This study investigated the relationship between the mental unwellness of middle school students in China and their academic performance in the subjects of Chinese, mathematics, and English. Additionally, this study explored the potential ameliorating effects of parental involvement variables  on the adverse impact of mental unwellness on academic performance. The examination of the effects of parental involvement also considered the differential effects of involvement by mothers and fathers. This study utilized national longitudinal representative data from the China Education Panel Survey (CEPS). Findings of the two-level Hierarchical Linear Modeling (HLM) analyses indicated that mental unwellness in Grade 7 negatively impacted academic performance in the aforementioned subjects in Grade 8. Nonetheless, these adverse impacts were alleviated when the parental involvement variables were taken into account. Furthermore, the results revealed that mother-child communication and father-child communication had moderating effects on the negative relationship between mental unwellness and academic performance in Chinese and English, respectively. This study contributes to the existing literature by shedding light on the beneficial effects of parental involvement and highlighting the differential involvement of mothers and fathers. Numerous studies have established a correlation between mental unwellness and academic difficulties in children and adolescents –4. On thThe investigation into the impact of social support has led to the formulation of two models—the main-effect model and the buffering model  , 18. TheThis study conceptualizes parental involvement as the active participation of parents in the educational and non-educational development of their children , 22, 23.What are the relationships between the mental unwellness of Chinese middle school students and their academic performance in the subjects of Chinese, mathematics, and English?Can the adverse effects of mental unwellness be alleviated or eliminated by types of parental involvement?Can different types of parental involvement moderate the relationships between middle school students’ mental unwellness and academic performance in Chinese, mathematics, and English?Researchers have oftentimes demonstrated a positive relationship between the mental unwellness of children and adolescents and their academic difficulties, though the direction of this relationship remains equivocal –4, 26–28Some other researchers have indicated that the academic difficulties experienced by children and adolescents serve as predictors of their mental unwellness. For instance, Moilanen et al.  state thTo address the issue of reverse causality, Fletcher  examinesHigher levels of parental involvement are generally associated with lower levels of mental unwellness in children and adolescents –8, 16. FParental involvement has long been believed by researchers to be positively associated with their children’s academic performance , 16, 23.On the other hand, it is important to acknowledge the multifaceted nature of parental involvement when researchers explore its impact on the academic performance of children and adolescents , 10, 37.Prior studies suggest that disparities exist between father involvement and mother involvement –15. For This study utilized secondary data from the China Education Panel Survey (CEPS), which was approved by the Renmin University of China. It is a nationally representative longitudinal dataset collected by the National Survey Research Center at Renmin University of China. The CEPS currently comprises two waves of publicly available data, which do not contain any information that can identify individual participants. Specifically, Grade 7  and Grade 9  students from 112 Chinese schools were first surveyed in the academic year 2013–2014 (Wave 1), and then those Grade 7  students were followed up in the academic year 2014–2015 (Wave 2). This study focused on the students who were in Grade 7 in Wave 1, as only they were followed up in Wave 2.w1w2sweight). Specifically, the weight being utilized was a panel weight calculated by the CEPS to adjust for student attrition between wave 1 and wave 2. Notably, the CEPS did not provide any school-level weights. After weighting, the results indicate that 49% of the students were female, 87.1% were Han (the predominant ethnic group in China), and 37.4% were from the center of the city/town , 88.9% were Han , and 32.2% were from urban areas . Further, recent data provided by the Ministry of Education of the People’s Republic of China [female = 7,569,306; Ntotal = 16,324,100), 88.1% were Han , and 39.5% were from urban areas . In general, the data used in this study exhibit similarities with the demographic characteristics of the population in 2013 and 2020.This study implemented listwise deletion and weighting (of China  show thaw2chn), mathematics (w2mat), and English (w2eng) of the 2014 fall semester. In China, the academic grading system often employs a scale of 0 to 100, where the pass grade is usually 60. Thus, to account for variations in the maximum scores of the academic subjects across different regions and schools, this study converted the full marks of mid-term tests to 100, using the raw full marks offered by the CEPS . Specifically, the raw full marks of Chinese (w2upchn) included 4 categories, i.e., 100 (40.2% of the 112 schools), 120 (38.4%), 130 (2.7%), and 150 (18.8%). The full marks of mathematics (w2upmat) included 4 categories, i.e., 100 (41.1% of the 112 schools), 120 (37.5%), 130 (2.7%), and 150 (18.8%). Then, the full marks of English (w2upeng) contained 5 categories 100 (42.9% of the 112 schools), 120 (37.5%), 130 (0.9%), 140 (0.9%), and 150 (17.9%). The weighted mean scores for Chinese, mathematics, and English were 68.436 , 63.025 , and 59.112 , respectively  to assess the mental unwellness of Grade 7 students (Wave 1). A sample item is “feeling blue in the last seven days,” with responses on a 5-point scale . While the CEPS does not provide information on the origin of its mental unwellness scale, its items are similar to certain items of the Center for Epidemiologic Studies Depression Scale (CES-D). For example, a CES-D sample item is “I felt that I could not shake off the blues even with help from my family or friends during the past week.” Studies such as Jiang et al. [The CEPS used five items . Responses were on a 6-point scale . Exploratory Factor Analysis (EFA) extracted one dimension . The Cronbach’s alpha was 0.744. A derived variable was obtained based on the mean of the three items, with a higher value indicating a higher level of parental non-academic activity involvement   see .b24a1, b24a2, b24a3, b24a4, b24a5) and father-child communication  among Grade 7 students, respectively. The items used by CEPS reflected the measurement of factual and emotional information exchange between children and their parents [Children’s perception of parent-child communication consists of mother-child communication and father-child communication. The CEPS used five items to measure mother-child communication , mathematics , and English  in Grade 7 (Wave 1), students’ gender , student ethnicity , whether a student is the only child , and parents’ educational level  (see The level-1 (student level) control variables included students’ academic performance in Chinese (ree) see .pla23, 37.4% center of the city/town, 10.7% outskirts of the city/town, 15.2% rural-urban fringe zone, 17.9% towns outside of the city/town, 18.8% rural areas) and school type . Notably, among the 112 schools being surveyed by the CEPS, there were 104 public schools, 1 private school subsidized by the government, 5 ordinary private schools, and 2 private schools for children of migrant workers. Due to the small sample size of the different types of private schools, this study grouped private schools together (see The Level-2 (school level) control variables were school location  analyses were employed to predict students’ academic performance in Grade 8. To answer research question 2, based on the HLM analyses, this study compared the effects of mental unwellness on academic performance in Chinese, mathematics, and English with and without controlling for parental involvement variables. Finally, to answer research question 3, this study carried out HLM analyses with interaction effects of different types of parental involvement and mental unwellness. The full HLM model is presented below.β1 = γ10…β12 = γ120Mental represents the standardized mental unwellness variable; Nonacademic represents the standardized parental non-academic activity involvement variable; MCOM represents the standardized mother-child communication variable; FCOM represents the standardized father-child communication variable; Mental * Nonacademic represents the interaction between mental unwellness and parental non-academic activity involvement; Mental * MCOM represents the interaction between mental unwellness and mother-child communication; Mental * FCOM represents the interaction between mental unwellness and father-child communication; ACA1 represents students’ academic performance in Chinese, mathematics, or English in Grade 7 (Wave 1); Control represents a set of level-1 control variables ; sLocation represents the dummy variables for school location (reference = center of the city/town); Stype represents school type (reference = private).Y represents middle school students’ academic performance in Chinese, mathematics, and English in Grade 8 (Wave2); The results of the Intraclass Correlation (ICC) obtained from the fully unconditional model (no predictors) of the HLM indicated the feasibility of using HLMs to address the research questions. The ICC, which is calculated as the ratio of the between-school variance to the total variance, demonstrated that 39.9% of the variance in Chinese test performance among middle school students was attributable to differences between schools , mathematics , and English  in Grade 8, though the correlations were weak. This suggests that higher levels of mental unwellness in Grade 7 were related to lower levels of academic performance in the aforementioned subjects in Grade 8. In comparison, children’s perceptions of parental non-academic activity involvement , mother-child communication , and father-child communication  in Grade 7 were all positively and significantly associated with their academic performance in Chinese, mathematics, and English in Grade 8, although the correlations were weak.As shown in r = 0.390, p < 0.05) and father-child communication . Furthermore, children’s perception of mother-child communication was moderately to strongly related to children’s perception of father-child communication .In the meantime, the correlations between students’ academic performance in Chinese, mathematics, and English in Grade 8 ranged from 0.666 to 0.712, indicating moderate to strong relationships. Additionally, children’s perception of parental non-academic activity involvement was weakly to moderately associated with children’s perceptions of mother-child communication (p < 0.05 (p < 0.05 (p < 0.05), while parental non-academic activity involvement  and father-child communication  were no longer associated with middle schoolers’ performance in Chinese in Grade 8  or father-child communication .Notably, the relationship between mental unwellness and academic performance in Chinese varied by the levels of mother-child communication, β = 0.296, p < 0.05 , Model 2p < 0.05 . In compBased on Model 2, the level-1 predictors