use of disposable plastics. We also demonstrate that, compared to image-based messages, text-based and infographic-based messages were more effective in promoting the reduction in the use of disposable plastics, but find no main effect of information source. Finally, our study highlights an important mediating role of negative emotions in promoting environmentally sustainable behaviors.


Skin aging is one of the most concerning issues that occur after menopause. The Genistein Nutraceutical (GEN) product, containing genistein, vitamin E, vitamin B3, and ceramide, has been formulated as a topical anti-aging product for improving the health of postmenopausal women’s facial skin. This study aimed to investigate the efficacy and safety of the GEN product on postmenopausal women’s facial skin. This randomized, double-blind, placebo-controlled trial randomly assigned 50 postmenopausal women to receive either the GEN product (n = 25) or the placebo (PLA) product (n = 25), topically applied twice daily for 6 weeks. The outcome assessments included multiple skin parameters related to skin wrinkling, color, hydration, and facial skin quality at baseline and week 6. The percentage mean changes or absolute mean changes, where appropriate, in skin parameters were compared between the two groups. The mean age of the participants was 55.8 ± 3.4 years. For skin wrinkling and skin color parameters, only skin redness was significantly higher in the GEN group when compared to the PLA group. Following the application of the GEN product, skin hydration increased while fine pores and their area decreased. Subgroup analysis of older women (age ≥ 56 years) with adequate compliance found significant differences between the two groups in the percentage mean changes of most skin wrinkle parameters. The GEN product has benefits for the facial skin of postmenopausal women, particularly those who are older. It can moisturize facial skin, lessen wrinkles, and enhance redness. In women, estrogen naturally declines after menopause, and several physiologic changes occur due to low estrogen levels . Skin isPhytoestrogens, or plant-derived compounds that mimic estrogen in the human body, can exert an anti-aging effect on the skin via estrogen receptors . Among tThe Genistein Nutraceutical (GEN) product, which contains genistein and some other ingredients, including vitamin E, vitamin B3, and ceramide, has been formulated as a topical alternative to anti-aging products for improving the health of postmenopausal women’s facial skin. Apart from the beneficial properties of genistein on the facial skin, it is well known that vitamin E, vitamin B3, and ceramide also have a significant impact on the improvement of facial skin conditions. The topical application of vitamin E is able to alter the biosynthesis of collagen and glycosaminoglycan in the skin owing to its antioxidant properties ,12. VitaThe present study aimed to assess the efficacy of the GEN product on postmenopausal women’s facial skin by means of a randomized, double-blind, placebo-controlled design. It was also designed to assess the product’s safety and participant satisfaction.This randomized, double-blind, placebo-controlled trial was conducted at the Clinical Research Center for Food and Herbal Product Trials and Development (CR-FAH), Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand, between May and July 2022. The clinical trial protocol and supporting documents were approved by the Research Ethics Committee of the Faculty of Medicine, Chiang Mai University (No. 045/2022). This study was prospectively registered with the Thai Clinical Trials Registry (TCTR20220209010) prior to enrollment.2, no significant laboratory abnormalities , and the ability to read and write Thai and give written informed consent. Those who (1) had used estrogen or isoflavones within the six months prior to study entry, (2) had a history of allergy to soy products, vitamin E, vitamin B3, or ceramides, (3) had an underlying disease for which soy products were contraindicated , (4) used medications , food supplements, herbs, vitamins, minerals, or other products that might significantly affect the skin within the three months prior to study entry, and (5) used narcotics or psychotropic substances, consumed excessive amounts of alcohol, or smoked cigarettes on a regular basis were excluded from this study. All the participants signed a written informed consent form prior to study entry.Postmenopausal women (aged between 48 and 65 years) who had stopped menstruation for at least 12 months were eligible for this study. Other inclusion criteria were as follows: a serum level of follicle-stimulating hormone (FSH) of >30 IU/L, a body mass index between 18 and 30 kg/mThis trial planned to enroll a total of 50 participants (25 in each group). The sample size of 25 per group was estimated to achieve a power of 80% and a level of significance of 5% (two-sided) for detecting an effect size of 0.9 with a dropout rate of 10%.w/w) as analyzed by high-performance liquid chromatography coupled with a diode array detector and a mass spectrometric detector  Co., Ltd., Chiang Mai, Thailand). The Genistein Nutraceutical (GEN) product consisted of 4% genistein, 1% vitamin E, 1% vitamin B3, and 0.2% ceramide. The placebo (PLA) product had a cream base without genistein, vitamin E, vitamin B3, or ceramide; it was packed in an opaque bottle identical to the GEN product.Genistein was purchased from Xi’an Rongsheng Biotechnology Co., Ltd. , with a purity of 99.03%  and were instructed to apply the product to their facial skin twice daily (0.6 mL in the morning and another 0.6 mL at night) for 6 weeks. The participants were instructed not to use any other cosmetics over the course of the trial, and they were also forbidden from ingesting any herbs or other nutritional supplements that were known to affect their skin condition.