Abies alba L.) branches, has comparable chondrogenic potential with the other tested extract and the polyphenols under inflammatory and non-inflammatory conditions. In our study, Belinal promoted significantly greater chondrogenesis compared to the untreated (p = 0.0289) and resveratrol-treated (p = 0.0468) MSCs from patients with hip osteoarthritis under non-inflammatory conditions. Under inflammatory conditions, chondrogenesis was significantly enhanced for MSCs treated with Belinal compared to the control (p = 0.0483). The other extract and the polyphenols did not show any significant effects on chondrogenesis under non-inflammatory or inflammatory conditions. None of the tested extracts and polyphenols showed significant effects on chondrogenesis in healthy donors, under either non-inflammatory or inflammatory conditions. Our data show that Belinal can boost the chondrogenesis of MSCs derived from patients with osteoarthritis, under both non-inflammatory and inflammatory conditions.Several plant polyphenols have been shown to reduce osteoarthritis symptoms due to their antioxidant, anti-inflammatory and immunomodulatory properties. We investigated the effects of two different polyphenolic extracts  and two different polyphenols  on the chondrogenic potential of bone-derived mesenchymal stem/stromal cells (MSCs) from healthy donors and patients with osteoarthritis. Our main aim was to determine whether Belinal, a commercially available polyphenolic extract from silver fir ( Osteoarthritis is the most widespread, chronic, progressive and debilitating joint disease of the elderly population, and as such, it represents a huge socio-economic burden . TherefoA growing body of research shows that the complex aetiology of osteoarthritis comprises genetic, mechanical and inflammatory factors . OsteoarPro-inflammatory cytokines are critical mediators of cartilage breakdown; however, it is not yet completely known whether it is the damage to the cartilage that triggers inflammatory responses, or vice versa . VariousMesenchymal stem/stromal cells (MSCs) are multipotent progenitors that reside in the various musculoskeletal tissues, also in the adult organism . MSCs caAs inflammation has been shown to have a pivotal role in cartilage degradation, particular interest has focused on the anti-inflammatory actions of natural compounds, such as polyphenols. Polyphenols represent a wide variety of compounds that are found in fruit, vegetables, red wine, tea, plant oils, cocoa and other sources. Their antioxidant, anti-inflammatory and immunomodulatory properties have been studied under many chronic inflammatory conditions, including osteoarthritis ,14. ThesPinus maritima, Pinus pinaster), and it has been shown to have antioxidative, anti-inflammatory and chondroprotective effects in vitro and in vivo. Moreover, in patients with osteoarthritis, clinical studies with Pycnogenol as an adjunct supplement have provided pain relief, the improvement of stiffness, and enhanced mobility [Abies alba L.). It has shown higher antioxidant activity in vitro than that of resveratrol, ascorbic acid and butylated hydroxytoluene, but similar activity to epigallocatechin gallate [Resveratrol is commonly recognized by the general public as an antioxidant in red wine, and it has been the most commonly investigated polyphenol in such studies. Resveratrol has shown anti-osteoarthritic effects due to its anti-apoptotic, anti-inflammatory and antioxidant properties . Pycnogemobility . Quercetmobility ,19. Beli gallate . Abigeno gallate . The Bel gallate , it is r gallate . Belinal gallate ,24, althBased on these data, we investigated whether the polyphenols in the Belinal extract can stimulate the in vitro chondrogenic potential of MSCs derived from patients with osteoarthritis. To this end, we initially screened for the effects of Belinal, the bark polyphenolic extract Pycnogenol, and the two most commonly used polyphenols, resveratrol and quercetin, on the viability and proliferation of MSCs. Secondly, we investigated the effects of selected concentrations of these two polyphenolic extracts and the two polyphenols on chondrogenesis under non-inflammatory and inflammatory conditions. These data show significant beneficial effects of Belinal on the chondrogenesis of MSCs derived from patients with osteoarthritis, under both non-inflammatory and inflammatory conditions. This study thus provides evidence that Belinal can be used to boost the chondrogenic potential of MSCs from patients with osteoarthritis, and it provides the basis for future in vitro and in vivo studies of the treatment of patients with this debilitating disorder.p = 0.0008; one-way ANOVA with Bonferroni correction). However, no significant differences were seen for cell proliferation between the tested serial dilutions of the polyphenolic extracts and the polyphenols (one-way ANOVA with Bonferroni correction). Based on these data, the concentration of 375 µg/mL was selected for further experiments.Cell viability and proliferation were determined for four serial dilutions  of the polyphenolic extracts and polyphenols tested (performed in triplicate), using flow cytometry A and then = 3) and donors post mortem without musculoskeletal disorders  were used to determine the effects of the polyphenolic extracts and polyphenols on the chondrogenesis of MSCs. In the MSCs from patients with hip osteoarthritis, Belinal showed significantly higher absorbance of the extracted Alcian blue (and thus increased chondrogenesis) in comparison with the control and the polyphenol resveratrol . For the chondrogenesis of the MSCs derived from healthy donors, there were no significant differences seen between the different treatments .Cell proliferation was screened to determine the optimal lipopolysaccharide (LPS) concentration for further experiments, using MTS assays A. No sigOsteoarthritis is a widespread joint disease in the elderly population that has a substantial socio-economic burden. Stem cell exhaustion and the decreased potency of their regenerative potential have been defined as hallmarks of aging . DecreasAs inflammation constitutes the major part of the osteoarthritis pathology, compounds with well recognized anti-inflammatory properties are of particular interest for the treatment of osteoarthritis. Since phytochemicals in natural remedies can have anti-inflammatory, anti-oxidant and anabolic effects, much is to be expected from their chondro-inductive and chondroprotective effects for the treatment of osteoarthritis . PolypheIn the present study, we investigated the effects of Belinal on the in vitro chondrogenesis of hMSCs derived from patients with osteoarthritis and from donors (post mortem) with no musculoskeletal disorders. The effects of Belinal were compared to those of the previously well recognized antioxidative and anti-inflammatory polyphenols resveratrol and quercetin, and the pine-bark polyphenolic extract Pycnogenol. We initially performed cell viability and proliferation screening to determine the optimal concentration of the polyphenolic extracts and polyphenols tested. Then, we performed in vitro chondrogenesis assays without and with pre-treatment with LPS, to simulate non-inflammatory and inflammatory conditions, respectively.The data from the cell viability and proliferation screening indicated the use of 375 µg/mL as the optimal concentration of the polyphenolic extracts and polyphenols for chondrogenesis in this MSC model. Moreover, these data showed significant benefits of Belinal for in vitro chondrogenesis for the MSCs from patients with hip osteoarthritis under non-inflammatory conditions. Of interest, no such benefits were seen when the MSCs from healthy donors underwent the same treatments. This might be because bone-marrow-derived MSCs from patients with advanced osteoarthritis have reduced chondrogenic potential in comparison with healthy donors .tert-butyl hydroperoxide-stimulated rat chondrocytes, via the attenuation of oxidative stress and apoptosis [Previous studies have used resveratrol at concentrations as high as 100 µM (22.8 µg/mL) to show its anti-inflammatory effects on interleukin-1β-induced inflammation in primary human chondrocytes in vitro . Pycnogepoptosis . In addipoptosis .The concentration of the polyphenolic extracts and polyphenols used in the present study was higher than those used in previous studies. However, the cell models used in previous studies were different from these trabecular bone-marrow-derived MSCs from patients with osteoarthritis. The most similar cell model was used in a study by Casado-Díaz et al. (2016), where they used bone-marrow-derived MSCs, although they obtained these from healthy volunteers . It appeMoreover, the screening of serial LPS dilutions in the present cell model did not show any significant differences in cell proliferation for the LPS treatments up to 1 µg/mL. Hence, the concentration of LPS for chondrogenesis under inflammatory conditions was selected based on previous studies using other cell models; e.g., 0.001 µg/mL LPS was used to pre-treat mouse macrophages, and 0.010 µg/mL LPS was used to pre-treat a murine microglial cell line ,26. The To sum up, using primary human MSCs from patients with osteoarthritis, we showed significant stimulation by Belinal of in vitro chondrogenesis in comparison with two polyphenols and a further polyphenolic extract with previously shown anti-inflammatory effects in in vitro models of osteoarthritis.The main advantage of our study was the inclusion of the primary human MSCs from patients with advanced hip osteoarthritis as well as healthy donors with no musculoskeletal disorders. Although these represent in vitro models, they more closely resemble the endogenous cells that would be the real targets of these compounds in vivo. Therefore, our study provides useful in vitro data that underpin the need for further trials on the effects of Belinal in osteoarthritis.There are at least two main disadvantages in this study. First, primary cell isolation was performed from different joints in the patients with osteoarthritis and healthy donors. This was due to the lack of ethical consent to sample hip tissue in post mortem donors, as knee is more easily accessed during autopsies. Secondly, we did not test the primary human MSCs from healthy donors under inflammatory conditions. Larger numbers of repeat experiments should be carried out on a larger scale with healthy donors and patients to be able to provide stronger evidence of the positive effects of the Belinal on chondrocyte differentiation. Moreover, the variance between the different donors, as already observed, is to be expected, as the cell populations from different donors might very well differ. The higher number of donors and the analysis of the cellular compositions of MSC populations would strengthen these studies.Further studies are also needed to highlight the influence of Belinal in inflammatory disorders, e.g., rheumatoid arthritis.The study design and the analyses are summarised in The primary hMSCs used in this study were derived from three patients with hip osteoarthritis who were undergoing routine total hip arthroplasty at the Valdoltra Orthopaedic Hospital, Ankaran, Slovenia. Hip osteoarthritis was diagnosed by clinical examinations and X-rays. The exclusion criteria included a history of inflammatory arthritis, metastatic cancer and disorders that can affect the bone. Two donors with no macroscopic evidence or history of musculoskeletal disorder  who had routine autopsies carried out at the Institute of Forensic Medicine, Ljubljana, Slovenia, were also included in the study. Trabecular bone was sampled from the femoral