explained 46.5% (p < 0.05). Further analysis incorporating parental involvement variables revealed that mental unwellness still had a negative association with academic performance in mathematics, β = -1.264, p < 0.05 (Model 2). Among the three parental involvement variables considered, only children’s perception of mother-child communication was positively related to academic performance in mathematics , while neither parental non-academic activity involvement  nor father-child communication  were found to be associated with academic performance in mathematics in Grade 8 , mother-child communication , and father-child communication . Based on Model 2, the level-1 predictors explained 53.3% . Nonetheless, no moderation effects were found for either parental non-academic activity involvement  or mother-child communication  on the aforementioned relationship.In regard to the moderation effects, the negative relationship between mental unwellness and English performance was weaker among students who perceived higher levels of father-child communication  and the national college entrance examination (gaokao), may help to reduce students’ academic burden, ease students’ academic stress, and alleviate parental educational anxiety, all of which can benefit students’ well-being.This study, utilizing longitudinal data, demonstrated the existence of adjustment erosion/social selection , 30–32. A closer look at the results reveals that the adverse effects of mental unwellness were most pronounced on the academic performance of middle school students in English, followed by mathematics. In comparison, the detrimental impact of mental unwellness on academic performance in Chinese was comparatively less severe. This may result from the fact that Chinese students live in a Chinese immersion environment, where they not only acquire knowledge of Chinese through formal school education but also learn through their everyday lives. In contrast, Chinese students mostly learn English and mathematics through formal education. After entering secondary education, they face an increased difficulty level in academic subjects, which can make them depend more on learning through formal education. Accordingly, the potential adverse impacts of mental unwellness on Chinese students’ academic performance in English and mathematics can be greater than in Chinese. Nevertheless, to gain a more thorough understanding, future researchers should investigate the underlying reasons extensively.The findings of this study indicate that the inclusion of parental involvement variables  diminished the negative impact of mental unwellness on middle school students’ academic performance in the subjects of Chinese, mathematics, and English. This implies that parental involvement can alleviate the adverse effects of mental unwellness on student academic performance. However, it should be noted that the parental involvement variables utilized in this study were unable to eliminate the negative impact of mental unwellness. Thus, future research is necessary to examine the effects of other types of parental involvement, as this study was limited by the availability of data and only investigated two types of parental involvement that are not directly academically related . It is pThe results of this study provide evidence that among the parental involvement variables examined, only mother-child communication was positively related to middle school students’ academic performance in the subjects of Chinese, mathematics, and English. The findings suggest that compared to parental non-academic activity involvement, parent-child communication has a more pronounced impact on the academic performance of middle school students (the main-effect model). Within parent-child communication, mother-child communication has a stronger effect on students’ academic performance than father-child communication. The findings of this study are in line with the research by Hsu et al. , who empThe moderation effects of parental involvement were observed to have an impact on the relationship between middle school students’ mental unwellness and their academic performance in both Chinese and English. Specifically, when students perceived a higher level of mother-child communication, the negative relationship between their mental unwellness and their academic performance in Chinese was weaker. In the meantime, when students perceived a higher level of father-child communication, the negative relationship between their mental unwellness and academic performance in English was weaker. Because parental involvement and parental support share similarities –21, the The findings of this study indicate that better prior academic performance, being female, and parental postsecondary education attainment were related to middle school students’ better academic performance in the subjects of Chinese, mathematics, and English. In the meantime, school location was found to have a differential impact on students’ academic performance across subjects. Results indicate that, compared to students from schools located in central urban/town areas, those from rural-urban fringe zones and rural areas exhibited significantly lower academic performance in mathematics and English. Additionally, students from schools located in towns outside of central urban/town areas displayed a lower level of English performance compared to their peers in central urban/town schools. These disparities could be attributed to socio-economic differences between developed and less developed areas. In China, students from urban areas generally enjoy better education quality than students from other areas . TherefoThe results of this study indicate a lack of correlation between school type and students’ academic performance in the subjects of Chinese and mathematics. On the other hand, school type was found to be associated with students’ performance in English, with public school students generally outperforming their private school counterparts. Nonetheless, private schools in China do vary. Because of the limited school sample size provided by the CEPS, this study grouped all types of private schools together, which masks the variability of the private schools. Therefore, the findings of the relationships between school type and students’ academic performance in Chinese, mathematics, and English should be interpreted with caution.This study is subject to several limitations. First, this study has been limited to an examination of two types of parental involvement  due to the limitations in the available data, thus rendering the effects of other types of parental involvement untested. Second, the available data have also precluded an assessment of the differential effects of parental non-academic activity involvement by mothers and fathers, thereby failing to account for the unique impact of each parent. Third, this study does not establish causal relationships between students’ mental unwellness and their academic performance in Chinese, mathematics, and English. That is, while mental unwellness may predict students’ academic performance (adjustment erosion), academic performance may also predict mental unwellness (academic incompetence). Fourth, the CEPS measured students’ mental unwellness using its own scale rather than the other well-established scales such as the Generalized Anxiety Disorder 7-item scale (GAD-7) and the Patient Health Questionnaire depression scale (PHQ-9). Fifth, limited items in the CEPS can be used to measure parental non-academic activity involvement and parent-child communication. For example, parental non-academic activity involvement can also be measured by parents supporting their children to participate in extracurricular activities, while parent-child communication can also be measured by children talking to their parents about their future plans. Finally, the data utilized in this study are relatively dated, which may impair the generalizability of the results obtained.


N = 253 participants with long COVID symptoms  completed a cross-sectional online survey on overall social stigma and the subfacets enacted and perceived external stigma, disclosure concerns, and internalized stigma. Data were analysed using multiple regression and controlling for overall burden of consequences of long COVID, overall burden of symptoms of long COVID, and outcome-specific confounders. In line with our preregistered hypotheses, total social stigma was related to more perceived stress, more depressive symptoms, higher anxiety, and lower mental hrqol, but—in contrast to our hypothesis—it was unrelated to physical hrqol after controlling for confounders. The three subscales of social stigma resulted in differential associations with the outcomes. Social stigma experiences go hand in hand with worse mental health in people with long COVID. Future studies should examine potential protective factors to buffer the effects of social stigma on people’s well-being.A considerable amount of people who have been infected with SARS-CoV-2 experience ongoing symptoms, a condition termed long COVID. This study examined nuanced experiences of social stigma in people with long COVID and their associations with perceived stress, depressive symptoms, anxiety, and mental and physical health-related quality of life (hrqol). A total of  The adverse effects of the COVID-19 pandemic on societies and individuals are manifold. One set of consequences receiving more and more attention are the persistent residual effects of acute infection with SARS-CoV-2, termed “long COVID” or “post-acute COVID-19” [Long COVID or post-acute COVID is a new medical condition that is one of the consequences of an acute COVID-19 infection . AlternaThe vast majority of studies on long COVID focus on the medical aspects of the disease, but more and more studies are also taking the psychosocial consequences of living with long COVID into account. One of these psychosocial consequences is social stigma ,6,7,8.Stigma was originally defined as a personal attribute people carry that leads to discriminative experiences . Given tSocial stigma is a common experience among people with various health conditions ,12. ReseThe negative emotional consequences of social stigma experiences are manifold: experiences of social stigma contribute to perceived stress and negatively impact people’s health and quality of life ,12. In pIt is important to note that outcomes of social stigma experiences are not universal but context- and population-specific ,11. For https://osf.io/ych3s) were tested: The aim of the present study was twofold: First, we examined the associations between social stigma experiences and various indicators of stress, mental health, and health-related quality of life in people with long COVID. For this, the following preregistered hypotheses  and the abovementioned outcomes. https://osf.io/sqjdu. Participants for this cross-sectional, correlational, online-survey study were recruited via social media , posts on online platforms, and mailings to a network of people with long COVID. Recruitment took place between June 2021 and October 2021 in Switzerland, Germany, and Austria. Inclusion criteria were being 18 years of age or older, understanding German, having had an acute COVID-19 infection 12 weeks before or longer, and experiencing persistent symptoms at the time of recruitment (long COVID). The Ethics Committee of the Faculty of Arts and Social Sciences of the University of Zurich reviewed and approved the study (reference number: 21.4.3). All participants signed an informed