(1)Skin wrinkling parameters, including depth of skin roughness , maximum roughness , average roughness , smooth depth , arithmetic average roughness , surface (the size of the wavy surface which was compared to a fully stretched flat surface), and volume , all of which were measured by the Skin Visiometer SV 700 ;(2)® CL 400 ;Skin color parameters, including brightness , redness , pigmentation , and individual typology angle , all of which were measured by the Skin-Colorimeter(3)® dual MPA 580, Courage and Khazaka electronic, Cologne, Germany);Hydration value, which was measured by the Corneometer (Cutometer(4)Facial skin parameters, including fine pores, large pores, and spots, all of which were measured by the Visioface 1000 D ; and(5)® PP34  .Acne parameters, including size and quantity, were all measured by the VisioporOutcome assessment was performed at baseline and at the end of the study. Efficacy outcome measures included five sets of skin parameters, as follows:Five dimensions of participant satisfaction  were assessed at the end of the study using the 10-point scale questionnaire, with a higher value indicating better satisfaction. Any adverse events seen by the investigators or self-reported by the participants were recorded. The weight of the product remaining in the bottle was measured to determine compliance. Participants who used the product at less than 50% of the prescribed dosage were considered poorly compliant.t-test; the mean difference (MD) between the two groups and its 95% confidence interval (95% CI) were then obtained. Subgroup analysis was performed to determine the effect of the product among postmenopausal women aged 56 years or older who had applied the product with adequate compliance.The intention-to-treat (ITT) and per-protocol (PP) approaches were used to assess the outcomes of this study. All participants who were randomly allocated were included in the ITT method, and the analysis was based on the group to which they were initially assigned. The analysis utilizing the PP approach excluded participants with poor compliance or loss of follow-up. Continuous variables are presented as mean ± standard deviation (SD). Percentage mean changes from baseline or absolute mean changes from baseline between the two groups were compared using the Student’s p-value of less than 0.05 was considered statistically significant.All statistical analyses were performed using IBM SPSS Statistics for Windows, Version 22.0. Armonk, NY, USA: IBM Corp., Release 2013. A A total of 57 women were initially assessed for eligibility, and seven individuals were excluded due to screening laboratory findings outside the normal range (n = 4) or a level of FSH lower than 30 IU/L (n = 3). Fifty postmenopausal women were randomly allocated to receive either the GEN product or the PLA product . The meaNo participants withdrew or were withdrawn from the trial throughout the study period. Five individuals (two in the GEN group and three in the PLA group) used the product less than 50% of the expected dosage and were defined as poorly compliant. Therefore, the ITT approach included 50 participants (25 in the GEN group and 25 in the PLA group) in the analysis, while the PP approach included 45 participants (23 in the GEN group and 22 in the PLA group) in the analysis .p = 0.044; PP: 9.35 ± 19.74% vs. −3.90 ± 11.37%, p = 0.009)  .p = 0.323; PP: MD = 5.84%, 95% CI, −3.81–15.48, p = 0.229).Skin hydration was significantly increased by 12.30 ± 15.66%  and 7.90 ± 15.54%  following the application of the GEN product and the PLA product, respectively. However, no significant difference in the percentage mean change in skin hydration was found between the two groups in both ITT and PP analyses  and −0.41 ± 0.79% , respectively, following the application of the GEN product, while no significant change was observed in the PLA group. There were no significant changes in other facial skin parameters following the application of either the GEN product or the PLA product. Also, no significant difference in any facial skin parameters between the two groups was observed in both ITT and PP analyses . No signp = 0.048), average roughness , smooth depth , arithmetic average roughness , and surface   with adequate compliance found significant differences between the two groups in the percentage mean changes of most skin wrinkling parameters, including skin roughness  .Most skin wrinkling parameters were significantly decreased following the application of the GEN product , whereas none of such parameters were significantly changed following the application of the PLA product .p = 0.030; PP: 9.39 ± 0.66 vs. 8.50 ± 1.74, p = 0.033), overall efficacy , and serum formulation   .During the six-week study