head in the patients with osteoarthritis, and from the medial tibia in the healthy (post mortem) donors. Approval for this study was obtained from the National Medical Ethics Committee of the Republic of Slovenia . Written informed consent to participate in this study was obtained from all patients.2/O2 atmosphere. The cells were culture expanded, and cells at passages 3 to 5 were used for all of the experiments. The MSC-like phenotype of these primary hMSCs was confirmed prior to the experiments, according to the International Society for Cellular Therapy [Primary hMSCs from trabecular bone were isolated as described previously . Briefly Therapy , as prev Therapy .Abies alba) wood contains lignans, which constitute approximately 10% of the extract and include isolariciresinol, hydroxymatairesinol, secoisolariciresinol, lariciresinol, pinoresinol, and matairesinol [The standardised plant polyphenolic extracts Belinal and Pycnogenol were obtained from Abies Labs and Plantex, respectively, and the purified polyphenols resveratrol  and quercetin  were from Sigma-Aldrich . Phytochemical investigations of Belinal  and Pycniresinol . All fouPrimary hMSCs were seeded at 2000 cells/well in two 96-well plates, with four replicates per plate. Viability screening was performed for four serial dilutions of the two polyphenolic extracts and the two polyphenols, as 1500, 750, 375 and 187 µg/mL. The cells were treated for 7 days, with fresh dilutions of the polyphenolic extracts and polyphenols prepared every second day. The negative controls included cells treated with the culture medium, and also cells treated with culture medium with the addition of the treatment solvents , in two replicates. For flow cytometry, the cells were detached  and the cell suspensions were stained using the fixable viability dye eFluor 780 . Data were acquired using a flow cytometer , with a recording of at least 10,000 single live cells. The unstained control included the same cells without the addition of viability dye, and was used to set up the gate. The data were analysed using the FlowJo v10 software .To monitor cell proliferation, a commercial cell proliferation assay was used , according to the manufacturer instructions. The reagent was added to the culture wells and incubated at 37 °C for 4 h. Absorbance was measured at 490 nm using a microplate reader .Escherichia coli  was determined using MTS cell viability assays. Micromasses were formed using 150,000 cells in 10 µL serum-free medium that consisted of high-glucose Dulbecco’s modified Eagle’s medium , 100 nM dexamethasone , 1% insulin–transferrin–selenium , 50 mg/mL ascorbic acid-2-phosphate , and 2% penicillin and streptomycin . The micromasses were allowed to attach to the well surface of 24-well plates pre-covered with 1.0 g/L gelatine  for 45 min. Then, 0.5 mL of serum-free medium was added. The next day, chondrogenic medium was added to each well, which consisted of the same serum-free medium with the addition of 10 ng/mL transforming growth factor (TGF)-β1 , and with the addition of each polyphenolic extract or polyphenol to be tested. The controls comprised micromasses without TGF-β1 and control micromasses with TGF-β1 but without any polyphenolic extracts or polyphenols. The assays were carried out for 7 days, with regular changes of the medium with freshly prepared medium, as before.Chondrogenic differentiation was carried out using micromass cultures for all three of the donors under both physiological and inflammatory conditions. For the induction of inflammatory conditions, LPS was used as a general activator of proinflammatory cytokines, instead of a more specific cytokine activator like IL-1 or TNF-α. The optimal concentration of LPS from Chondrogenesis was assessed using Alcian blue staining . The micromasses were fixed using ice-cold methanol  for 30 min at −20 °C. The Alcian blue was filtered and 500 µL was added per micromass, for 12 h. The micromasses were washed three times with distilled water and the 24-well plates were scanned to visualise the stained micromasses. The Alcian blue was then extracted with 100 µL 6 M guanidine hydrochloride  in distilled water, over 6 h at room temperature, with gentle rocking. The extracted solutions were transferred (100 µL) to 96-well plates. Guanidine hydrochloride  was included as the blank. The absorbance of the extracted Alcian blue was measured at 630 nm using a microplate reader .E. coli O111:B4  was dissolved in phosphate-buffered saline at the stock concentration of 1 mg/mL. The cell proliferation screening to determine the optimal LPS concentration for further experiments was performed using MTS assays. Serial dilutions of LPS  were added to primary hMSCs from the patients with hip osteoarthritis, which were left for 18 h to simulate inflammatory conditions. After 18 h, the cell medium was changed and the tested polyphenolic extracts and polyphenols were added at 375 mg/mL. The MTS assays were performed as described above. Based on the results of these MTS assays, the optimal concentration of LPS was selected as 0.01 µg/mL for the condition of chondrogenesis under inflammation.Lipopolysaccharide from Therefore, micromasses were formed from MSCs derived from the patients with hip osteoarthritis and were pre-treated with 0.01 µg/mL LPS for 18 h to simulate inflammation. Then, the micromasses were treated with the polyphenolic extracts and polyphenols to be tested, and with the addition of TGF-β1, for 7 days. Alcian blue was used to determine chondrogenesis as described above.www.graphpad.com). p values < 0.05 were considered as statistically significant. The Figures were created using Mind the Graph (https://mindthegraph.com).To compare the data between the polyphenolic extracts, Belinal and Pycnogenol and the polyphenols resveratrol and quercetin, either one-way ANOVA or two-way ANOVA with Bonferroni post hoc for multiple testing was used. The statistical analyses were performed using GraphPadPrism, version 6 for Windows (GraphPad Software, La Jolla, CA, USA, In conclusion, these data show significant beneficial effects of Belinal, a water-extractable fraction of silver fir branches, on in vitro chondrogenesis of MSCs derived from patients with osteoarthritis, under both non-inflammatory and inflammatory conditions. This study thus provides evidence that Belinal can be used to boost the in vitro chondrogenic potential of MSCs from patients with osteoarthritis and suggests further in vitro and in vivo studies using this polyphenolic extract.


Objective: This study aims to explore the association between life events and coping styles, and how resilience and self-esteem mediate the association.Methods: A cross-sectional study was conducted among 981 left-behind adolescents (LBAs) in five junior high schools in Hunan Province, China, from April 13 to April 20, 2020. We utilized self-designed sociodemographic questionnaire, Adolescent Self-Rating Life Events Checklist, Resilience Scale Chinese Adolescent, Rosenberg Self-Esteem Scale, and Simplified Coping Style Questionnaire to assess the mental health of LBAs. Statistic description, Pearson correlation analysis, and structural equation model were adopted to analyze the data.Results: Results revealed that life events could negatively predict resilience  and self-esteem  and positively predict LBAs' positive coping style  and negative coping style . Self-esteem could also positively predict the resilience of LBAs ; resilience could negatively predict the negative coping style  and positively predict the positive coping style . Life events not only have direct effects on negative coping style (β = 0.21) and positive coping style (β = 0.28) but also have indirect effects on coping styles by affecting resilience (β = −0.29) and self-esteem (β = −0.39). The total effect of life events on coping styles was 0.32, where 34.37% was mediated by resilience and self-esteem.Conclusion: We proved that resilience and self-esteem mediated most of the effects of life events on coping styles. The findings had important implications for interventions to promote mental health of LBAs, particularly the enhancement of resilience and self-esteem. The widening gap in social and economic development between China's urban and rural areas and the relaxation of immigration restrictions have led to a large number of rural laborers leaving the countryside and seeking better job opportunities in cities. Children had to separate from their parents because of financial constraints and the transient nature of the work in urban areas . AccordiLife events refer to events or situations that challenge, threaten, damage, or exceed the physical and mental capacity of individuals . CompareCoping style is the process of managing external or internal demands and an important intermediary regulating factor in the process of psychological stress . Stone aResearchers found that some children develop well in some stressful situations, even beyond the level of normal children; resilience acts as an intermediary between adverse situation and good adaptation . Kumpfervia resilience.Self-esteem is also an important factor affecting psychological stress response . The AdoAlthough some researchers have investigated the relationships between life events, resilience, self-esteem, and coping styles, few researches have explored the protective factors of coping styles among LBAs in China, as well as the comprehensive relationships between these variables. In particular, few studies have divided coping styles into positive and negative coping styles in-depth discussion among LBAs. Considering parent–child separation is long-standing and widespread in the context of Chinese social environment, rapid change or reduction of the life events of LBAs may not be possible, whereas resilience and self-esteem can be changed under appropriate intervention –54. Accovia resilience.Based on literature review, a theoretical hypothesis model, as shown in A cross-sectional study was conducted among LBAs in Hunan Province, China, from April 13 to April 20, 2020. The number of LBAs in Hunan Province accounts for 10.1% of the total number of LBAs in China, making it one of the provinces with the largest number of LBAs in the country . From HuThe inclusion criteria for participants were as follows: (a) one or both of their parents are out-migrant workers for at least 6 months; (b) aged between 11 and 18 years; and (c) they are conscious and have volunteered for the study. Correspondingly, the exclusion criteria were as follows: (a) participants who are illiterate; (b) participants with insanity or severe mental disorders and inadequate communication ability assessed by psychiatrists; and (c) participants with hearing or speech dysfunctions.Two-step stratified random sampling was used in the study. First, we randomly selected one city in each of five administrative regions of Hunan province. Second, one rural junior high school in each sampled city was chosen randomly. In total, five rural junior high schools were included. Before data collection, we received the permissions from the headmaster and head teacher of each school. With the help of the head teachers, we gathered the participants in several classrooms and handed out questionnaires to them without the presence of teachers. After all questionnaires were collected, we divided the participants into LBA or NLBA group according to the answer to the question, “Did one or both of your parents out-migrant for work for at least 6 months?” and selected the LBAs' questionnaires for analysis. Finally, we recruited 1,000 LBAs in the study, and 19 students refused to participate or returned incomplete questionnaires. Thus, 