Background: Grief is a multi-faceted experience including emotional, social, and physical reactions. Research in ICD-11 prolonged grief disorder (PGD) in different cultural contexts has revealed different or potentially missing grief symptoms that may be relevant.Objective: This study thus aimed to explore the prevalence of somatic symptom distress and its associations with grief and negative affect in a culturally diverse sample of bereaved individuals with symptoms of PGD.Methods: Based on cross-sectional survey data from the Measurement and Assessment of Grief (MAGIC) project, this study included 1337 participants  from three regions , who experienced a loss of a significant other. Associations between somatic symptom distress , symptoms of PGD , anxiety , depression  as well as demographic and loss related characteristics were investigated. Three hundred and thirteen participants (23.4%) scored above the proposed cut-off for clinically severe PGD.Results: ‘High’ or ‘very high’ levels of somatic symptom distress were more frequent in a possible PGD group (58.2%), than in a non-PGD group (22.4%), p < .001, as divided per cut-off in the IPGDS. In a multiple regression analysis, PGD symptoms were significantly but weakly associated with somatic symptom distress  beyond demographics, loss-related variables, and negative affect. Negative affect (anxiety and depression) mediated the relationship of PGD symptoms with somatic symptom distress and the indirect effect explained 58% of the variance.Conclusions: High levels of somatic symptom distress can be observed in a substantial proportion of bereaved across cultures. Our findings suggest that PGD is related to somatic symptom distress partly and indirectly through facets of negative affect. 30.8% of bereaved adults showed ‘high’ or ‘very high’ levels of somatic symptom distress.Anxiety and depression partially mediate relationship of PGD symptoms with somatic symptom distress.Findings encourage practitioners to consider somatic symptom distress in psychotherapeutic treatment of PGD. However, some individuals may experience ongoing symptoms of grief that are highly distressing and impairing, as described by the prolonged grief disorder (PGD). Recently, a representative sample study from Germany found prevalence rates of 3.3% and 4.2% in bereaved, depending on the diagnostic system  and associated somatoform disorders in various groups of bereaved individuals, including refugees   and can be used to conduct different analysis or re-analysis of the current data set. The original published report conducted a psychometric validation of the IPGDS scale and a network analysis to examine core symptoms of PGD in different cultural contexts . The data were collected at more than 23 universities across 5 countries between August 2019 and June 2020. Data were collected through social media platforms and institutional emailing lists in each country using snowball sampling. The following procedure was used in each country: a standard email that contains information on the study as well as a link to the online survey – created in Qualtrics and containing a consent form at the beginning of the survey was sent via weblink to local universities as well as local hospice and bereavement institutions  with the request to forward this information to potentially eligible clients/patients. In addition, information about this study was provided on websites (e.g. Department of Psychology website), via university-wide list serves, and via campus advertisements and flyers. The online survey link and study coordinator’s contact information (email and phone) were included in all of the recruitment materials distributed so potential participants can access the online survey directly or contact the study coordinator prior to study participation.This cross-sectional, survey-based study is part of a large research collaboration  to study grief symptoms in international cross-cultural samples led by the University of Arizona, Network for International Collaborative Exchange (NICE) , Cyprus (n = 73), Turkey (n = 287), Iran (n = 87), and the USA (n = 848). The country groupings are based on cultural and location similarities. For example, Greece and Cyprus are Southern European, primarily orthodox, Greek-speaking while Iran and Turkey are Central-Western Asian and Muslim countries. It should be noted that we collected data from a small sample (13 participants) from 10 other countries. We did not include this data in the subgroup analysis by region as they did not fit into the country grouping criteria. Ethical approval was provided by the University of Arizona. Additionally, the study was approved by the institutional ethics review board at each participating university before data collection. Inclusion criteria comprised adults (18 + years old), ability to read and write in the presented language of the survey  and experienced the death of a loved one. The