period, only one individual in the GEN group experienced mild skin rashes on both cheeks on Day 5, which resolved after oral antihistamine therapy for 2 days (Days 6–7). The GEN product was withheld for 3 days (Days 6–8) until completely resolved and continued after that until the end of the study. No skin rash occurred again after re-exposure to the GEN product.The present study demonstrated that the combination of genistein, vitamin E, vitamin B3, and ceramide in a topical skincare product could improve certain features of facial skin appearance in postmenopausal women. This clinical trial was executed to the gold standard, with a randomized, double-blind, placebo-controlled design in which study creams were coded and randomly assigned at the source, while the research participants and the investigators were kept blind about the coding until study completion. Facial skin outcomes  were measured objectively and quantitatively using multiple well-established tools.Our analysis revealed that postmenopausal women, particularly those who are older, could benefit from the GEN product in terms of skin wrinkles. It is well known that genistein and 17-β-estradiol share some similarities in their chemical structure, and genistein is characterized as a partial agonist for the beta isoform of the estrogen receptor (ERβ) ,21,22. IWe also observed an increase in skin redness among those who applied the GEN product when compared to the others. Since skin color changes may provide some clues to intervention responses, it is important to consider the sources of variance associated with the individuals, the environment, and the colorimetric principles. Melanin, hemoglobin, carotene, and bilirubin are the main chromophores that determine skin color , and theThe present study demonstrated that the GEN product, when topically applied for 6 weeks, was considered safe. Only one participant experienced mild skin rashes on both cheeks, possibly owing to vigorous scrubbing and over-exfoliating the skin. This adverse event was unlikely to be associated with the GEN product because it did not occur again after a rechallenge with the GEN product.The results of the present study should be interpreted in light of the study’s context and limitations. First, the intervention was evaluated over a six-week duration in postmenopausal women. Therefore, the findings do not provide information about applications that would last for a longer period of time. Some benefits of the GEN product might have been seen if the study had been undertaken for a longer duration ,26,27,46The GEN product has advantages for postmenopausal women’s facial skin, especially for those who are older. It can moisturize facial skin while also reducing wrinkles and enhancing redness. The GEN product is safe and achieves high levels of participant satisfaction with regard to product usage. Further research is required to close the gap identified in this study, and it ought to be conducted over a longer duration with more research participants. It may be beneficial to investigate further how different genistein-based formulations might affect the product’s efficacy when combined with other skin-lightening and/or antioxidant ingredients.


Oryzias latipes is an important model organism for physiology, genetics, and developmental studies, and has also emerged as a relevant vertebrate model for aquatic ecotoxicology. Knowledge regarding its associated microbiota on the other hand is still scarce and limited to adults, despite the relevance of the associated microbiome to the host’s biology. This study provides the first insights into the establishment of bacterial microbiota during early developmental stages of laboratory-reared medaka using a 16S-rRNA-sequencing-based approach. Major shifts in community compositions are observed, from a Proteobacteria-dominated community in larvae and juveniles to a more phylum-diverse community towards adulthood, with no obvious difference between female and male specimens. Major bacterial taxa found in adults, including genera Cetobacterium and ZOR0006, establish progressively and are rare during early stages. Dominance shifts are comparable to those documented in another major model teleost, the zebrafish. Results from this study provide a basis for future work investigating the influence of medaka-associated bacteria during host development. In recent years, toxicology and ecotoxicology have embraced the so-called microbiome revolution ,2. A hosOryzias latipes is a major nonmammal vertebrate model organism used for genetic, developmental, physiological, and ecotoxicological assays [The medaka fish l assays . Thanks l assays ,11,12). l assays ,13.Microcystis aeruginosa was shown to trigger major changes in gut-bacterial microbiota composition [Cetobacterium was, for example, extremely stable whatever the changes; meanwhile, genus ZOR0006, the most abundant member of the Firmicutes, dropped in abundance upon exposure to cyanobacteria and their metabolites in all experiments.The medaka was already employed in several ecotoxicological experiments that investigated the response and influence of its associated microbiota ,15. A 28position  and a doposition . The gutposition . In thesThese examples confirm the potential of lab-reared holobionts, such as the medaka, for ecotoxicological