981 questionnaires were available, and the response rate was 97.32%.N = [Uασ/δ]2. Uα was the value of υ corresponding to the test level α, σ was the overall standard deviation, and δ was the allowable error. Based on the preliminary experiment, the standard deviation was σ = 1.47. Taking α = 0.05, δ = 0.1, the minimum sample size was 830. Considering the 20% of the loss of access rate and sampling error, the sample size expanded to 1,000 , 10,000–20,000 RMB (intermediate), >20,000 RMB (high), and unclear], source of income, father or mother as out-migrant worker, working duration of parents, educational level of parents, occupation of parents, and contact frequency.Life events were measured by the Adolescent Self-Rating Life Events Checklist (ASLEC) , which cResilience was examined by the Resilience Scale Chinese Adolescent . The scaSelf-esteem was determined by the Rosenberg Self-Esteem Scale . The scaCoping style was measured by the Simplified Coping Style Questionnaire (SCSQ) . The scaThe ethics approval was obtained from the ethics committee of our university (no. E201946) before data collection. The permissions to collect the data were guaranteed by the rector and head teacher of each school. Prior to filling out any questionnaires, all participants were informed about the purpose of the study and signed an informed consent form, which informed that the whole study process was carried out completely voluntarily, anonymously, and confidentially. Further, participants have the rights to decline the study at any time without any penalty.via Pearson correlation analysis were examined. Third, the SEM was used to determine the hypothetical mediation model, and the relationship between variables was determined by using AMOS 23.0. Specifically, a hypothetical model of coping style to life events was constructed. We set life events as exogenous explicit variables, resilience and self-esteem as endogenous explicit variables, and positive coping styles and negative coping styles as endogenous latent variables. We then estimated the path coefficient and evaluated the data fit of each model.Data were put in and analyzed using SPSS 20.0 and AMOS 23.0. First, we checked for missing values, outliers, and normality and excluded 10 missing data before data analysis. Second, summative score values for each scale were calculated, and the relationships between variables 2 statistics [χ2/degrees of freedom (df )] and root mean square error of approximation (RMSEA). If the χ2/df was <3, the model would be regarded as a good fit; if it was between 3 and 5, the model would be considered an acceptable fit. Furthermore, if the RMSEA value was <0.05, it would indicate that the model had reached a close fit, and if the value was <0.08, the model would be counted as a good fit. Additionally, fit indices were the comparative fit index (CFI), goodness-of-fit index (GFI), normed fit index (NFI), relative fit index (RFI), and Tucker–Lewis index (TLI). If the CFI, GFI, NFI, RFI, and TLI values were 0.90 or higher, it would indicate that the model achieved a good fit . Of the total participants, 51.9% were males, and 48.1% were females. Less than two-thirds of LBAs' parents were out-migrant workers (58.1%), whereas 33.2% of LBAs' fathers were out-migrant workers, and only 8.7% of LBAs' mothers were out-migrant workers. Most of the fathers migrated to work in other cities for about 6–10 years (39.0%), whereas most of the mothers migrated to work in other cities for less than a year (50.6%).As shown in F = 2.813, P = 0.038) had significant differences in life event scores, and further Student–Newman–Keuls pairwise comparisons showed no difference. There were significant differences in life events between fathers' different working duration . The life event scores in order, from high to low, were 6–10 years, <1 year, and 2–5 years. Similarly, there were significant differences in life events between mothers' different working duration . The life event scores in order, from high to low, were <1 year, 2–5 years, and 6–10 years.The analysis of variance results showed that the different annual income of families , and the RMSEA was 0.064. Furthermore, the CFI, GFI, NFI, RFI, and TLI values were higher than 0.900 .First, the initial hypothetical model  showed uP < 0.001) and self-esteem  and positively predict LBAs' positive coping style , and negative coping style . It indicated that the more serious the negative life events perceived by LBAs, the lower their own level of resilience and self-esteem, and the more they are likely to choose negative coping style, while positive coping style also exists. Further, self-esteem could positively predict LBAs' resilience , indicating that the higher the self-esteem level of LBAs, the higher their resilience. Moreover, resilience could negatively predict the negative coping style  and positively predict the positive coping style , indicating that the richer the resilience resources of the individual, the more they are able to avoid negative response, yet the more they tend to adopt a positive response.Obviously, resilience and self-esteem partially mediated the relationship between life events and coping styles. The direct effect value from life events to negative coping style was 0.21, and the total mediating effect was (−0.29) × (−0.21) + (−0.39) × 0.62 × (−0.21) = 0.11; the total effect was 0.32. The mediation effect amount was 34.37%, and the mediation effect amounts of the two indirect pathways were 19.03 and 15.86%, respectively.via resilience.In the current study, we employed SEM to investigate the relationships between life events, resilience, self-esteem, and coping styles among LBAs in China. Although some researchers have investigated some relationships between two or three variables –39, 51, Our research showed that life events of LBAs are significantly related to coping styles, which supported hypothesis 1. The factors of life events were significantly positively correlated with negative coping styles, which was consistent with Wang's research –39, 51. Most studies –66 reveaThe current study showed that life events not only have direct effects on the negative coping style and positive coping style but also have indirect effects on coping styles by affecting resilience based on the SEM. This means that resilience mediated the relationship between life events and coping styles, which supported hypothesis 2. First, the study proved that life events, as an environmental factor that is not conducive to the normal development of individual psychology, have a significant negative effect on the mental health conditions of left-behind children, such as resilience. The longer the parent–child separation, the more frequent life events the LBAs may encounter, and the lower the level of mental health. Therefore, LBAs' parents can avoid working outside for a long time or increase the opportunities to come back home and communicate with children, and school can also hold various activities to enrich LBAs' lives and let them feel the love and support from teachers and classmates to reduce the negative impact of life events on the mental health of LBAs.Therefore, results showed that those with a higher level of resilience tend to cope more positively with life events, which were consistent with the previous studies –70. ExisResults also indicated that those with a higher level of self-esteem were more likely to cope with life events positively, and self-esteem cannot play the separate mediating role between life events and coping styles, relying on the mediating role of resilience, which were consistent with hypothesis 3. Self-esteem, as individuals' evaluation and perception of their own sense of life meaning and value, is considered to have an important role in maintaining mental health and promoting positive coping style . PreviouAlthough the study offered a preliminary conceptual framework of relationships between life events, resilience, self-esteem, and coping styles by using SEM, we acknowledged that the study has several limitations. First, the study used convenience sample to collect data, and the results could be biased toward those adolescents who were in good mental health conditions and motivated to share their perceptions. We still believe that it is unlikely that including the entire LBA population would have made much difference to the study results as the sample size was quite representative and the response rate was relatively high. Second, only LBAs in the rural junior high school in one province were selected in the current study. The sample may limit the generalizability of the results to wider age groups or geographical area. Future studies are recommended to include children in different stages of development and different locations in China or other countries. Third, although the researchers did their best to explain the questionnaire before collection, and participants were asked to finish questionnaires without the presence of teachers, our data were based on self-reports and may lead to information bias. Objective data collection method and data from parents, caregivers, teachers, and peers could complement our data. Fourth, although SEM is generally referred to produce information about causal relationships, the lack of use of longitudinal data prevents the interpretation form reflecting true causality. Therefore, further longitudinal or experimental studies should be conducted to better investigate causalities and the long-term effects of life events on coping styles of LBAs. Finally, the current study only focused on the relationships of life events, coping styles, resilience, and self-esteem among LBAs; future researchers could also identify the relationships between these psychological characteristics among NLBAs and compare the differences between LBAs and NLBAs.via resilience. Our research provided preliminary insight into the mechanisms that have a significant influence on the relationship between life events, resilience, self-esteem, and coping styles among LBAs. The present study provides a basis for policy makers, educators, or practitioners to develop target school activities or intervention programs designed to promote LBAs to adopt positive coping style toward life events by enhancing their resilience and self-esteem.We found that all the hypotheses we proposed have been supported. Results indicated that life events negatively affected resilience and self-esteem and positively affected coping styles in LBAs, whereas resilience and self-esteem appeared to play a protective role. Meanwhile, results showed that resilience and self-esteem acted as mediators between life events and coping styles. Resilience can directly mediate life events and coping styles, whereas self-esteem mediates life events and coping styles The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The study involving human participants were reviewed and approved by the ethics committee of Central South University (No: E201946). Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.JZ is the primary investigator of the study and provided comments and ideas, and revised this paper. JL and Y-pC did data analysis, developed, and revised the manuscript. Y-fL provided comments and ideas, and helped revised the manuscript. S-lY, C-xT, Y-xT, and B-yY helped conduct the study, including developing survey, sampling, data analysis, and proof reading the manuscript. M-mL and MV provided substantial critical suggestions for manuscript revision, interpretation of data for the work, and revised the manuscript. All authors read and agreed on the final revision of the manuscript.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.