consent form.This study was part of a larger project on the psychosocial consequences of long COVID. A detailed description of the larger project can be found at Means and standard deviations of all measures are reported in Perceived social stigma was assessed using a COVID-specific stigma questionnaire [enacted stigma  and the four items of perceived external stigma  loaded on the same factor. Given the similarity between the two subfacets of (perceived) enacted stigma and perceived external stigma, these items were collapsed into one scale . Furthermore, the two items of the subscale disclosure concerns  and the three items of internalized stigma  were each averaged to build two different subscales. ionnaire  that wasAnxiety and depressive symptoms were measured as indicators of mental health with the Hospital Anxiety and Depression Scale (HADS) [e (HADS) . The HADPerceived stress was measured with the Perceived Stress Scale (PSS-4) [ (PSS-4) , which iHealth-related quality of life was measured with the SF-12 [M = 50 ± 10).he SF-12 ,22. Two overall burden of long COVID symptoms with the question, “Overall, how burdensome have you perceived the disease with all its symptoms?” and the perceived overall burden of consequences of long COVID with the question, “Overall, how burdened have you been by the consequences of the long COVID disease?”, with a response format for both questions ranging from 0  to 10 (extremely burdened).Furthermore, we assessed the perceived sociodemographic information was assessed for all participants.Moreover, standard Statistical analyses were conducted with SPSS 28 . We ran bivariate correlations between all the main constructs of the study see  and betwhttps://osf.io/ych3s) was to determine bivariate correlations and implement regression analyses, as well as sensitivity analyses to control for potential confounders, such as age, sex, and illness severity. We present the bivariate correlations in Our preregistered analysis plan , n = 95 (37.5%) were German, n = 8 (3.2%) were Italian, n = 7 (2.8%) were Austrian, and 1 participant (0.4%) was a French citizen. Participants’ mean age was 45.49 , with n = 224 (88.5%) being women. The majority  were married or in a committed relationship, n = 52 (20.6%) of participants were single, n = 33 (13.1%) were divorced or widowed, and n = 2 (0.8%) did not respond. The majority of participants reported having a university degree , while n = 111 (43.9%) had completed vocational training, n = 4 (1.6%) of participants were still undertaking their studies/vocational training, and n = 9 (3.6%) did not respond to this question. In terms of income, n = 18 (7.1%) reported having a total household income of less than CHF 2000 (approx. USD 2200), n = 27 (10.7%) reported having an income between CHF 2001 and 4000 , n = 50 (19.8%) reported a total household income between CHF 4001 and 6000 (approx. USD 4401–6500), n = 48 (19.0%) reported a total household income between CHF 6001 and 8000 (approx. USD 6501–8700), n = 31 (12.3%) reported a total household income between CHF 8001 and 10,000 , n = 33 (13.0%) reported an income greater than CHF 10,000 , n = 44 (17.4%) did not know or preferred not to say, and the responses of n = 2 (0.8%) were missing.A total of n = 227 (89.7%) of the participants had either tested positive for COVID-19 or had had the antibodies detected via a COVID-19 antibody test. Moreover, n = 205 (81%) participants reported that their long COVID condition had been confirmed by medical staff. The most frequently reported symptoms of long COVID were fatigue , difficulties concentrating , and attention deficits . The least reported symptoms were sleep apnoea , loss of weight , and pulmonary fibrosis . The most reported pre-existing illnesses were diseases of the musculoskeletal system  and psychological disorders . Most participants contracted COVID-19 either in March 2020  or between October 2020 and March 2021 . Concerning the subjective rating of acute COVID-19 symptom severity, n = 53 (20.9%) reported having had strong symptoms, n = 145 (57.3%) reported having had moderate symptoms, n = 53 (20.9%) reported having had mild symptoms, and n = 1 (0.4%) reported having had no symptoms. A total of n = 34 (13.4%) were hospitalized during acute COVID-19 infection. Overall, On average, participants in this study reported relatively low levels of social stigma experiences see . The mear = −0.19, p = 0.001) and overall burden of long COVID symptoms  were negatively correlated with age, indicating that older participants were less likely to report internalized stigma and a lower burden of long COVID symptoms than younger participants. Sex was negatively related to physical health-related quality of life , indicating that women were more likely than men to report lower physical health-related quality of life.As displayed in As hypothesized (H1), total social stigma was positively related to perceived stress when controlling for the effects of the burden of consequences resulting from long COVID and the burden of long COVID symptoms see . A 1 uniThe analyses examining hypothesis two (H2) on the positive associations between total social stigma and depressive symptoms and anxiety confirmed H2. For depressive symptoms, a 1 unit change in total social stigma resulted in a significant increase of 0.85 units in depressive symptoms while controlling for anxiety, the burden of consequences resulting from long COVID, and the burden of long COVID symptoms see . A totalFor anxiety, a 1 unit change in total social stigma resulted in a significant 0.80 unit change in anxiety while controlling for depressive symptoms, the burden of consequences resulting from long COVID, and the burden of long COVID symptoms see . OverallFor physical and mental health-related quality of life, total stigma was only positively related to mental health-related quality of life and not to physical health-related quality of life in the regression analyses. Thus, H3 had to be partially rejected. Only the confounders mental health-related quality of life and sex, as well as the burden of consequences resulting from long COVID and the burden of long COVID symptoms, were significantly negatively related to physical health-related quality of life, resulting in a total explained variance of 23%. With regard to the three stigma subscales, only enacted and perceived external stigma see  were sigFor mental health-related quality of life, another picture emerged: total social stigma was negatively related to mental health-related quality of life after controlling for physical health-related quality of life and the two forms of burden from long COVID see . A 1 unin = 48 participants reported that their long COVID had not been officially confirmed by a physician, we ran sensitivity analyses by rerunning the analyses with only the sample of n = 205 participants who reported that their long COVID had been confirmed by a physician. The pattern of results was unchanged (see Finally, given that nged see .This cross-sectional study examined the associations between social stigma experiences among people with long COVID and perceived stress, depressive symptoms, anxiety, and physical and mental health-related quality of life. The results confirmed two out of three of our preregistered hypotheses: higher reports of social stigma were related to more perceived stress, more depressive symptoms, higher anxiety, and lower mental health-related quality of life. Contrary to our assumptions, social stigma was unrelated to physical health-related quality of life after controlling for confounders.Overall, mean social stigma levels were rather low, indicating that participants were not heavily confronted with social stigma. However, for those experiencing social stigma, the hypothesized detrimental associations with stress and lower mental health and mental health-related quality of life were confirmed. This aligns with previous research on the undesired associations between social stigma and stress, mental health indicators, and health-related quality of life ,19,20. TThe exploratory analyses with the social stigma subfacets in this study provided nuanced insights into the associations between social stigma experiences and the different outcomes. This is important because outcomes of social stigma experiences are context- and population-specific ,11. WhilAnother point to consider in future studies is that stigma experiences due to long COVID might not emerge in isolation. Instead, different kinds of stigma experiences across different domains—e.g., health-related stigma, such as for long COVID, and stigmatization due to age, sex, race, or socioeconomic status—could potentially co-occur and, if so, are likely to interact, potentially leading to more severe consequences in particularly vulnerable people . This shThis study has several strengths. For example, the nuanced assessment of social stigma, together with the assessment of a set of different stress, mental health, and health-related quality of life outcomes, produced a comprehensive picture of the associations under study. This is particularly important because experiencing social stigma differs for different populations ,12. ThusAside from these positive aspects, several limitations need to be considered when interpreting the results. The cross-sectional nature of this study imposed several limitations. First, causality could not be inferred. As a consequence, an alternative explanation for the associations found in this study could be that worse mental health and quality of life could increase the likelihood of perceptions of social stigma. Second, social stigma and mental health outcomes are likely to interact dynamically in the form of a downward spiral . For exaAnother limitation was that the measure of social stigma was adapted from the context of acute COVID-19 infections  but was This sample comprised a substantial number of participants who reported that their long COVID had not been officially confirmed by a physician. At the time of this study, there were long waiting lists for specialized consultations for long COVID in Switzerland, Germany, and Austria. Thus, we did not want to limit participation to only people with an official confirmation of their long COVID. However, there might also have been other reasons for not receiving this official confirmation by a physician. This is why we ran sensitivity analyses to examine the robustness of the results when excluding all participants without confirmation from a physician. The results were robust, thus indicating that having an official confirmation of long COVID did not make a difference for the associations between social stigma, including the subfacets, and the various outcomes considered in this study.Finally, online surveys come with specific challenges, such as lack of control over who completes the survey and inattentiveness . OverallSocial stigma experiences went hand in hand with greater perceived stress, more depressive symptoms, higher anxiety, and lower mental health-related quality of life in people with long COVID while controlling for the burden of overall consequences and the burden of symptoms of long COVID and outcome-specific confounders. Moreover, considering different subfacets of social stigma, such as enacted and perceived external stigma, disclosure concerns, and internalized stigma, provided a nuanced picture of different aspects of social stigma and various stress, mental health, and health-related quality of life outcomes in this cross-sectional study with people with long COVID.