approaches that would include investigation of the microbiome’s response. However, establishing relevant model holobionts for microbiome-aware ecotoxicology requires a better understanding of host–symbiont interaction than currently available. Lab-reared animals are, for example, known to harbor only a limited fraction of the microbiota found in their wild counterparts, and that difference needs to be characterized ,20. BesiTo support the relevance of the medaka fish as a model organism for microbiome-aware ecotoxicology, the aim of the present study is to understand how its bacterial microbiota is established throughout the different life stages in a laboratory setting. For this, bacterial partners are characterized using a 16S-rRNA-sequencing-based approach, starting from eggs to larvae, juveniles, up to sexually mature male and female specimens. Microbiota diversity and composition are explored in order to identify potential shifts in community structure as well as eventual differences between males and females. Altogether, this study provides new data regarding microbiota acquisition in an important model organism and will help account for microbial responses during early life stages.Oryzias latipes) were maintained in freshwater-containing aquaria in the temperature-controlled medaka facility of the MNHN. Water parameters were monitored on a regular basis and adjusted . Water was recirculated and replaced with freshwater  and 1/3 filtered) by automated pumps. Fish were fed three times daily (~3–5% of the fish biomass per day) with Nutra HP 0.3 . Reproduction was induced by shifting the light/dark cycle from 12:12 h to 15:9 h. Eggs were collected on day 0 postfertilization . For later stages, other eggs were placed in a Petri dish containing Yamamoto medium , then placed in an incubator at 28 °C until hatching. Larvae were collected at hatching , and the remaining larvae were transferred to freshwater-containing aquaria with a light/dark cycle of 15 h:9 h for 1 week in a small tank, then in a larger one for the following 3 weeks.Adult specimens of medaka fish . After d30 posthatching (d40 dpf), fish were transferred to a new aquarium, with a 12:12 h light/dark cycle, and specimen sampling occurred at d30 (5 specimens). Later samplings allowed sex determination and occurred at d90 posthatching  and d120 posthatching . Reproduction was then induced by shifting the light/dark cycle from 12:12 h to 15:9 h after d90, confirming that the remaining fish had reached sexual maturity. Upon sampling, fish were anesthetized in 0.1% tricaine methanesulfonate  buffered with 0.1% NaHCO−1). An extraction-blank control sample was also performed. The V3-V5 region of the 16S rRNA encoding gene was amplified using primers 341F and 926R [DNA was extracted from water filters. For fish samples, batches of 3 pooled eggs forming the same clutch were extracted to maximize the amount of recovered DNA. DNA from whole individual specimens collected at d10, d14, and d30 were extracted due to small specimen size. Finally, DNA was extracted from dissected guts for d90 and d120 specimens. Extractions were performed using the ZymoBIOMICS DNA Mini-prep kit with a FastPrep 5G beat beater disruption  and quality-controlled, chimeras were removed, and Amplicon Sequence Variants (ASVs) were obtained with DADA2  using QIIME2 . ASVs weEggs hatched 10 days (d10) postfertilization, and fish larvae started feeding at d14 dpf. Juveniles sampled until d30 posthatching could not be assigned to male or female. Female and male specimens were distinguishable starting d90 posthatching, and fish were able to reproduce at d120.Despite several attempts, no PCR product was obtained from any of the nine batches of three eggs, nor from any of the nine tested larvae collected at d10 dpf. Attempts to further pool eggs of larvae specimens did not yield PCR products either. Products were obtained from all specimens from d14 onwards, yielding a total of 650,196 paired-end quality-filtered reads  or in the Shannon index .Species richness was highest in water  and p = 0.17 (WU), 12 females and 11 males sampled at d90 and d120 and analyzed together). Males and females clustered together in the graphs  and Weighted UniFrac (WU) distances clearly separated water samples from fish samples; among fish samples, the different sampling dates were clearly separated, with high explanatory power on the first two axes  and below 1% in the water samples , which was less abundant at d14 and far less abundant after d30. Two other ASVs, affiliated to Hyphomicrobium (Alphaproteobacteria) and to the Gemmataceae (Planctomycetes) also peaked at d30 and were absent before this date and rare after.At the ASV level, some taxa were present throughout all dates, including one gammaproteobacterial ASV affiliated to genus  samples . Howeverch rarer . Other ACetobacterium. This genus represented a median of 1.5 to 2.8% of reads in water communities at all dates. However, it was almost absent from animals before d90, but then became by far the most abundant taxon in most specimens (median 75.5 ± 12.6% at d90 and 52.6 ± 19.5% at d120). At d120, Fusobacteria (again mostly represented by genus Cetobacterium) and Proteobacteria were still abundant, but there was also a clear increase in Firmicutes, mostly consisting of 7 closely