survey was conducted anonymously via the online-platform Qualtrics. Eligible participants were informed beforehand about the study goals, risks, and benefits and gave their written consent to participate in the study. All participants were provided with debriefing forms according to the Helsinki Declaration. The completion time of the survey was approximately 40 minutes.The main data sets were obtained from Greece .The survey was divided into 12 sections including informed consent, demographics, loss variables, scales to assess symptoms of grief and further sequelae of distress , and a final debriefing as well as the opportunity to submit questions to the researchers. If versions of the scales were not standardized or available in the required language, they were translated following recommended translation protocols. For example, for the Persian and Greek versions of the scales, forward translation was performed by two fluent native Greek and Persian speakers. Back translation was performed by a master translator and consensus was obtained. The Turkish translation used the five-step translation process where (1) two translators did forward translation, (2) two translators did backward translation, (3) all translators discussed the translation, (4) external individuals provided feedback, and (5) the final translation was produced. The details can be found here: https://osf.io/tyz3u/).The full list of self-report measures and all data is publicly available on the Open Science Framework , which has been previously validated in cross-cultural samples  . This measure was translated into Persian for the current study.Negative affect in terms of depressive symptoms was assessed by the Patient Health Questionnaire, depression scale  , age, education , and occupation . Loss-related variables included relationship to the deceased  and cause of death .2.2.5.t-tests (Welch’s t-test in case of variance heterogeneity), and univariate analysis of variance (ANOVA) were used to test group differences. We conducted zero-order correlations and partial correlations (controlled for negative affect) for the association between symptoms of PGD and somatic symptom distress. We detected no signs of multicollinearity or autocorrelation of residuals. Using studentized excluded residuals, 10 outliers (0.7%) were identified and regression and mediation analyses were repeated excluding these values. Since the main results of these analyses were not altered and other indices for detecting outliers  were unremarkable, we thus report the results based on the total sample. Multicategorical predictors were dichotomized, including education (A-level vs. non-A-level), cause of death , and relationship to the deceased . For subgroup analyses, the residence regions of participants were grouped into USA (n = 826), Turkey/Iran (n = 320), and Cyprus/Greece (n = 179). To investigate predictors of somatic symptom distress, demographic characteristics , loss-related variables , PGD symptoms, and negative affect  were entered into a hierarchical multiple regression model. A three-step approach was chosen. In the first step, PGD symptoms were included as a predictor. In the second step, demographic and loss-related variables were added, and in the third step, anxiety, and depression. In this way, we were able to test if the effect of PGD symptoms on somatic symptom distress remains after controlling for covariates and also examine the particular improvements in explained variance when adding variables of negative affect. Regression analyses were also stratified by gender and region as subgroup analyses. A possible mediation of the association of PGD symptoms and somatic symptom distress by both anxiety and depression was further tested with the PROCESS macro, v3.5.3  with missingness > 90% per questionnaire were deleted. Possible PGD status (non-PGD/PGD) was determined using the proposed cut-off score by Killikelly et al. . Chi-squ3 Hayes, , using a2.3.SD 8.59, range 18–82), 1018 (76.1%) were female and 876 (65.6%) had no higher education. In the US-American sample, 584 of 825 respondents (70.7%) reported a Caucasian descent, 80 (9.7%) Asian (American), 58 (7%) African (American) descent, or others . In the overall sample, most participants (78.2%) had lost a second-degree relative or friend and natural causes of death were most common (79%). Three hundred and thirteen participants (23.4%) scored above the proposed cut-off for clinically severe PGD. Possible PGD was significantly more frequent in individuals with no higher education (i.e. less than A-level) and when the deceased was a second degree relative in contrast to other relationships. No significant differences were observed for age, gender or occupation. Prevalence of possible PGD significantly differed between cultural groups and was highest among individuals from Cyprus/Greece (43.6%) compared to the USA (21.1%), and Turkey/Iran (18.4%).3.3.1.SD = 