Marine polyphenols, including eckol(EK), dieckol(DK), and 8,8’-bieckol(BK), have attracted attention as bioactive ingredients for preventing Alzheimer’s disease (AD). Since AD is a multifactorial disorder, the present study aims to provide an unbiased elucidation of unexplored targets of AD mechanisms and a systematic prediction of effective preventive combinations of marine polyphenols. Based on the omics data between each compound and AD, a protein–protein interaction (PPI) network was constructed to predict potential hub genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to provide further biological insights. In the PPI network of the top 10 hub genes, AKT1, SRC, EGFR, and ESR1 were common targets of EK and BK, whereas PTGS2 was a common target of DK and BK. GO and KEGG pathway analysis revealed that the overlapped genes between each compound and AD were mainly enriched in EGFR tyrosine kinase inhibitor resistance, the MAPK pathway, and the Rap1 and Ras pathways. Finally, docking validation showed stable binding between marine polyphenols and their top hub gene via the lowest binding energy and multiple interactions. The results expanded potential mechanisms and novel targets for AD, and also provided a system-level insight into the molecular targets of marine polyphenols against AD. Moreover, the disruption of neuronal synapses, accumulation of oxidative stress, mitochondrial dysfunction, and neuroinflammation are associated with AD occurrence and development [Alzheimer’s disease (AD) is a neurodegenerative disease of the central nervous system and the most prevalent cause of dementia in the elderly. Approximately 50 million people worldwide live with dementia, and owing to the aging population, the number of patients is expected to triple by 2050, increasing the risk of disability, disease burden, and healthcare costs . TypicalMarine algae have attracted considerable attention as natural sources of bioactive ingredients that play an important role in the development of functional foods . In addiβ-secretase (BACE1), which are known as AD-related enzymatic targets [α, IL-β1) and mediator (PGE2) through the NF-κB/MAPK signaling pathway in the PC12 cell line [Marine polyphenols are unique metabolites of brown algae species not found in terrestrial plants. In particular, phlorotannins are an important class of marine polyphenols, constituting approximately 5–12% of their dry weight . They ar targets ,10,11,12ell line . A recenell line . Marine Network pharmacology is an emerging and promising integrated strategy that is based on systems biology, bioinformatics, and multivariate pharmacology. This strategy is a novel approach to analyzing candidate mechanisms for multifactorial diseases, identifying new targets, and expanding new indications at a systematic level, which can comprehensively reflect the mechanism of potential candidates on disease networks, thus supporting lead compound discovery for complex diseases and natural products .The present study demonstrated the novel implications on EK, DK, and BK of important genes and pathways that could potentially be exploited against AD. Based on the intersection targets between each marine polyphenol and AD, a protein–protein interaction (PPI) network was constructed to discover hub genes and subnetworks. For discovering the various mechanisms of the three compounds for AD prevention, Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were subsequently conducted. Finally, the relationship between key targets and each compound was validated by molecular docking simulation. Overall, the results were first to explore the novel target genes, and their AD-related mechanisms, of marine polyphenols via network pharmacology. The workflow involved in the present study is illustrated in The chemical structures of marine polyphenols, EK, DK, and BK are presented in Proteins form dynamic PPI networks to attain multifunctionality and various cellular signaling pathways. To obtain the PPI networks of EK, DK, and BK with AD, the core targets of 92 (EK), 54 (DK), and 69 (BK) were imported into the STRING database and visualized using Cytoscape software (version 3.10.0). As shown in To obtain an intersection of the top 10 hub genes of the three compounds in AD, an UpsetR diagram was constructed . The comp-values , cellular component (CC), and molecular function (MF), in ascending order of p-values . The corp-values . In the p-values a. The CCGO analysis of DK-anti-AD showed that 155 BP terms, 42 CC terms, and 59 MF terms were enriched . The corThe core target of BK-anti-AD was found to be associated with 268 BP terms, 62 CC terms, and 71 MF terms . As showp < 0.05  to validate the network pharmacology results. The results of the molecular docking and binding energies are presented in AD is a complex multifactorial neurodegenerative disorder and the most common form of dementia. Despite all scientific efforts and many protracted and expensive clinical trials, no new drug has been approved by the FDA for treatment of AD since 2003. Indeed, more than 200 investigational programs have failed or have been abandoned in the last decade . TherefoMarine polyphenols have attracted a lot of attention, primarily due to their polypharmacologic mechanisms of action. However, there have been challenges in attaining complete knowledge of their multi-targets, which is lacking compared to terrestrial polyphenols. The present study might suggest a new perspective on the mechanisms of action in AD prevention compared with previous experimental studies.Phlorotannins are marine polyphenols formed by the polymerization of phloroglucinol monomer units, which are characterized by three hydroxyl groups bound to a benzene ring skeleton. Even though phlorotannin content can vary depending on the species, region, and extraction technique, HPLC analysis has shown that EK, DK, and BK are the major compounds in edible brown algae, accounting for more than half of the total phlorotannins . Structuβ and tau [β, the autophagy system, and oxidative stress [κB-dependent inflammatory gene transcription [β production, Aβ-mediated oxidative stress, and NF-κB signaling pathway activation [Based on the node degree, the average shortest path length, closeness centrality, and clustering coefficient in the PPI network and modules, the hub genes were ranked. It is noteworthy that major hub genes of EK and BK such as AKT1, SRC, EGFR, and ESR1 are mainly involved in AD prevention. Furthermore, the docking results demonstrated a strong binding affinity between AKT1, the identified core target, and both EK and BK. Several recent experimental studies have demonstrated the potential of AKT1 as a novel AD target that differs from traditional core targets related to A and tau ,24,25,26e stress ,21,22,23tivation ,13. AlthAKT encoded by AKT1 is a serine/threonine kinase participating in the phosphoinositide 3-kinase (PI3K) signaling pathway, which is well-known to regulate various cellular functions such as growth, proliferation, migration, and differentiation . Furtherβ and tau through the proteasome system [DK showed somewhat different but very interesting results. The compound regulated specific target genes including vascular endothelial growth factor A (VEGFA) and heat-shock protein 90 alpha family class A member 1 (HSP90AA1), which are key regulators involved in angiogenesis. Regarding the study of VEGFA in AD, higher-baseline CSF VEGFA levels are associated with slower rates of hippocampal atrophy and slower rates of cognitive decline, particularly among individuals with elevated levels of AD biomarkers . Moreovee system . Other se system ,35.Previous studies have demonstrated that a compound with a binding energy less than −7.0 kcal/mol has a high binding affinity for the target protein . The preβ deposition, tau hyperphosphorylation, neuroinflammation, etc., the importance of MAPKs in AD pathogenesis is being increasingly recognized [GO results indicated that the core genes of the three compounds were related to peptidyl-serine phosphorylation, which is one of the most predominant post-translational modifications that are fundamental in mediating various cellular functions . Furthercognized ,39. Accocognized ,41.Taken together, the present study, based on network pharmacology and docking analysis, systematically demonstrated the potential mechanisms of marine polyphenols against AD. Furthermore, the key target genes and signaling pathways of our compounds in AD prevention have never been reported in previous studies, suggesting a novel framework for further investigation of the anti-AD effects of marine polyphenols.http://www.swisstargetprediction.ch, accessed on 13 June 2023) and STITCH  database to predict compound-related target genes. Only the genes from “Homo sapiens” were selected for the following analysis.The SMILE features of EK, DK, and BK were obtained from the PubChem database. These features were then imported into Swiss Target Prediction  database, with the search term “Alzheimer’s disease.” GeneCards is a human gene database that performs gene-centric data integration from approximately 150 web sources, including genomic, transcriptomic, proteomic, genetic, clinical, and functional information. The final AD target genes were obtained after removing the repetitive elements. Then, the targets of each marine polyphenol and AD were overlapped using Venn diagram . The AD-related genes were retrieved from GeneCards , the PPI interaction was constructed to investigate the interaction relationship between the targets that contribute to AD prevention among EK, DK, and BK. “Homo sapiens” was set as the species and the confidence level (≥0.04) was used as the criterion for evaluating the interaction score. In addition, the network topology parameters were analyzed via Network Analyzer in CytoScape3.10.0 software. The key topological parameters such as degree, average shortest path length, closeness centrality, and clustering coefficient were used to rank the core targets of the PPI network. The intersections of the top 10 hub genes of marine polyphenols were visualized by UpSet R package tool .Using STRING11.0 database . The results of GO analysis were described in the BP, CC, and MF categories based on p < 0.05. Moreover, the results were visualized in a histogram form using the SRplot . In the KEGG study, the top 20 pathways were finally selected and the results were output in the form of bubble plots.GO and KEGG pathway enrichment analyses were performed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) database  and ChemSpider  databases, and structurally optimized and converted to PDB format using UCSF Chimera 1.17.1. The 3D structures of AKT1 (PDB ID 3o96) and VEGFA (PDB ID 6zfl) were downloaded from the RSCB PDB database , and water molecules and original ligands were removed using UCSF Chimera. AutoDockTools-1.5.6 was used to add polar hydrogen atoms and charges, the ligand rotation bond was established and the receptor and ligand were saved in PDBQT format. AutoDockVina was used to dock and search for the optimal conformation, which was determined by the binding mode and its corresponding binding affinity. Interactions between the compounds and targets were analyzed using LigPlot+ and Discovery Studio Visualizer 2021. For the redocking steps, the ligand was extracted and redocked into the original ligand binding pocket in the target protein, then the RMSD was calculated for each docking pose with respect to the native pose [To confirm the accuracy of the network pharmacology prediction, the active compounds were used as ligands to perform molecular docking with the key target proteins which were identified by network pharmacology. The 3D structures of the ligands were obtained in SDF format from the PubChem (In conclusion, the result of this investigation increases the understanding of the molecular drivers that underlie AD progression and the prevention of the disease by marine polyphenols including EK, DK, and BK, and the identified key genes and pathways constitute potential therapeutic targets. Although more extensive experimental validation is required for the prevention and/or treatment of AD, these findings are expected to provide novel directions for the development of marine polyphenols and their potential use as a promising source of anti-AD agents.