Type 1 diabetes mellitus (T1DM) is a chronic, immune-mediated disease characterized by the destruction of insulin producing cells and persistent hyperglycemia. At present, the drugs for type 1 diabetes mellitus can reduce blood glucose rapidly and effectively, but there are risks of hypoglycemia, large fluctuation of blood glucose, and chronic complications. Related research found that compared with continuous hyperglycemia, blood glucose fluctuations are more harmful to the chronic complications of diabetes. Blood glucose variation is closely related to the occurrence and development of chronic complications of diabetes. Sancai powder (SC) is made on the basis of 3 ancient Chinese medicine formulas, which has the effect of lowering blood glucose. There have been reports on the clinical study of SC in the treatment of diabetic patients, but there is no systematic evaluation of SC in the treatment of type 1 diabetes, so it is necessary to summarize and evaluate the existing evidence.This study will be conducted according to Preferred Reporting Items for Systematic Reviews and Meta-analysis Protocols. We will search 3 English databases and 4 Chinese databases. Two methodologically trained researchers will read titles, abstracts, and full texts, and independently select eligible literature based on inclusion and exclusion criteria. After assessing the risk of bias and extracting data, we will conduct a meta-analysis of the results, including: standard deviation of blood glucose level, coefficient of variation, mean blood glucose, postprandial blood glucose fluctuation, hypoglycemia index, glycated hemoglobin, overall impact rate, and adverse effects. The heterogeneity of the data will be tested by Cochrane x2 and I2. Based on reliable subgroup effect guidance, we established 3 hypotheses for subgroup analysis: disease status at baseline, duration of intervention, and type of concomitant medication. Sensitivity analysis will be carried out to assess the stability of the results. The publication bias assessment will then be performed by funnel plot analysis and Egger test. Finally, we will use the “grading, evaluation, development and evaluation of recommendations” system to assess the quality of evidence.The results of this systematic review and meta-analysis will be published in a peer-reviewed journal.In our study, the evidence of SC in the treatment of reducing blood sugar fluctuation in type 1 diabetes will be comprehensively summarized and carefully evaluated. It will provide more options for clinical treatment of the disease.INPLASY202050052 Although China's type 1 diabetes registration study has confirmed that China is one of the countries with the lowest incidence of type 1 diabetes in the world, due to the large population in China, the actual number of type 1 diabetes patients is not small. The study shows that there are at least 13,000 new cases of diabetes every year in China, of which 4271 are type 1 diabetes patients. According to data from the International Diabetes Federation world diabetes map 2017, the number of newly diagnosed children with type 1 diabetes in China ranks fourth in the world, and the top 3 are followed by the United States , India , and Brazil (7600). Treatment of T1DM puts a heavy burden on the government's finances.Type 1 diabetes mellitus (T1DM) is an autoimmune disease, which mainly occurs in children and adolescents, and exists in all ages. The main causes of the disease are genetic factors, environmental factors, autoimmune response, virus infection, and drug exposure. In this way, the immune response of the body is disturbed, and the autoantigen is activated, which destroys the B cell of the islet of Langerhans, and the insulin of the body is absolutely insufficient, leading to diseases. It is characterized by rapid onset, long duration, multiple complications, as well as thirst and polyuria.,6 Blood sugar fluctuations are closely related to the occurrence and development of chronic complications of diabetes.–10 Related studies have shown that insulin resistance, oxidative stress, and inflammatory factors can cause blood sugar instability in diabetic patients.,12 This increases the risk of various complications of diabetes. The main treatment methods of TIDM include islet/pancreatic cell transplantation, insulin injection, weight loss, stem cell therapy, and immune intervention. These therapies have their own limitations. Due to the presence of autoimmune reactions, islet/pancreatic cell transplantation cannot fundamentally solve the problem and is expensive. Although insulin injections are convenient and fast, many patients have difficulty persisting with daily injections, which can easily cause hypoglycemia and exacerbate blood sugar fluctuations. Weight loss therapy is not suitable for non-obese type 1 diabetic patients. Stem cell therapy and immune intervention therapy have great potential, but are not yet mature, and further research is needed,,15 so high-quality research is urgently needed. This study will systematically collect clinical studies of sancai powder (SC) treatment of T1DM, and conduct a systematic review and meta-analysis to provide a reliable basis for clinical practice.In addition to controlling high blood sugar, patients with type 1 diabetes should pay attention to blood sugar fluctuations. The harm of blood sugar fluctuations to chronic complications of diabetes is even greater than that of persistent high blood sugar.SC comes from the Chinese Medicine ancient book “Wen Bin Tiao Bian”,which is composed of 6 traditional Chinese medicines (TCMs) such as Tian Dong, Raw Dihuang, Ginseng, Coptis, Cinnamon, and Dark Plum. Designed to balance the yin and yang of the human body. SC has clear efficacy in treating diabetes. Modern pharmacological studies have shown that the 6 herbs in its group have hypoglycemic effects. In addition, raw dihuang can inhibit the production of inflammatory factors, and ginseng has both anti-oxidant and anti-inflammatory effects. Coptis can improve insulin action on target cells and relieve insulin resistance. Cinnamon can increase insulin sensitivity and reduce insulin resistance. In addition to reducing blood sugar, dark Plum also has antioxidant effects. SC strictly follows the theory of compatibility of TCM prescriptions and SC in multiple mechanisms, multi-targeted way to lower blood sugar levels, relieve glycemic excursions. SC has been widely used in the prevention and treatment of diabetes in China, but there is currently no meta-analysis on SC to improve blood glucose fluctuations in type 1 diabetes. Therefore, the urgent need for high-quality research. In this study, we will systematically collect SC clinical study of type 1 diabetes treatment, and a systematic review and meta-analysis to provide reliable evidence for clinical practice.2To investigate:1.Efficacy and safety of SC on Chinese patients with type 1 diabetes.2.Whether SC can effectively reduce the blood sugar variability of patients with type 1 diabetes in China.33.1https://inplasy.com/inplasy-2020-5-0052/. The article will be reported in accordance with the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols checklist.The protocol for this systematic review was registered on INPLASYand is available in full on 3.2Those randomized controlled trials that treated T1DM with SC in combination with placebo or conventional treatments will be included in our study. Conventional treatments include oral hypoglycemic medications and insulin injections. Of course, insulin pumps are among the routine treatments. TIDM diagnosis based on World Health Organization (1999) criteria. In order to obtain as many references as possible and reduce bias, we do not have any restrictions on factors such as age, gender, and duration of illness.3.3Studies that met the following criteria will be excluded for the meta-analysis:1.increased blood sugar is not due to T1DM being excluded.2.In some studies, the authors uses SC and other medicines together in experimental group, since SC is not the main intervention in these studies, these studies will be excluded.3.Studies whose outcomes are not directly related to reduce glycemic excursions or lower blood sugar.4.Repeatedly published articles.3.43.4.1Standard deviation of blood glucose level and coefficient of variation .3.4.21.MBG, PPG, hypoglycemia index, glycated hemoglobin.2.Total effective rate; the judgement of effectiveness takes into account the improvement of examination indicators and the relief of clinical symptoms.3.Hyperglycemia or hypoglycemia that may occur during the study.4.Have any adverse effects of medication throughout the process.3.5We will search 4 Chinese databases, including articles knowledge service platform, VIP information resource integration Service platform , the China National Knowledge Infrastructure database, China Biomedical CD (Sino Med), as well as 3 English databases, including Embase, PubMed, Cochrane Library Controlled Trials Register, Chinese, and English restricted languages. In addition, to identify unpublished studies or other relevant documents, we will also search Google scholar, Baidu scholars. Last but not least, the Chinese Clinical Trials Registry, and ClinicalTrials.gov will also be searched. A manual search will be performed in the Chengdu University of TCM library. We will use a search strategy that combines MeSH words with free words. Two authors will search and screen all the citations independently. The process of search is presented in the following Table 3.6First, the electronic references downloaded from the above database will be managed by us in the Mac version of endnote X7 . In the second part, 2 researchers trained in methodology read titles, citations and abstracts, and selected citations according to inclusion and exclusion criteria. By reading the full text, they will further determine those articles that meet the conditions. In case of any difference, they will make a final decision by consensus. A flowchart has been drawn to show the process of study selection Fig. .3.7We will extract the data of these qualified articles into Microsoft Excel. For each study, the following data will be extracted: first author of the article, year of publication, country, intervention in the experimental group, intervention in the control group, number of patients in each group, age, and gender of patients, BMI, lifestyle, course of disease, Time of illness, results, and safety data. If there is not enough data in the study, we will contact the corresponding author for more detailed data. When the details of the method are not mentioned in the paper to be extracted, we will contact to provide further instructions.3.8 Two reviewers will independently assess the risk of bias and any differences will be resolved through discussion by all reviewers.The 2 reviewers will use a mature and reliable way to assess the risk of bias in the study: the Cochrane Collaboration tool. In this tool, 7 items were used to evaluate the risk of experimental bias: random sequence generation (selection bias), assignment concealment (selection bias), blindness of participants and personnel (performance bias), blind (detection) bias of result evaluation, incomplete result data (loss bias), selective reporting (reporting bias), and other biases. Each project is divided into “low