related ASVs belonging to genus ZOR0006. This genus was present in all specimens from d90 onwards  and considerably increased at d120 (5.3 ± 10.0%), as observed in water samples, while it was absent from earlier stages. Besides these main ASVs, other ASVs were detected in most specimens starting at d90 and were then stable, including one affiliated to Shewanella and several others that were present in low abundances. No abundant ASV was sex-specific.Major changes occurred at d90. The main ASVs responsible for the shift towards increased abundance of Fusobacteria were affiliated to genus Despite these changes in abundances, many of the identified ASVs were shared between different life stages . When coOryzias latipes is a major vertebrate model organism for ecotoxicology as well as molecular biology, and addressing the composition of its associated microbiota at all life stages is necessary in the context of the emerging holobiont approach [The medaka fish approach ,32. Indeapproach ,33,34. IDespite repeated attempts and pooling of several specimens, no PCR product could be obtained from eggs and d10 dpf larval stage (at hatching), suggesting either the absence of bacteria or their occurrence at levels below the detection limit of the chosen PCR strategy. These stages correspond to developmental steps that predate mouth opening and the start of external food uptake. Thus, at these stages, colonization of the digestive tract through oral uptake cannot have started yet. It was previously shown that a very low abundance of bacteria occurred on the skin and gill of adult lab-reared medaka fish, based on the need to pool several samples to obtain a positive PCR response and on fluorescence in situ hybridization assays . This lack of efficient colonization of external epithelia could be linked to the relatively clean rearing conditions, which involve regular water renewal by a mixture of osmosed and filtered water, certainly leading to low bacterial abundance in the water. This assumption is also supported by the very low DNA yields obtained from water filters and from d0 and d10 samples here. Regardless, this certainly is specific to our lab-reared conditions. Indeed, colonization of the egg and skin surface is well-documented in aquaculture settings; for example, for commercial species such as the European Seabass and Gilthead Seabream, and in environmental settings as documented from the brown trout and some coral reef fish ,36,37. IPseudomonas), and some of which may also include potential opportunistic pathogens  [Bacterial diversity levels tend to increase only marginally at the latest d120) compared to earlier stages, but remain within the same order of magnitude, and evenness does not vary much, indicating that no huge variation in the overall level of fish microbiome diversity occurs during development. The first two stages in which bacteria are detectable, namely d14 and d30, were analyzed as whole specimens, their gut being too small to be dissected and analyzed. In these, Proteobacteria are dominant, mostly Gamma- then Gamma- and Alphaproteobacteria. Temporal shifts in dominant ASVs are also observed, mostly involving ASVs related to waterborne bacteria, many of which belong to groups previously documented in fish eggs  ,36,38. Iwanella) . Change wanella) . Howeverwanella) . Moreovewanella)  as well wanella) , Atlantiwanella) , and twoCetobacterium become dominant, and Firmicutes also become abundant, mostly consisting of genus ZOR0006. Major changes in community composition were also documented between 35 and 75 dpf in the zebrafish, with dominance of Fusobacteria and Firmicutes from 75 dpf onwards [Cetobacterium and ZOR0006 are two of the most dominant gut-associated bacteria identified in all our previous studies on the medaka from the MNHN facility [Cetobacterium is a major nonpathogenic gut-resident bacterium in many fish species, including zebrafish and tilapia [In the present study, from d90 onwards, Fusobacteria of the genus  onwards . Cetobacfacility ,17,18. C tilapia ,41,42,43 tilapia . It was  tilapia ,17. Its  tilapia ,18. Inte tilapia . Besides tilapia ,33, and Specimens from d90 and d120 could be assigned to pubertal or mature male or female. Their associated communities were not significantly different at a given date though, either in terms of diversity or in composition. Sex-dependent effects are documented in microbiota composition of vertebrates, assumed to be due to hormones, reproductive status, and immune response ,45. On tBacterial communities associated to lab-reared medaka were not detectable until after mouth opening, possibly due to the clean environment. After that, community compositions changed with time, with shifts in community compositions that resemble those documented in lab-reared zebrafish, another major model teleost. They converge towards the composition found in guts of adults upon sexual maturity, with no significant differences between males and females at least until day 120. Future studies investigating effects on the medaka holobiont during its development, for ecotoxicology as well as other research fields, must thus consider the changes that occur in microbiota composition, which is largely a function of life stage. This will be particularly important in tests that involve the earliest stages.