The aim of this work is to determine the changes in essential-oil composition under different irradiation intensities of 60Co and to select an appropriate irradiation dose with GC–IMS. Dosages of 0, 5, and 10 kGy of 60Co were used to analyze turmeric (Curcumae Longae Rhizoma) volatile oil after 60Co irradiation . The odor fingerprints of volatile organic compounds in different turmeric volatile oil samples were constructed by headspace solid-phase microextraction and gas chromatography–ion mobility spectrometry (GC–IMS) after irradiation. The differences in odor fingerprints of volatile organic compounds (VOCs) were compared by principal component analysis (PCA). The results showed that 97 volatile components were detected in the volatile oil of Curcuma longa, and 64 components were identified by database retrieval. With the change in irradiation intensity, the volatile compounds in the three turmeric volatile oil samples were similar, but the peak intensity was significantly different, which was attributed to the change in compound composition and content caused by different irradiation doses. In addition, the principal component analysis showed that JH-2 and JH-3 were relatively correlated, while JH-1 and JH-3 were far from each other. In general, different doses of 60Co irradiation can affect the content of volatile substances in turmeric volatile oil. With the increase in irradiation dose, the peak area decreased, and so the irradiation dose of 5 kGy/min was better. It is shown that irradiation technology has good application prospects in the sterilization of foods with volatile components. However, we must pay attention to the changes in radiation dose and chemical composition. Curcumae Longae Rhizoma, a rootstock plant called turmeric belonging to Fam. Zingiberaceae in China, is extensively cultivated in South China, such as in the Sichuan, Guangxi, Guangdong, Yunnan, and Zhejiang provinces. Turmeric has been widely used in traditional Chinese medicine to promote Qi circulation, dissolve blood sludge, induce menstruation, and relieve pain. It can be used to relieve pain in the chest and hypochondriac regions, as well as treat amenorrhea, mass formation in the abdomen, rheumatic pain of the shoulders and arms, traumatic swelling, and pain . Modern research shows that the abundant volatile oil and curcumin in turmeric (up to about 4%) have anti-tumour, anti-inflammatory, and antioxidant effects .60Co-γ irradiation ray sterilization method, widely used in the sterilization of Chinese medicinal materials, is a new disinfection and sterilization process in the 20th century, which is based on the high-energy rays produced by X-ray, γ ray, and other ionizing radiation to inhibit the continuation of pests and diseases and to effectively kill insects. Sterilization at room temperature and high efficiency, with simple operation and other characteristics, dosages of 5, and 10 kGy are most widely used [60Co, which can be of great reference value to the quality problems of traditional Chinese medicine and its preparation products. The quality of traditional Chinese medicine will have an impact on its efficacy. With the development of modern medicine, the quality standards of traditional Chinese medicine are also constantly improving. However, the source of traditional Chinese medicine is complex, and it is easy to be contaminated by bacteria in the process of production and storage. Traditional dry-heat sterilization, damp-heat sterilization and ethylene oxide gas sterilization technologies easily destroy the effective components of drugs, affecting the efficacy or residual organic solvents. The sterilization operation should remove microorganisms and ensure the quality and efficacy of drugs . 60Co-γ ely used ,4. HowevThe traditional detection method of volatile compounds in turmeric uses gas chromatography-mass spectrometry (GC–MS) , but it 60Co rays of different intensities . A total of 64 components were detected using GC–IMS technology to compare the changes before and after irradiation. Finally, the composition changes of volatile oil under different irradiation intensities were given to provide a certain reference for the production and sterilization of turmeric and its preparations. This study provides a sound basis for the use of 60Co-γ ray irradiation sterilization technology during the preparation of medicinal herbs. GC–IMS, which has the advantages of simple operation, strong separation ability, short detection cycle, and retaining the original flavor of samples to the greatest extent, can be successfully applied to foods.In this study, the volatile oil of turmeric was extracted by steam distillation and irradiated, with Turmeric was collected from Baise, Guangxi Province, China, and identified by Prof. Zhaoming Xie at the Hunan Academy of Traditional Chinese Medicine. A voucher specimen (HNATCM2022-006) was deposited in the herbarium of the Hunan Academy of Traditional Chinese Medicine.The essential oil was extracted through steam distillation by referring to the Chinese Pharmacopoeia 2020 edition (part 4) volatile oil determination method  for determination. We added 500 g of dried turmeric with an appropriate amount of water and a few glass beads into a 1000 mL round-bottom flask, treating the solution by slowly heating it to a boil, followed by keeping it slightly boiling for 6 h. The upper oil phase  was gathered and sealed and stored away from light at 4 °C for further use.1 is the total mass of the extracted oil, and m0 is the initial mass of the turmeric used in each extraction.After extraction, the extraction rate of volatile oil is calculated according to the following formula:2SO4 and then divided into three equal parts for 60Co irradiation. The dose rates were 0, 5, and 10 kGy/min. The 60Co γ radiation source was located at Hunan Radiological Technology Application Research Center . The resulting curcumin EOs was dehydrated with anhydrous NaIn the experiment, the volatiles were concentrated and separated by headspace solid-phase microextraction, with reference to Wang  and othe®, G.A.S., Berlin, Germany). Gas chromatography (GC) was performed under the following conditions: carrier gas, nitrogen (99.99%); column, mxt-5 (15.0 m length × 0.53 mm ID × 1 μm thickness); running time, 50 min; flow rate, initial 2.0 mL/min, holding for 2 min, linearly increasing to 100 mL/min within 18 min, and holding for 20 min. Ion mobility spectroscopy (IMS) was carried out under the following conditions: drift gas, nitrogen (99.99%); flow rate, 150 mL/min; IMS detector temperature, 45 °C.The components of volatile compounds were identified by chromatography–ion mobility spectroscopy . We compared the fingerprint of the gallery plot plug-in to intuitively and quantitatively compare the differences in volatile organic compounds between different samples. A dynamic PCA plug-in was used for dynamic principal component analysis, cluster analysis of samples, and rapid determination of unknown and unknown samples.Three-dimensional topographic plots of turmeric irradiated by three different doses are shown in A two-dimensional topographic spectrum (planform of the 3D plot) was also obtained for its difficulty to observe the differences between three-dimensional groups B. In thiAccording to The fingerprint of VOCs corresponding to each sample with different irradiation intensities is displayed in The substances in the green box in 60Co-γ irradiation were determined and analyzed. By using headspace sampling and GC–IMS technology, the compounds can be qualitatively analyzed according to the GC retention time and ion migration time of volatile substances. A total of 64 volatile compounds were identified by GC–IMS analysis and built-in NIST database retrieval. The results showed that the contents of various volatile substances were different under different irradiation intensities. The volatile oil in turmeric is responsible for the aroma of turmeric, while curcumin  is responsible for its bright yellow color [60Co-γ ray irradiation did not have a great impact on the effectiveness of turmeric volatile oil.The main active components and volatile components of turmeric volatile oil exposed to different doses of ow color ,13. Someow color , includiow color . The resow color ,17,18, aow color ,20,21, aow color , treatmeow color ,24. Most60Co were used to analyze turmeric (Curcumae Longae Rhizoma) volatile oil after 60Co Irradiation . With the increase in irradiation dose, the peak area decreased. It is of great significance to explore the sterilization dose of irradiation sterilization of different traditional Chinese medicine varieties using 60Co. Dosages of 5 and 10 kGy are the most widely used. There is a maximum level and limit for radiation which should be discussed in the future. It could be better to do it for 0, 2.5, 5, 7.5, and 10.Dosages of 0, 5, and 10 kGy of In this study, the author found that gas chromatography–mass spectrometry can effectively identify volatile odor compounds such as alcohols, ketones, aldehydes, esters, and terpenes. Food ,26, agri60Co irradiation of turmeric (Curcumae Longae Rhizoma). This study provides a sound basis for the use of 60Co-γ ray irradiation sterilization technology during the preparation of medicinal herbs.In this study, the volatile components of turmeric volatile oil samples with three different irradiation intensities were analyzed by GC–IMS. A total of 97 volatile substances were detected, and a total of 64 components were determined by database retrieval, such as dimers of some substances. The volatile organic compounds in three turmeric volatile oil samples, mainly including terpenes, esters, aldehydes, alcohols, and ketones, were found. The results showed that the chemical components of the three turmeric volatile oil samples were similar, but the contents were quite different, suggesting that the irradiation intensities might have an impact on the volatile organic compounds of turmeric volatile oil. JH-1 and JH-3 have great differences in irradiation strength, and the difference in the principal component analysis is also large, indicating that the difference in chemical composition is the largest. This study provides a scientific basis for the dose control of irradiation sterilization of turmeric and its volatile oil. With the increase in irradiation dose, the peak area decreased, and so the irradiation dose of 5 kGy/min was better for It is shown that irradiation technology has good application prospects in the sterilization of foods with volatile components, However, attention must be paid to the changes in radiation dose and chemical composition. GC–IMS which has the advantages of simple operation, strong separation ability, short detection cycle, and retaining the original flavor of samples to the greatest extent, can be successfully applied to foods.