risk”, “high risk” or “unclear risk”.4 When P < .05 and I2> 50%, statistical heterogeneity is considered important. If P > .05 and I2 <50%, the included studies are homogeneous and the difference between the 2 is negligible. If there is significant heterogeneity, the random effect model is used to aggregate the data; if there is no significant heterogeneity, the fixed effect model is used. If a large amount of heterogeneity does not allow quantitative synthesis, the results will be presented in the form of tables and charts.Reference management will be completed by MAC's endnote X7 (Thomson Reuters), while Review Manager 5.3 software  and MAC's stata14.0 software  will be used to create forest maps and perform group analysis and sensitivity analysis. For continuous variables, the effect is expressed as mean difference and 95% confidence interval. For binary variables, the impact will be expressed as a risk ratio and a 95% confidence interval and will be calculated using the Mantel–Haenszel (MH) method. The heterogeneity of the data will be studied by Cochrane x2 and I2 tests.5 We will perform subgroup analysis based on these subgroup hypotheses. Then, we will evaluate the credibility of the subgroup analysis according to reliable subgroup analysis guidelines. If there is enough research, then meta-regression will be conducted to further explore the source of heterogeneity.When there is great heterogeneity between studies, we will conduct subgroup analysis and meta-regression to explore heterogeneity. The patient's condition at baseline, the type of medication used during treatment, and the duration of intervention are the 3 hypotheses we established for the subgroup analysis.6The purpose of conducting sensitivity analysis is to ensure the stability of the research results. Each study included in the analysis will be excluded one by one, and then re-analyze and summarize the data, and compare the difference between the regained effect and the original effect. In this way, the impact of a single study on the overall results and whether the results are reliable will be assessed.7,22 In the case of P < .05,we think there is a publication bias.When there are more than 10 studies in this analysis, the publication bias will be evaluated in the form of funnel chart analysis, and the statistical survey will be conducted by the method of Egger test.8 Finally, the evidence will be shown from 5 domains: certainty assessment, number of patients, effect, certainty, and importance. In the Grading of Recommendations Assessment, Development, and Evaluate system system, the quality of evidence can be defined as “high”, “moderate”, “low”, and “very low”.At this stage, we will use a tool that is widely used in quality assessment: Grading of Recommendations Assessment, Development, and Evaluate system to summary finding tables for each out comes.9There are no patients and the public in this study10This meta-analysis does not require moral approval. The existing research evidence of SC will be comprehensively evaluated in our research. Through this strategy, we will provide clinicians with more evidence-based medical support for SC treatment of TIDM. Finally, our results will be published in peer-reviewed journals.11–27 With the increasing application of SC in clinical and a series of clinical studies have been carried out about it. We should systematically and comprehensively summarize its existing clinical evidence. In this study, we will conduct this systematic review and meta-analysis to provide more evidence-based medical support for clinical use of SC.T1DM is a disease should not be underestimated, as the disease progresses, patients often face the risk of various complications of diabetes. In addition, T1DM has been a huge burden on national health care.Although SC has been widely used in the treatment of diabetes in China, this is the first systematic review and meta-analysis of SC in the treatment of type 1 diabetes. The results of the study may help us develop a better way to reduce blood sugar fluctuations in patients with type 1 diabetes, thereby providing patients and clinicians with more choices. The limitation of this analysis is that the number of studies that meet our screening criteria is not many, but in order to obtain as many studies as possible, we do not have any restrictions on factors such as age, gender, and duration of illness.In this study, we will conduct subgroup analysis and regression analysis in order to obtain more accurate and reliable conclusions to explore the possible heterogeneity between studies. In order to avoid meaningless post-analysis, we will conduct a subgroup analysis based on a predetermined set of hypotheses to assess reliability standards. Finally, in order to provide better guidance for clinical use, we will classify the available evidence.Conceptualization: Weiwei Yu, Dongqi Zhou, Li Zhang.Data curation: Dongqi Zhou, Li Zhang.Formal analysis: Weiwei Yu, Rumeng Chen.Funding acquisition: Ziping Gao.Investigation: Dongqi Zhou.Methodology: Peishuai Zhang, Rumeng Chen.Project administration: Ziping Gao.Resources: Lisha Sun.Software: Peishuai Zhang, Weiwei YuSupervision: Rumeng Chen.Writing – original draft: Weiwei Yu, Dongqi Zhou, Li Zhang.Writing – review & editing: Weiwei Yu, Dongqi Zhou, Li Zhang.


Antipathella subpinnata colonies from three mesophotic shoals situated along the Ligurian Coast of the Mediterranean Sea  in the spring of 2017. At the Portofino shoal, samples of A. subpinnata and the gorgonian Eunicella cavolini were collected in November 2016 and May 2017. Bacterial communities were profiled using 16S rRNA gene amplicon sequencing. The bacterial community of E. cavolini was consistently dominated by Endozoicomonas. Contrastingly, the black coral microbiome was more diverse, and was primarily composed of numerous Bacteroidetes, Alpha- and Gammaproteobacterial taxa, putatively involved in all steps of the nitrogen and sulfur cycles. Compositional differences in the A. subpinnata microbiome existed between all locations and both time points, and no phylotypes were consistently associated with A. subpinnata. This highlights that local conditions may influence the bacterial community structure and potentially nutrient cycling within the A. subpinnata holobiont. But it also suggests that this coral holobiont possesses a high degree of microbiome flexibility, which may be a mechanism to acclimate to environmental change.Black corals are important habitat-forming species in the mesophotic and deep-sea zones of the world’s oceans because of their arborescent colony structure and tendency to form animal forests. Although we have started unraveling the ecology of mesophotic black corals, the importance of the associated microbes to their health has remained unexplored. Here, we provide in-depth assessments of black coral-microbe symbioses by investigating the spatial and temporal stability of these associations, and make comparisons with a sympatric octocoral with similar colony structure. To this end, we collected samples of  Antipatharians, commonly known as black corals, can be found throughout the world’s oceans from tropical to polar latitudes . This taMacro-organisms live in symbioses with microbes (an assemblage termed the ‘holobiont’), which are known to play important roles in host health, and contribute to their host’s adaptation and acclimation to environmental change . In coraAntipathes dichotoma   and two Pacific Antipathes spp. found a similar microbial community composition at the phylum level  , and a rluetkeni . Howeverluetkeni , but a moroflexi . Two recum level . Howeveraberrima . These baberrima .L. glaberrima holobiont as well  is the most common mesophotic black coral species in the Mediterranean Sea, but it has also been reported in the Atlantic Ocean (–2) aggregations     and Eunih, 1887) , which ah, 1887)  suggest the case . E. cavobpinnata . Besidesbpinnata . In contn detail , 2018b, A. subpinnata. In addition, we provide a comparison with the gorgonian E. cavolini to assess differences between the microbiota of these two sympatric species. We show that, in contrast to the gorgonian, the bacterial communities of A. subpinnata are highly diverse and differ significantly between locations and between autumn and spring time points. Interestingly, no bacteria were consistently associated with this species across space and time, suggesting that local environmental conditions are the main drivers of the microbiota associated with this black coral.Here, we describe the spatial and temporal patterns in the bacterial communities of the arborescent black coral Antipathella subpinnata, samples (one per colony) were collected by technical diving in Portofino   and spring . Here, samples of 6 colonies of Eunicella cavolini were also collected at both time points. To characterize the spatial stability in the microbiome of A. subpinnata, samples were collected from two other locations off the Ligurian coast near Bordighera  and Savona   in a Niskin bottle. The collected seawater was sequentially filtered through 3 and 0.2 μm Whatman Nucleopore Track-Etched filters (Sigma-Aldrich) and the 0.2 μm filter was kept in RNAlater at 4°C. An overview of the sample collection can be found in To assess the effect of seasonality on the microbiome of m depth)  in autum 14.3°C)  in sprinEunicella cavolini and a small population of A. subpinnata is located on the steep westernmost side of the shoal  on the margins of this animal forest  was used according to the manufacturer’s protocol, with the exception that the bead beating was performed at 30 Hz for 1 min using a CryoMill  at room temperature. Extracted DNA was shipped to Macrogen  for sequencing library construction using the 341F (5′-CCTACGGGNGGCWGCAG-3′) and 805R (5′-GACTACHVGGGTATCTAATCC-3′) primers that target the V3-V4 regions of the 16S rRNA gene , and 2x CT = 15.0 ± 0.23) and 0.148 ± 0.015 ng/μl (CT = 19.6 ± 0.08) of bacterial DNA in seawater and coral samples, respectively. The negative DNA extraction kit controls contained 0.02 ± 0.05 pg/μl (CT = 32.4 ± 1.28) of bacterial DNA. As such, the potential contamination was estimated to be low and had likely no to little impact on our results.Negative DNA extraction kit controls  were not included in the sequencing run, although DNA extraction kits are known to be contaminated with microbial DNA. This may affect 16S rRNA gene amplicon sequencing results , especia1)  . The raw1)  using thA. subpinnata samples collected in spring 2017 were identified as outliers (two samples from Portofino and one sample from Bordighera) during sample quality assessment of the dataset during differential abundance analysis (see below), and were also removed from the OTU table.Unassigned OTUs, and OTUs classified as chloroplast or mitochondria were excluded from the dataset. The most abundant OTU, OTU1 , was also removed from the dataset after it was identified as a match to the mitochondrial 12S rRNA gene of black corals.  