7.19) and 412 participants (30.8%) showed ‘high’ or ‘very high’ levels of somatic symptom distress (see t(559.69) = 5.55, p < .001, d = 0.36) and significantly differed between the three regions , F = 30.39, p < .001, η² = 0.04. ‘High’ or ‘very high’ levels of somatic symptom distress were more frequent in the possible PGD group (58.2%), than in the non-PGD group (22.4%) χ²(4) = 161.60, p < .001, see ρ =  .069, p = .223).In the overall sample, the mean score in the SSS-8 was 8.29 , while the partial correlation of symptoms of PGD and somatic symptom distress, controlling for negative affect, was much weaker, yet significant . The results of the regression analyses are listed in R² = .23, model 2: R² = .25, model 3: R² = .58). In the full model, the predictive effect of PGD symptoms  was small but significant and lower than for depression  and anxiety , which formed the strongest predictors of somatic symptom distress. Only gender  was identified as another significant predictor. When analyses were stratified by gender PGD symptoms remained a significant predictor of somatic symptom distress in female individuals  but not in male participants , see Supplementary Material B.Zero-order correlations between symptoms of PGD and other measures of psychopathology were significantly large  and from Cyprus/Greece  but not in participants from Turkey/Iran , see Supplementary Material C.Furthermore, when stratified by region, PGD symptoms remained a significant predictor of somatic symptom distress in the full model in participants from USA  and depression  mediated the relationship between PGD symptoms and somatic symptom distress (see p = .003), indicating a partial mediation of somatic symptom distress .When controlling for demographic and loss-related variables, anxiety , while the other results were largely comparable to the total sample.However, when stratified by region, this direct effect was no longer significant in participants from the USA or Cyprus/Greece , indicating full mediation. In participants from Cyprus/Greece, the confidence interval indicated no significant indirect effect of anxiety on somatic symptom distress  and this was particularly high in females, the Cyprus/Greek sample, and the probable PGD group. Additionally, PGD symptoms predicted symptoms of somatic symptom distress while controlling for demographics, loss-related variables, or negative affect . Our findings also show that depression and anxiety partially mediate the association between PGD symptoms and somatic symptom distress.Around two-thirds of individuals with possible PGD reported high or very high levels of somatic symptom distress in the SSS-8, which is remarkably higher than prevalences in the general population . Network analysis revealed that the Turkey/Iran and Greece/Cyprus groups had stronger network connections with somatic symptoms such as feeling paralyzed and having no energy. In this current study, across cultural groups, somatic symptom distress and symptoms of PGD were most prevalent in individuals from Cyprus/Greece. This subsample had a higher proportion of deceased being first degree relatives, as well as older, and higher educated participants. In this case, the network theory could explain these differences in cultural groups by including environmental factors in the network structure. Environmental or cultural factors may act as a catalyst that facilitates the change from an acute response, such as the normal grief response, to a chronic condition, PGD . In the current study we included all participants who had lost a loved one regardless of time since loss. Our main aim was to explore the relationship between severity of PGD symptoms and somatic symptoms in a general population sample. Future studies should examine the relationship between different PGD diagnostic criteria (e.g. more than 6 or 12 months since loss) and somatic symptoms in a clinical sample. Thirdly, the sample could be considered heterogeneous, e.g. regarding time since loss, and a self-selection effect for survey participation cannot be ruled out. Among the strengths of the current study are the inclusion of validated and reliable instruments, particularly for ICD-11 PGD, extending previous evidence based on a different assessment of negative affect (Thege et al., 4.2.Elevated somatic symptom distress can be observed in a substantial proportion of bereaved individuals across cultures. Our results provide support to the idea that to some extent, somatic symptom distress ‘masks’ anxiety and depression, looming in the background of grief. However, the latter also showed unique influences on somatic symptom distress, which should be explored further. A psychosomatic perspective can help to improve the understanding of the multi-faceted symptom network of PGD across cultures and encourage practitioners to consider somatic symptom distress in psychotherapeutic treatment.Click here for additional data file.