Louisiana ranks among the bottom five states for air pollution and mortality. Our objective was to investigate associations between race and Coronavirus Disease 2019 (COVID-19) hospitalizations, intensive care unit (ICU) admissions, and mortality over time and determine which air pollutants and other characteristics may mediate COVID-19-associated outcomes. In our cross-sectional study, we analyzed hospitalizations, ICU admissions, and mortality among positive SARS-CoV-2 cases within a healthcare system around the Louisiana Industrial Corridor over four waves of the pandemic from 1 March 2020 to 31 August 2021. Associations between race and each outcome were tested, and multiple mediation analysis was performed to test if other demographic, socioeconomic, or air pollution variables mediate the race–outcome relationships after adjusting for all available confounders. Race was associated with each outcome over the study duration and during most waves. Early in the pandemic, hospitalization, ICU admission, and mortality rates were greater among Black patients, but as the pandemic progressed, these rates became greater in White patients. However, Black patients were disproportionately represented in these measures. Our findings imply that air pollution might contribute to the disproportionate share of COVID-19 hospitalizations and mortality among Black residents in Louisiana. Coronavirus Disease 2019 (COVID-19) severity and mortality have been associated with several vulnerability factors, including comorbidities, environmental exposures, natural disasters, sociodemographic factors, and residence in congregate settings , while c2.5) with SARS-CoV-2 infection prevalence [2) concentrations for prevalence [3) concentration for mortality [2.5 and NO2. However, evidence was mixed, with some studies showing no association for NO2 [3 [2.5 [Soon after the start of the pandemic, some evidence emerged of an association between long-term average air pollution concentrations and the prevalence or severity of COVID-19. Notably, significant associations were observed for long-term average concentration of particulate matter (PM) having a diameter smaller than 2.5 μm  [Louisiana parishes routinely score well below the national average for quality of life, morbidity, and mortality indices such as low birthweight, child poverty, and median household income . Based oGiven the disproportionate impact of COVID-19 on communities of color in Louisiana and the U.S., the goals of this research were to investigate the association of race and COVID-19 outcomes over time and to identify if exposures to air pollution and other characteristics, if any, may mediate associations of race with COVID-19 hospitalizations, ICU admissions, and mortality. We combined datasets from a Louisiana hospital system distributed across the Industrial Corridor and an air pollution database to include both individual and environmental level risk factors. We investigated factors including race, insurance status, comorbidity, and pollutant exposure for four waves of COVID-19 between 1 March 2020 and 31 August 2021.In our cross-sectional study, we evaluated associations between race and COVID-19 hospitalizations, ICU admissions, and mortality and tested for factors that may mediate relationships. We used the Franciscan Missionaries of Our Lady (FMOL) Health System COVID-19 registry to identify patients at ten Louisiana locations distributed across the Industrial Corridor . The stuA total of 13,454 patients aged eighteen years or older who tested positive by a polymerase chain reaction (PCR) test for SARS-CoV-2 were identified using the Epic healthcare software between 1 March 2020 and 31 August 2021. This period is broken down by waves: 1 March–10 June 2020 (First Wave), 11 June–6 October 2020 (Second Wave), 7 October 2020–30 June 2021 (Third Wave), and 1 July–31 August 2021 (Fourth Wave). These were chosen to minimize both cases and mortality at the beginning and end of each period using the Johns Hopkins database for Louisiana . Patient-level variables included hospital department, SARS-CoV-2 test date, SARS-CoV-2 test result, age, insurance status , self-reported race, self-reported ethnicity, sex, admission date, discharge date, length of hospital stay, admission status, ICU stay, ICU admission date, ICU discharge date, length of ICU stay, discharge dispatch, body mass index (BMI), presence of comorbidities, census tract, and census block group. Specific comorbidities were not listed consistently in the database, so they were simply recoded as presence (1) or absence (0) of any comorbidities for each patient in the database. To minimize bias in the patient database, negative PCR tests were not included in the database because tests were often obtained for non-medical reasons . Records were complete for hospitalization and ICU admission; records were missing for mortality for 171 Black patients and 128 White patients. Data with missing hospitalization, ICU, or mortality information were removed from the dataset. The final sample size was 11,331. Ethnicity data were missing for 9977 patients. A total of 113 patients (<1%) responded that their ethnicity was “Hispanic or Latino/a”, “Mexican, Mexican American, or Chicano”, or “Other Hispanic, Latino/a, or Spanish origin”, while 1271 patients responded that they were “Not of Hispanic or Latino/a or Spanish Origin”. Therefore, ethnicity was not included in the statistical analyses.2.5 and hazardous air pollutants (HAPs) known to have respiratory health effects: 1,3-dichloropropene, 2,4-toluene-diisocyanate, acetaldehyde, acrolein, acrylic acid, arsenic, beryllium, cadmium, chlorine, chloroprene, chromium, diesel PM, formaldehyde, hexamethylene-1,6-diisocyanate, hydrazine, hydrochloric acid, naphthalene, nickel, polycyclic organic matter (POM), propylene, and triethylamine. Oil and gas wells and refineries, which are prevalent naphthalene sources, and a neoprene plant, a chloroprene source, fall within the hospital service area  or with disease-related variables  were screened via χ2 testing was performed to check the relationship between race and each variable, and between each variable and health outcomes. Potential mediators and potential covariates in the association between race and health effect were identified. Associations of each variable with both race and health effect indicated that the variable is a potential mediator. Variables associated with just health effects but not with race were identified as covariates to be controlled in the mediation analysis. Mediation analysis was then used to test if a portion of the race–outcome relationship could be accounted for by each intermediate variable after adjusting for all potential mediators, covariates, and confounders [We used mediation analysis to test for environmental risk factors, called third variables, that might explain widely reported racial disparities in the COVID-19 outcomes. Mediation analysis is used here because it tests for causal associations from the explanatory variable (race) to third variables  and then to the outcome  to determine if the pollutants are responsible for the association ,27,28. Pfounders ,27,28. Sdplyr, tidyr, bit65, and data.table) and for the merger of geographic data with air pollution emissions data and output of shape files containing emissions burdens . The R package mma was used to perform the mediation analysis [We used the R software v4.0.5 for data organization , but average BMI for both groups was in the obese range (BMI > 30). Length of hospital and ICU stays were both significantly higher among White patients, although that difference diminished for Medicare recipients and those without insurance. More Black patients had Medicaid (61.9%) or were uninsured (61.6%), while more White patients had private insurance (62.5%) or Medicare (59.4%). Among the twenty-two pollutants tested, emissions burden was statistically significantly higher for Black patients in seventeen compounds and for White patients in three compounds, with no significant difference for two pollutants, hydrazine and propylene.Of the 11,331 patients in the final sample, 5708 (50.4%) identified as non-Hispanic Black, and 5623 (49.6%) identified as non-Hispanic White . In comp2, but the proportion of hospital and ICU admissions among