Three decontam (A. subpinnata and E. cavolini) samples separately because of the large difference in microbial DNA concentration in the samples. In case of contaminants, a bimodal distribution of the decontam scores is expected (P∗ < 0.05) range  had a very low prevalence (present in 2–3 samples), indicating low classification accuracy and sensitivity . The unfiltered OTU table, sample metadata and representative sequences of each OTU are provided in the H, evenness: Simpson’s E) were calculated from the OTU table using QIIME v1.9 (phyloseq package (DESeq2 package (version 1.20.0)  between time points and sampling sites. Overall effects of location and time point on the coral-associated bacterial communities were investigated using the adonis function in the R-package vegan   in R (ve 1.20.0) , were usge vegan . Core mi2)  were included. Differences in the relative abundances between locations and time points of functional groups overall were analyzed using a negative binomial generalized linear model using the R-package MASS (multcomp (The PICRUSt2 (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) pipeline  was used2) , the rel2) . OTUs thage MASS  followedmultcomp .Antipathella subpinnata and the gorgonian Eunicella cavolini were distinct from the surrounding seawater at each location and time point  between the three sampling locations , and diversity (Shannon-Wiener H) are provided in A. subpinnata and seawater, except at the Savona shoal (p = 0.0001). In addition, the prokaryotic communities of both A. subpinnata and the seawater showed significant spatial differences (p < 0.02), however, no difference was found in the microbiota of A. subpinnata collected near Portofino and Bordighera. At the Portofino location, significant differences in the richness of the microbiota were found between the coral species and seawater. A similar pattern was observed in microbiota diversity, with the exception that the diversity in the seawater and in the A. subpinnata-associated microbiota was similar. No temporal differences were observed in any of the alpha diversity metrics.Outcomes of the analyses of the three alpha diversity metrics richness (observed OTUs), evenness , Bacteroidetes, Firmicutes, Cyanobacteria, Planctomycetes and Verrucomicrobia  was also found commonly associated with A. subpinnata (relative abundance ∼0.05%) from the Bordighera (9/10 samples) and Portofino  populations, but it was absent in the Savona population , Chlamydiales OTU45, Rickettsiaceae OTU102, Mycoplasma and Endozoicomonas (Endozoicomonas OTUs (OTU2 and 7) were the main core bacterial symbionts of the sympatric gorgonian E. cavolini , Planctomycetes, Alteromonadales and Thiotrichales compared with May 2017, but lower levels of Endozoicomonas and Rickettsiales bacteria  were also differentially abundant in the seawater , DMSP-demethylating , which may have been particularly related to the higher relative abundances of Endozoicomonas in May compared with November  the composition of the black coral microbiota in the context of coral microbial ecology, (2) the putative functions of the prokaryotes associated with A. subpinnata, (3) the potential causes of the high variability in the microbiota of this black coral, and (4) the implications that the lack of a ‘core microbiome’ may have for this coral species as well as for the field of coral microbial ecology in general.We present one of the first in-depth assessments of the bacterial communities associated with black corals, which provide crucial forest-like structural habitat in the mesophotic and deep sea, by profiling its associated microbial community on both spatial and temporal scales. Our findings show that A. subpinnata was dominated by Proteobacteria , Bacteroidetes and to a lesser extent by Firmicutes, Cyanobacteria, Planctomycetes, and Verrucomicrobia. When placing these results in context with the five other studies on black coral-associated microbes published to date , possesses an associated microbial community that is composed mostly of Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria (A. subpinnata and Bacillus), Actinobacteria  and Gammaproteobacteria . This also suggests that many of the bacterial symbionts of black corals may be amenable to cultivation, allowing more detailed studies of their function.Our study shows that the microbiome of the black coral  to date , some inbacteria . In addibacteria  isolatedA. subpinnata and other black corals were also found to share significantly more characteristics of the microbial communities associated with other Hexacorallia than with Octocorallia. The holobiont of Mediterranean A. subpinnata showed similarities with the Mediterranean corals Oculina patagonica and Cladocora ceaspitosa. These shallow scleractinian corals also harbor relatively high levels of Bacteroidetes and Alpha-, Delta- and Gammaproteobacteria  and lower levels of Verrucomicrobia, Planctomycetes, Cyanobacteria and Firmicutes  and sulfur (DMSP metabolism)  and retain (via nitrification and ammonification) nitrogen within the holobiont. Nitrogen fixation has been shown in the deep-sea coral L. pertusa  . This leommunity . The pre pertusa  and a co pertusa . Howeverplankton .Thalassospira, Vibrio, Pseudoalteromonas, Psychrobacter and Bacteroidetes cycling is therefore of high importance for a holobiont. In addition to the uptake of inorganic phosphate, phosphonates are another main source of phosphorus in the marine environment. Although primarily a microbial process, some marine invertebrates  possess genes representing the complete pathway for phosphonate synthesis  and controidetes . These tA. subpinnata shows that sulfur cycling likely takes place within the black coral holobiont. Although the metabolic activities of these microbes are unknown, the relatively high levels of sulfide-oxidizing bacteria in comparison with sulfate-reducing bacteria may indicate a net loss of sulfur.We also found several microbial taxa linked to the degradation of dimethylsulfoniopropionate (DMSP), a sulfur compound shown to be produced at high levels by coral animals . The sulA. subpinnata as Vibrio bacteria were often present at low abundance in its microbiota. These bacteria are commonly found in corals, particularly in the surface mucus layers    have beeA. subpinnata holobiont. This suggests that functional redundancy may be present within the A. subpinnata microbiota. However, it also implies that, rather than having a true ‘core microbiome,’ this black coral holobiont may ensure the presence of important core functions within its microbiome, with a potential microbiome regulatory role for some of its common microbial associates.Overall, it appears that each step in the cycling of nutrients may be performed by a number of different bacterial taxa present in the A. subpinnata were found to be distinct at each of the three locations and different between the spring and autumn. As all colonies were visually healthy at the time of collection, a cause for these differences is difficult to identify. However, it was unlikely to be related to the trapping of bacterioplankton in the large amounts of mucus produced by this black coral  handles petroleum products, harbors a large fleet of commercial fishing vessels and is frequented by cruise and cargo ships, whereas the ports of the other two localities are primarily used for yachting. It is tempting to speculate that these microbes may convert these hydrocarbons into a bioavailable carbon source for the coral host as has been previously hypothesized for deep-sea corals from the Red Sea  . ParticuA. subpinnata: plankton. It was demonstrated recently that A. subpinnata in the Portofino area feeds primarily on mesozooplankton in spring, but nano- and picoplankton (incl. the bacterioplankton) in autumn , which has been linked to tolerance of another coral in the ‘microbiome regulator’ spectrum to environmental change  at all locations. This suggests that some degree of fidelity still exists within the composition of the holobiont, but not at the 97% phylotype/OTU level. Instead, black corals may rely on a ‘functional core’ (i.e., a complement of metabolic and other molecular functions that are performed by the microbiome but are not necessarily provided by the same organisms, sensu stricto criterion for functional relevance as the composition of a holobiont may depend on multiple factors, including age, sex, life history and environment. Functional and/or metagenomics studies are, however, required to investigate whether changes in the microbiota affect the functioning of the coral holobiont.However, it should be noted that 90–95% of the prokaryotic community of A. subpinnata here or the other species studied in-depth, L. glaberrima (A. subpinnata may explain this lack of a bacterial ‘core microbiome.’ In agreement with Drosophila melanogaster found that phylosymbiosis may also be driven by short-term changes in the microbiota (Rigorous sampling at multiple locations, depths and over time has revealed that numerous scleractinian and gorgonian species possess a bacterial core microbiome, i.e., they consistently associate with certain bacteria regardless of space and time. Black corals, however, seem to largely lack this feature, as no core microbiome has been detected in aberrima . The higcrobiota . ConsequA. subpinnata, have limited microbiome regulatory capacities. Yet, microbiome flexibility may also allow these corals to tailor their microbiota to local, and potentially changing, environmental conditions by selecting the most beneficial microbes from the surrounding environment. This could thus be another strategy that may have contributed to the black corals’ success in colonizing the mesophotic zone and deep sea.The composition of the bacterial communities of black corals is more similar to the microbial community associated with the reef-building scleractinian corals than the structurally similar, but more distantly related, and sympatric gorgonian corals. The potential lack of a core microbiome and major spatial and temporal differences observed shows that environmental factors largely determine the compositional differences of the black coral-associated microbial communities. However, whether the functioning of the microbiota has changed or that there is functional redundancy remains to be assessed through functional and metagenomics studies. This may suggest that black corals, such as The datasets generated for this study can be found in the NCBI Sequence Read Archive: PRJNA506661.JW, MC, MB, and CF-P designed the study. MC, FE, and MB collected and processed the samples. MC performed the DNA extractions. JW analyzed the sequencing data. JW, MC, MB, and CF-P wrote the manuscript. All the authors reviewed, edited and approved the final manuscript.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 the selected clinical information and retinal vessel measures were used to develop different discriminative models for QDBS and PSIC using logistic regression. Discriminative efficacy and model performances were evaluated by plotting a receiver operating characteristic curve. As compared to QDBS, the PSIC patients had a lower incidence of insomnia problems  and a higher tortuosity index . Moreover, the area under the curve of the logistic model showed that its discriminative efficacy based on both retinal and clinical characteristics was 86.7%, which was better than the model that employed retinal or clinical characteristics individually. Thus, the discriminative model using AI-assisted retinal characteristic analysis showed statistically significantly better performance in QDBS and PSIC syndrome differentiation among stroke patients. Therefore, we concluded that retinal characteristics added value to the clinical differentiation between QDBS and PSIC.We aimed to investigate the efficacy of an objective method using AI-based retinal characteristic analysis to automatically differentiate between two traditional Chinese syndromes that are associated with ischemic stroke. Inpatient clinical and retinal data were retrospectively retrieved from the archive of our hospital. Patients diagnosed with cerebral infarction in the department of acupuncture and moxibustion between 2014 and 2018 were examined. Of these, the patients with Qi deficiency blood stasis syndrome (QDBS) and phlegm stasis in channels (PSIC) syndrome were selected. Those without retinal photos were excluded. To measure and analyze the patients' retinal vessel characteristics, we applied a patented AI-assisted automated retinal image analysis system developed by the Chinese University of Hong Kong. The demographic, clinical, and retinal information was compared between the QDBS and PSIC patients. The  Since 2012, stroke has been a leading cause of death and disability in China, and its incidence has been increasing at a rate of 8.7% per year . One of According to the “Guidelines for the Diagnosis and Treatment of Common Diseases in the Traditional Chinese Medicine” , the synTCM treatment selection for stroke is based on the TCM syndrome types. Patients with QDBS are characterized by hemiplegia, weakness of limbs, numbness of the body, deviation of the tongue, swelling of hands and feet, pale complexion, shortness of breath, lack of strength, palpitation and spontaneous sweating, deviated and pale tongue with a thin white coating, and a fine gentle pulse, therefore improving Qi and promoting blood circulation is the first principle of treatment . On the An essential component of TCM diagnosis is an overall observation of human symptoms, which is defined as the TCM syndrome. However, due to the lack of standard and objective evaluation criteria, such diagnoses, can be influenced by the doctors' personal experience, which puts the repeatability and reliability of the diagnosis in question . In lieuOne of the critical pathological changes of stroke is cerebral vascular change . AccordiThis study was approved by the Ethics Committee of the Shenzhen Traditional Chinese Medicine Hospital  and was performed in accordance with the guidelines of the Declaration of Helsinki (1964). All patients provided written informed consent for their participation in the study.In the case-control study, a total of 328 ischemic stroke patients from the Shenzhen Traditional Chinese Medicine Hospital were included. Patients were divided into 2 groups according to their TCM syndrome types: the QDBS group and the PSIC group. The patients' demographic and clinical data, including age, sex, medical history, physical examination, laboratory test, and electrocardiography results, were collected by trained doctors.The inclusion criteria were as follows: the subject was in the recovery stage of ischemic stroke, aged between 30–80 and was adequately able to maintain his/her posture while sitting for the duration of the retinal photography procedure. If the subjects were found to be suffering from any of the following conditions, they were excluded from the study: clinically unstable and requiring close monitoring, moribund, had an eye disease that severely affected retinal vessel structures, or was physically or subjectively unable to comply with magnetic resonance (MR) examination. In addition, if the patient was suspected to have cerebral diseases or conditions that may potentially alter retinal vessel morphology, he or she was excluded as well. Eventually, 196 of the 328 patients with ischemic stroke were included in our study .Risk factors related to cerebral infarction, such as hypertension, diabetes, dyslipidemia, high homocysteine, coronary heart disease, atrial fibrillation, smoking history, drinking history, and sleep disorders, were collected in this study. Hypertension was defined as a systolic blood pressure greater than 140 mmHg and a diastolic blood pressure above 90 mmHg or the use of antihypertensive medication for up to 2 weeks prior to the start of the study. According to diagnostic criteria from the National Diabetes Data Group, diabetes mellitus was defined as a fasting serum glucose level of more than 6.99 mmol/L, a nonfasting value of more than 11.1 mmol/L, or a history of treatment for diabetes . Based ond day of hospital admission using the SmartScope Ey4 Camera . To ensure the compatibility of the parameters, all the retinal images were scaled to 1365∗1024 pixels and saved in the jpg format. A patented, fully automatic retinal image analysis was applied to measure the retinal vessel characteristics, including vessel diameter, vessel branching angle and bifurcation measures, vessel tortuosity, and fractal dimensions [Retinal images were taken on the 2mensions –24.t-tests to compare the continuous data and chi-squared tests to compare the categorical data. A p value of <0.05 was considered statistically significant. We used nonparametric test (Mann-Whitney test) if the normality test showed rejection of normality assumption. For categorical data, exact Fisher's test was used if the expected count was less than five. A logistic regression model was used to build classification models. The steps used to establish these models were drawn from the method proposed by Hosmer and Lemeshow [We reported data as mean and standard deviation (mean ± SD) for continuous variables and as proportions for categorical variables. To analyze the manually measured clinical and retinal characteristics, we used 2 sample independent Lemeshow  for selep=0.019), fewer insomnia problem , and a higher tortuosity index syndrome . The drinking status presented a certain difference between the two syndromes with borderline significant difference (p=0.051). On the other hand, no statistically difference was found between the two groups in terms of the other clinical risk factors such as hypertension, diabetes, coronary heart disease (CHD), lipid level, or homocysteine level  and drinking history (p=0.039) were significant. The percentages of correct classification for PSIC and QDBS were 39.5% and 80.8%, respectively. The model that used retinal characteristic variables alone included AVR (p=0.053), bifurcation coefficient of venule (p=0.003), hemorrhage (p=0.025), and arterial occlusion (p=0.033), and a composite score of retinal interactions, showed that the percentages of correct classification were 70.4% and 80.8% for PSIC and QDBS, respectively. A final logistic regression model combined both clinical and retinal variables included insomnia status (p=0.020), AVR (p=0.099), BCV (p=0.006), hemorrhage (p=0.019), arterial occlusion (p=0.030), and a composite score of interactions (p=0.001). The percentages of correct classification were 76.5% and 84.8% in PSIC and QDBS, respectively. The odds ratios (OR) and the corresponding 95% confidence intervals (95% CI) of each of the significant variables are shown in The stepwise logistic regression method was used to build classification models that included only clinical variables, only retinal variables, and combination of both clinical and retinal variables together. For the clinical model, the insomnia status (p=0.004) for model 1 with clinical variables alone, 0.857  for model 2 with retinal variables alone, and 0.867  for model 3 with both clinical and retinal characteristics. Compared with model 1, the AUCs of model 2 and model 3 have statistically significant increased . The increase in AUC from model 2 to model 3 was not statistically significant (The AUCs were 0.624 (95% CI: 0.542–0.706, nificant .In practice, the four clinical TCM diagnosing techniques, that is, observation, auscultation and olfaction, interrogation, and pulse feeling and palpation are combined to identify a specific disease. However, obtaining a correct diagnosis using these techniques is highly dependent on the domain knowledge of the TCM physicians. For instance, most of the diagnostic information is gathered by the naked eye and subjective feeling during assessment of each physician. Therefore, our proposed method of using an objective AI-based retinal characteristic analysis is considered a highly valuable approach in the clinical practice of traditional Chinese medicine.At present, there is a vast difference between the procedure of standardization of the TCM syndrome classification of syndrome type and the method of statistical processing. The clinical methods suitable for standardizing TCM syndrome classification include cluster analysis, factor analysis, principal component analysis, artificial neural networks, regression analysis, and discriminant analysis –28. CollIn recent years, there have been many clinical studies on the correlation between objective indicators and the TCM syndrome types of ischemic stroke, of which the indicators in question have been biochemical indicators, imaging indicators, and scale evaluations –31. In 2Insomnia and alcohol consumption are related to the occurrence ischemic stroke and are considered to be its risk factors. Insomnia is a common symptom that is associated with increased risk of mortality in first-time stroke patients . A high A cross-sectional study based on two community populations in southeastern United States explored the positive relationship between fundic vascular anomalies and cerebral infarction and confirmed it via MRI . This stIn this study, we differentiated between the two TCM syndromes of ischemia stroke using different retinal characteristics. A model that facilitated this differentiation model displayed higher diagnostic efficacy based on both retinal vessel characteristics and clinical variables, rather than being dependent only on clinical variables. In fact, the model based on retinal variables alone is almost as good as the combined model with both clinical and retinal variables. Therefore, the retinal vessel features that were obtained by us could be used for interpretation and guidance of stroke syndromes in TCM clinical practice.Several potential study limitations should be considered. First, all the participants included in this study were recruited in the same hospital. A multicenter clinical study should be designed to fully investigate our results. Second, in addition to QDBS and PSIC, the other five TCM syndrome types should be investigated in future studies. Third, due to the small sample size of our study, a test data was not conducted in this study. Future studies on this topic should include larger sample sizes. However, we have carried out a leave-one-out cross-validation for the final model. The percentages of correct classification for PSIC and QDBS were 77.8% and 74.7%, respectively.In this study, we developed a logistic regression diagnosis model by combining clinical variables and retinal characteristics. We collected information on clinical variables and used the features extracted automatically from retinal images to create an objective method of diagnosis of ischemic stroke. This model is effective for distinguishing between the two TCM syndromes of ischemic stroke. Therefore, we concluded that retinal characteristics are useful for clinically differentiating between QDBS and PSIC.