Black patients were 16% and 36% greater, respectively, than the share of the population identifying as Black.For the study duration, hospitalizations were significantly higher among White patients (53.4%), while ICU admissions were significantly higher among Black patients (52.4%). Information regarding mortality  was available for 11,032 (97.3%) cases . For theThe mediation analysis figures  illustra2.5 and chromium exposures may have increased the effect among Black patients. However, these exposures may have added uncertainty to the race–hospitalizations total effect because the different sign of these mediation coefficients widened the confidence intervals around the total effect. Age and, with a smaller contribution, presence of comorbidities were significant mediators of the race–hospitalization relationship  for the 2.5 and chromium emissions burden potentially contribute to a greater effect in Black patients but widened the confidence intervals around total effect. Age was a mediator of the race–ICU admission effect during each wave. During the third wave, the total effect between race and ICU admission was near zero, but there was a greater direct effect on Black patients and greater indirect effect of PM2.5 emissions on Black patients balanced by greater indirect effects of age, cadmium emissions, and nickel emissions on White patients. The fourth wave produced a large total effect for the race–ICU admission model that included a direct effect comprising more than half of the total effect and indirect effects from age, insurance status, sex, and emissions of POM.The model for race–ICU admission for the entire study period  includedThe mediation analysis results indicate that for the total duration and for each wave, there was a greater total effect in White patients, with age consistently a significant mediator of the total effect of race on mortality . The dirA complicated picture of racial disparities in COVID-19 hospitalization, ICU admission, and death emerges from these results. For the entire study period, hospitalization and mortality rates among those who were diagnosed with COVID-19 in Louisiana’s Industrial Corridor were greater for White patients than for Black patients, while ICU admission rates were higher for Black patients. These proportions shifted towards White patients by late 2020. However, the proportion of those diagnosed with COVID-19 as well as those hospitalized, admitted to the ICU, and who died remained disproportionately higher for Black patients compared with the patients’ residential areas, despite the 7.9-year age difference between Black and White patients. For example, across the entire study period, COVID-19 mortality among Black patients was 25% greater than what would be anticipated based on the proportion of the patient population identifying as Black, while COVID-19 mortality among White patients was 14% below what would be anticipated based on the patient population identifying as White. Among the population of those who had to be hospitalized due to COVID-19, most of the association of race could be explained by mediators, i.e., third variables. Age was the strongest mediator, accounting for the largest share of the association between race and COVID-19 hospitalization. In each wave, the average age of Black patients was 8–9 years younger than the average age of White patients. In fact, life expectancy for Black Louisiana residents is 3.4 years shorter than for White Louisiana residents . These f2.5 explained ICU admissions and mortality among Black patients and was 5.2 times greater among Black patients compared with White patients. Several studies [2.5 with COVID-19 using data from the first few months of the pandemic, but they either used a nationwide domain or studied different parts of the country. Sidell et al. [2.5 and NO2 concentrations and between COVID-19 infection and 1-year average O3 concentrations for the second, third, and fourth waves and entire study duration. However, the magnitude of the associations declined over the third and fourth waves, especially for PM2.5. Uncertainties persist about the influence of air pollution on COVID-19 outcomes over the course of the pandemic. Terrell and James [2.5 concentration with COVID-19 mortality for Louisiana, and Xu et al. [2.5 concentrations were not associated with COVID-19 mortality.Findings that naphthalene and chloroprene explained part of the associations between White race and ICU admissions and that naphthalene also explained part of the associations of White race with hospital admissions and mortality were surprising given that their burdens among Black patients in Louisiana were 8.9 and 4.5 times higher, respectively, than for White patients. Chlorine was found to explain ICU admissions among Black patients, and hydrochloric acid was found to explain mortality among Black patients. These findings are consistent with chlorine’s burden being 17 times greater and hydrochloric acid’s burden being 8.0 times greater among Black patients than White patients. Terrell and James  noted hi studies ,12,13 fond James  calculatu et al.  noted foThere were some limitations specific to this dataset. These analyses reflect the data and results of the full population that interfaced with the FMOL Health System based primarily in the Industrial Corridor. This selective population was not representative of all Louisiana COVID-19 hospitalizations and thus limits some generalizability of our results for the full state. The most recent HAP emission data were from 2017. Additionally, vaccination status was not included in the dataset but could have affected severe outcomes during the last two waves.Mediation analysis showed a clear relationship between race and outcome at the beginning of the pandemic, but race appeared less influential over time. Mediation analyses highlighted the uncertainty in the race–outcome relationships across waves. Although several air pollutants were associated with race, with higher emissions burdens among predominantly Black census tracts, air pollution did not appear to consistently mediate the total race–outcome relationship for most waves. Uncertainties in the mediation analyses raise questions about unmeasured confounding. VanderWeele  assertedThe wave-by-wave results of this study indicate that the role of race in the associations of COVID-19 outcomes has evolved over the course of the pandemic in Louisiana. Early in the pandemic, the association of race with hospitalization, ICU admission, and mortality appeared to be mediated by age. However, the younger age profile of Black COVID-19 patients contradicts findings of enhanced risk to older patients , suggest2.5 in Black patients on ICU admissions and greater effect of hydrochloric acid in Black patients on mortality were not surprising because their burdens among Black patients were 17, 5.2, and 8.0 times higher, respectively, than among White patients. Our results suggest that disparities in environmental conditions may have exacerbated inequities in COVID-19 impacts among Black patients.Burden from air pollutants may have explained some of the race–outcome associations. Findings that greater effect of chlorine and PM


Hemophagocytic Lymphohistiocytosis (HLH) is a rare clinical condition characterized by sustained but ineffective immune system activation, leading to severe and systemic hyperinflammation. It may occur as a genetic or sporadic condition, often triggered by an infection. The multifaceted pathogenesis results in a wide range of non-specific signs and symptoms, hampering early recognition. Despite a great improvement in terms of survival in the last decades, a considerable proportion of patients with HLH still die from progressive disease. Thus, prompt diagnosis and treatment are crucial for survival. Faced with the complexity and the heterogeneity of syndrome, expert consultation is recommended to correctly interpret clinical, functional and genetic findings and address therapeutic decisions. Cytofluorimetric and genetic analysis should be performed in reference laboratories. Genetic analysis is mandatory to confirm familial hemophagocytic lymphohistiocytosis (FHL) and Next Generation Sequencing is increasingly adopted to extend the spectrum of genetic predisposition to HLH, though its results should be critically discussed with specialists. In this review, we critically revise the reported laboratory tools for the diagnosis of HLH, in order to outline a comprehensive and widely available workup that allows to reduce the time between the clinical suspicion of HLH and its final diagnosis. It may develop in the context of a familial disorder (FHL) or as a sporadic condition, in association with diverse triggers, such as infections, autoinflammation, acquired immune deficiencies, or malignancies. The identification of familial cases is crucial to allow a definitive cure by hematopoietic stem cell transplantation (HSCT). Clinical manifestations may include persistent fever, hepatosplenomegaly, multi lineage cytopenia, coagulopathy, hyperferritinemia, and central nervous system involvement. These signs and symptoms are the consequence of an exaggerated cytokine release and organ infiltration by activated lymphocytes and macrophages. Patients may rapidly deteriorate and develop multi organ failure and death. Thus, prompt recognition and treatment are essential for patients’ survival.Historically, FHL has been associated to impaired cytotoxicity due to biallelic mutations in FHL-related genes. Nowadays, the advent of Next Generation Sequencing (NGS) and improvement of functional studies led to re-define HLH as a syndrome with etiopathogenetic heterogeneity, blowing the distinction between familial and sporadic cases. Starting from an historical perspective up to the current understanding of HLH, in this review we critically revise this complex condition and the available diagnostic tools, with the final aim to describe a comprehensive clinical and laboratory approach for the management of patients with suspected HLH.2The first description of HLH dates 1939, when Scott and Robb-Smith reported 4 patients with unremitting fever, lymphadenopathy, and hepatosplenomegaly Figure 1PRF1, was identified in December 1999 and PRF1-mutated FHL was then named FHL type 2 (FHL-2) (SH2D1A affecting the SLAM associated protein (SAP) signaling have been recognized as the cause of a clinical syndrome phenotypically described in 1977 by Purtilo et al.   cell cytotoxic activity, which was observed in affected patients, compared with healthy relatives, in the 1980s –8. The f FHL-2) . At the  . At the, XLP-1) –15. Late, XLP-1) . In receDiagnostic guidelines were defined for the first time in 1991 and included fever and splenomegaly, hemophagocytosis in bone marrow, spleen, or lymph nodes, cytopenias, hypertriglyceridemia and/or hypofibrinogenemia . At that3PRF1, FHL-3 (UNC13D), FHL-4 (STX11), FHL-5 (STXBP2), Griscelli Syndrome type 2 (RAB27A) and Chediack-Higashi syndrome (LYST)  –15. Secoe (LYST) . In the e (LYST) .RHOG loss-of-function variants were recently reported in a patient with early-onset HLH and defective lymphocyte exocytosis but reports of additional patients are needed to gain insights into penetrance (NLRC4 or hemizygous mutations in XIAP) and mutations involved in immune response to viral infections (i.e. hemizygous mutations in SAP) have been related to HLH  by a target cell stimulate the formation of secretory lysosomes, which are delivered to the intercellular space by exocytosis. These organelles contain cytotoxic proteins such as perforin and granzymes . Perforinditions .PRF1 mutations with reduced protein levels  result in the reduction of CD107a externalization after proper stimulation in vitro, by resulting in cytotoxicity defects -labeled K562 target cells when co-cultured with NK cells at different ratios . Unfortu isotope . Further isotope . For the5.3SH2D1A mutations usually do not display defects in the degranulation machinery. The SH2D1A gene encodes the SLAM-associated protein, also called SAP, which is principally expressed in NK and T cells, and is likely to regulate signal transduction initiated by SLAM protein family , induced by alterations in the RC4 gene . Less coRC4 gene . FurtherRC4 gene . To detedeletion . Howeverdeletion , 67. Sinanalysis .5.4+ and CD8+ T cells from a cohort of 93 patients with active HLH  without rheumatologic or malignancy conditions  were found to be increased in secondary HLH and MAS. CD4dimCD8+ T cells were highly represented together with laboratory parameters associated with severe MAS, confirming their involvement in the pathogenesis of secondary HLH and MAS . The study demonstrated increased levels of these cytokines during active MAS and secondary HLH .In the last years, IL-18 and CXCL9 are being increasingly adopted in clinical settings. CXCL9 is used to monitor IFN-γ activity in HLH and MAS, in the context of IFN-γ neutralizing antibodies  clinical trial. Together with sCD25, it has been suggested in HLH recognition . FurtherRecently, a novel cytometric bead array (CBA) was developed to measure IFNγ, TNF-α, IL-2, IL-4, IL-6, and IL-10 . Taking + and CD8+cells  . High sC6Despite the correlation between phenotype and gene variants has been reported in HLH , 87, thePRF1, UNC13D, STX11, STXBP2, RAB27A and LYST leading to impaired cytotoxicity. Other genes involved in control of infection and dysregulated inflammasome activity have been associated to HLH as a predominant manifestation. Among these, X-linked lymphoprolipherative diseases type 1 and 2 due to hemizygous pathogenic variants in SH2D1A and BIRC4 respectively, are the most known syndromes , which combines 15 HLH-associated genes and was launched in September 2013. Gadoury-Levesque et al. used this approach to perform a definite genetic diagnosis in 1892 patients with suspected HLH. The group obtained a diagnostic rate of 10.4%. This low value can be explained, by one hand, by lax inclusion criteria of patients, and on the other by the exclusion of newly reported HLH-associated genes, such as d CDC42 . Tesi etd CDC42 .UNC13D, BIRC4, and RAB27A , which gives the opportunity to perform more comprehensive genetic analyses and to identify novel genes predisposing to HLH or HLH-mimicking phenotypes. WES is a method designed to perform a target resequencing of all protein-coding nuclear genes in the genome. Since non-coding sequences are generally characterized by variants with a mild or no effect on phenotype, the advantage of WES is to be directed on the small percentage of coding genome (1%) that is more likely to carry pathogenic variants . On the d RAB27A , 100–102d RAB27A .Overall, genetic studies remain crucial for HLH diagnosis and allow familial counseling and prenatal diagnosis. The combination of immunological screenings with novel technologies, such as WES and WGS, will allow us to understand the significance of genetic variants, filling the gap between pathophysiological mechanisms and targeted treatments of HLH.7HLH is a rare syndrome with multiple clinical manifestations. Without prompt recognition, it is invariably fatal. Despite recent improvements in survival, around 40% of patients with HLH still die from refractory disease or toxicity. Thus, a rapid and effective diagnostic approach is crucial for early treatment start and HSCT decision.PRF1 gene, whereas a defective degranulation assay is suggestive of mutations in UNC13D, STXBP2, STX11, and RAB27A. Other diagnostic tests, including cytokines dosage, provide supporting information (i.e. high IL-18 levels for inflammasome related diseases), but are not always timely accessible for clinical decisions. Under these conditions, cytofluorimetric tests have a high diagnostic value. Cardiac evaluation with echocardiography should be performed as soon as possible both to evaluate the cardiac function and to rule out coronary signs of Kawasaki disease. Additional investigations should address rarer conditions associated to HLH including other immunodeficiencies and LPI (Lysinuric Protein Intolerance), that is the only metabolic disease really mimicking FHL.In recent years, many novel approaches have been proposed to investigate patients with clinical and laboratory findings consistent with HLH. Their rationale and reliability has been widely analyzed in this review, and some of them might gain a pivotal role in HLH management in the future. However, there are extensively validated investigations that remain now essential in the diagnostic approach. In the view of the clinical and genetic heterogeneity of the syndrome, it may still be challenging for the clinician to make quick handling decisions when facing a patient with suspected HLH. For this reason, we recommend a comprehensive diagnostic approach to HLH that integrates clinical, functional, and genetic criteria, possibly in the setting, or with the advice, of a highly specialized team Figure 2Nevertheless, a strong clinical suspicion of genetic HLH, even without functional defects nor mutations of FHL-related genes, should direct toward NGS investigation to identify rarer subtypes of genetic HLH beyond cytotoxicity defects, such as those involving impaired control of infections or dysregulated inflammasome activation Table 1.in vitro to clarify their biological impact. Despite WES is being increasingly adopted and easily accessible worldwide, we strongly recommend centralization of samples in reference laboratories to allow a reasoned approach starting from clinical evaluation towards functional and genetic analysis. Personnel expertise is indeed crucial to guide the diagnostic algorithm and to correctly interpret the results, with the final aim to support clinical and therapeutic decisions. Indeed, genetic findings do not always impact on patient management, including HSCT. In the last decades, great progress has been made, but novel technologies and therapeutic strategies are developing and will hopefully improve patient’s outcome in the next years.Thus, besides optimizing costs and timing, WES is also useful for expanding the knowledge of genetic variants associated with HLH. It is worth noting that the massive amount of information deriving from WES output should be interpreted with caution. The variants of uncertain clinical significance should be considered on a case-by-case basis, depending on the clinical phenotype, and should be validated by functional studies confirming the defect In conclusion, as the Italian reference center for HLH, we suggest the integration between clinical evaluation, functional and genetic analysis to optimize diagnosis timing and accuracy, by following a reasoned clinical-based approach open towards novel diagnostic tools. To effectively realize this integration, we must keep in mind the knowledge collected so far by looking beyond at the same time, with the final aim to unravel the tangle of HLH spectrum, by allowing an increasingly personalized approach to each patient.ES designed the study. AC and ES wrote the manuscript. AC, LB, FP, IT, MC, and ES reviewed and edited the manuscript. All authors contributed to the article and approved the submitted version.