Background: Anxiety has been suggested as a potentially modifiable risk factor for dementia, but results are still controversial. Our main objectives are to develop an updated meta-analysis of prospective population-based studies on the relationship between anxiety and risk of dementia, and to estimate the population fraction of dementia attributable to anxiety (PAF). Methods: We searched for cohort studies listed on PubMed or Web of Science from January 2018 to January 2020 that reported risk estimates for the association between anxiety and incident dementia. These were added to cohort studies published before January 2018 that were used in a previously published meta-analysis. Fully adjusted RRs were pooled using random effects models. We estimated the proportion of incident dementia attributable to anxiety by using PAF. Results: The meta-analysis included nine prospective cohorts from eight studies, representing 29,608 participants. The overall relative risk (RR) of dementia was 1.24 (95% CI: 1.06–1.46) and the PAF of dementia due to anxiety was 3.9%. Conclusions: Anxiety is significantly associated with an increased risk of all-cause dementia. The treatment or prevention of anxiety might help to reduce dementia incidence rates, but more research is needed to clarify whether anxiety is a cause of dementia rather than a prodrome. Dementia has been considered a public health priority by the World Health Organization (WHO), due to the growing number of subjects suffering the disease around the world and its burden for patients, their families and society . Due to A recent systematic review shows that around 35% of dementia is attributable to the combination of several modifiable risk factors, including cardiovascular risk factors, depression or educational attainment . AnxietySince our meta-analysis in 2018, further papers on the link between anxiety and risk of all-cause dementia have been published ,17,18. TThis study was conducted in accordance with the PRISMA guidelines for reporting systematic reviews and meta-analyses .In October 2019, we conducted a search of all cohort studies reporting the association between anxiety and risk of all-cause dementia published at MEDLINE via PubMed and Web of Science (the search was repeated in January 2020 with the same results). Briefly, the search strategy included the following terms: ) using both medical subject headings and free text . We onlyWe extracted the following information from each study included in the meta-analysis (MA): country, sample size, number of prevalent cases of anxiety, the number of incident cases of all-cause dementia, percentage of women, mean age of the sample, instrument used to measure anxiety, tool and clinical criteria used to diagnose dementia, covariates included in the adjusted models, statistical model, adjusted RR estimates, and time of follow-up.We used the Newcastle-Ottawa scale (NOS) for cohort studies  to analyd was performed to illustrate the effect size of the differences in the risk of all-cause dementia between anxiety and non-anxiety groups. The effect size for overall RR and its confidence interval were calculated according to Sánchez-Meca et al. [Relative risk (RR) was used as measure of association. Risk estimates from fully adjusted models were preferentially pooled in our analyses. Cohen’s a et al. , and wasa et al. . Q statistic was used as a measure of heterogeneity  and quantified with the 2I statistic (high heterogeneity was considered as ≥75%) [N value was used as indicator of publication of bias [The Hedges as ≥75%) . We perfas ≥75%) , taking  of bias . This st of bias ,27 and i of bias . In the  of bias .Finally, we estimated the proportion of incident all-cause dementia attributable to anxiety by calculating the PAF  and its All the statistical analyses were performed with STATA statistical software  and R . The flowchart of the search strategy and study selection process is shown in The eight included studies were published between 2009 and 2018, with nine prospective cohorts  and a tThe studies used different scales to classify anxiety. Three used the State-Trait Anxiety Inventory (STAI) ,32,33, aThe criteria used to diagnose dementia were more uniform across the studies, being based on the DSM in all but one, which used the modified Telephone Interview for Cognitive Status (TICSm) .The studies also differed in the types of covariates that were included in the adjusted models. We calculated the pooled RR taking into account the RR estimated from the full adjusted models. The adjusted RR values ranged from 0.81 (95% CI: 0.50–1.30)  to 2.74 Four studies presented low risk of bias (7–9 from a maximum of nine points) ,16,18,35p=0.009). However, the effect size was small, with a Cohen’s d equal to 0.12 (95% CI: 0.03–0.21).Q=15.76, df = 8, p = 0.046; 2I=49.2%, 95% CI: 0–76%)  . However: 0–76%) , indicatAfter conducting meta-regression analyses, we found no significant association between the outcome and age, gender, sample size, follow-up duration, or methodological quality see . This inN was 30, indicating that 30 studies with a null result would be needed to reduce the overall RR to a non-significant value. This would indicate absence of publication bias. Furthermore, the ‘trim and fill’ method to adjust for publication bias had a marginal effect on the pooled RR  (p = 0.029).The fail-safe 02–1.42) , which rPrevalence rates of anxiety were pooled across eight of the nine selected cohorts , considered a precursor of cognitive decline and dementia in adults older than 50 . These sOur study has several strengths. As a MA of all available up-to-date studies of anxiety and the risk of all-cause dementia, it has greater power than individual studies and previous meta-analyses with fewer studies. Moreover, we included cohort studies mostly comprising cognitively intact community samples or studies that controlled for baseline cognition in their analyses, thus minimizing the risk of recall and selection bias. The long periods of follow-up in the studies allow a sufficient number of incident dementia cases to be observed. All the studies included in the MA reported adjusted risk estimations, thus helping to provide more accurate results. Finally, all included studies except one  controllSome limitations should be considered when interpreting our results. In our MA, studies assessed anxiety with different tools, and only two used clinical criteria to determine clinically relevant anxiety ,18. MostOur results have several clinical implications. Anxiety is a common yet treatable mental disorder , despiteEpidemiological research has an important role in the study of potential modifiable risk factors of dementia, given the current absence of treatment for this disease. Our finding of a 24% increased risk of all-cause dementia for subjects with anxiety is comparable to that reported for low education as a risk factor of dementia (HR = 1.28) . ConsideIn conclusion, this meta-analysis adds to previous evidence on anxiety as a significant risk factor of all-cause dementia. Considering that anxiety is a common yet treatable condition among older adults, its treatment and prevention might help reduce the incidence and prevalence of dementia, as well as its personal, familiar and social burden. However, more research is needed to finally clarify whether anxiety is a cause of dementia or rather an early sign of the disease itself.


The classification of colorectal cancer (CRC) lymph node metastasis (LNM) is a vital clinical issue related to recurrence and design of treatment plans. However, it remains unclear which method is effective in automatically classifying CRC LNM. Hence, this study compared the performance of existing classification methods, i.e., machine learning, deep learning, and deep transfer learning, to identify the most effective method. A total of 3,364 samples  from Harbin Medical University Cancer Hospital were collected. All patches were manually segmented by experienced radiologists, and the image size was based on the lesion to be intercepted. Two classes of global features and one class of local features were extracted from the patches. These features were used in eight machine learning algorithms, while the other models used raw data. Experiment results showed that deep transfer learning was the most effective method with an accuracy of 0.7583 and an area under the curve of 0.7941. Furthermore, to improve the interpretability of the results from the deep learning and deep transfer learning models, the classification heat-map features were used, which displayed the region of feature extraction by superposing with raw data. The research findings are expected to promote the use of effective methods in CRC LNM detection and hence facilitate the design of proper treatment plans. Colorectal cancer (CRC) has a higher recurrence rate than all other cancers  Burges, , decisioDeep learning  before surgery using Philips Achieva, with a 16-channel torso array coil. MRI sagittal T2WI scan sequence was performed with the following parameters: TR/TE 3000/100 ms, the number of signal frequency (NSA) 2, and the layer thickness 4.0 mm, and layer spacing 0.4 mm. The position of the rectal lesions was determined by the sagittal position, which was perpendicular to the intestinal canal lesions, with a transverse T2WI scan: TR/TE 3,824/110 ms, NSA 0–3, layer thickness 3.5 mm, and interval 0.2 mm. According to the sagittal lesion position, patients with parallel pathological changes received coronal T2WI scans: TR/TE 3,824/110 ms, NSA 3, and layer spacing 0.2 mm. Then, the objective LN in the sagittal, transverse, and coronal images were located. All images used in this study were marked as CRC LNs and classified as negative or positive by experienced radiologists. All patches were manually segmented by experienced radiologists, and the image size was based on the lesion to be intercepted. In this study, intensity features were extracted by a gray-level histogram (GLH) Otsu, , and theEight classical supervised machine learning methods, including AdaBoost (AB)  The traditional feature extraction methods are not effective in CRC LNM classification. (2) The pre-trained model of deep learning has strong transferability. Deep transfer learning applied to a small medical image dataset is better than traditional methods, and it does not need to train the model from scratch. (3) The weights of the pre-trained model realize better initialization of the parameters.In conclusion, this study showed that deep transfer learning is better than deep learning and machine learning mainly because the pre-trained CNN extracts features are more discriminative than those extracted by a CNN and artificially-extracted features. Deep transfer learning has been a popular method for image classification in recent years, and it was proved to be the best classification method among all the methods selected in this study. It could extract underlying features from raw data, does not need to select features or use raw data as input, and is less prone to user bias. A pipeline of transfer learning will be established in future studies, and the optimal deep transfer learning model for CRC LNM classification will be found.The original contributions presented in the study are included in the article/supplementary materials, further inquiries can be directed to the corresponding author.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.