Genetic deletions at Xp22.31 are associated with the skin condition X linked ichthyosis (XLI), and with a substantially increased risk of atrial fibrillation/flutter (AF), in males. AF is associated with elevated thrombosis, heart failure, stroke and dementia risk.Through: (a) examining deletion carriers with a diagnosis of AF in UK Biobank, (b) undertaking an online survey regarding abnormal heart rhythms (AHRs) in men/boys with XLI and female carriers of XLI-associated deletions and (c) screening for association between common genetic variants within Xp22.31 and idiopathic AF-related conditions in UK Biobank, we have investigated how AHRs manifest in deletion carriers, and have identified associated risk factors/comorbidities and candidate gene(s). Finally, we examined attitudes towards heart screening in deletion carriers.STS (7 065 298–7 272 682 bp in GRCh37/hg19 genome build) in males, and of common GI disorder and asthma/anaemia risk variants around PNPLA4 (7 866 804–7 895 780 bp) in males and females, respectively. Deletion carriers were overwhelmingly in favour of cardiac screening implementation.We show that AHRs may affect up to 35% of deletion carriers (compared with <20% of age-matched non-carriers), show no consistent pattern of onset but may be precipitated by stress, and typically resolve quickly and respond well to intervention. Gastrointestinal (GI) conditions and asthma/anaemia were the most strongly associated comorbidities in male and female deletion carriers with AHR, respectively. Genetic analysis indicated significant enrichment of common AF risk variants around Our data suggest AHRs are frequently associated with Xp22.31 deletion, and highlight subgroups of deletion carriers that may be prioritised for screening. Examining cardiac function further in deletion carriers, and in model systems lacking steroid sulfatase, may clarify AF pathophysiology. Deletions at Xp22.31 are associated with a substantially increased likelihood of diagnosis of atrial fibrillation/flutter in middle-aged individuals, but how abnormal heart rhythms (AHRs) manifest in this population is unclear.STS as a candidate gene for AHR and PNPLA4 as a candidate gene for comorbidities. We also show that deletion carriers are in favour of cardiac screening.We show that AHR in Xp22.31 deletion carriers are common, comparatively mild, largely responsive to treatment, tend to be precipitated by stress, and are comorbid with gastrointestinal conditions, asthma and anaemia. We identify The study guides future work in Xp22.31 deletion carriers and suggests that work in steroid sulfatase-deficient model systems may provide insights into the pathophysiology of atrial fibrillation. The feasibility of cardiac screening in individuals carrying Xp22.31 deletions or diagnosed with X linked ichthyosis  might be investigated.Prenatal screening estimates indicate that genetic deletions within Xp22.31 occur in around 1 in 1500 males and 1 in 750 females.Using the large UK Biobank sample recruited from the general population of the UK, we have recently shown that middle-aged men carrying deletions of 0.8–2.5 Mb within Xp22.31 -related phenotypes and comorbidities in men and boys with XLI, and in female carriers, through an online survey. These initial analyses were intended to enhance our understanding of the nature and course of, and risk factors for, AF in individuals with Xp22.31 deletions with a view to improving AF prediction within this genetic cohort. Convergent findings across our two distinct participant groups (UK Biobank and online samples) would be expected to be robust and generalisable. Third, we aimed to gauge opinion on screening for cardiac abnormalities in patients with confirmed XLI/Xp22.31 deletion. Finally, we aimed to identify candidate genes within the Xp22.31 deletion interval by screening for common risk variants for idiopathic AF and associated conditions in the UK Biobank sample; this would provide clues regarding biological mechanisms for any phenotype–genotype associations and would suggest future studies geared towards clarifying these.STS) from the UK Biobank sample were compared between individuals diagnosed with AF and those not diagnosed with AF.The UK Biobank sample comprises almost half a million participants recruited between 2006 and 2010 at ages 40–69 years from the UK general population for which anonymised genotype and extensive phenotype data are available.Adult (>18 years) men with a confirmed diagnosis of XLI, confirmed adult female carriers and parents of boys with a confirmed diagnosis of XLI were recruited via relevant charities, online patient support groups and social media; diagnosis/carrier status was typically confirmed on the basis of a combination of: family history, assessment of skin condition and biochemical/genetic testing. Participants were directed to an online survey which was open from 2 September to 22 December 2021, and anonymised data were returned to the study team upon completion.The survey was designed in Qualtrics10.1136/jmg-2022-108862.supp1Supplementary data2 or Fisher’s exact test; ORs are presented as a measure of effect size. Where multiple medical phenotypes were assessed, Benjamini-Hochberg False Discovery Rate correction was applied.For each group, the proportion of individuals reporting AHRs was calculated, and the nature of those abnormalities characterised. Subsequently, each participant group was divided into individuals with and without self-reported AHRs, and the two subgroups were then directly compared across demographic measures and medical phenotypes to identify factors co-segregating with heart arrhythmias within these populations. Continuous variables were compared between groups with unpaired t-test or Mann-Whitney U test depending on normality of the data, and categorical data were analysed by ΧA total of 363 693 white British and Irish individuals remained in our UK Biobank sample after removing related individuals and those who have since chosen to withdraw from the study. Diagnoses were coded according to ICD-10. The UK Biobank contains 3 917 799 imputed SNPs on the X chromosome. These imputed data were quality controlled (QC) by removing rare SNPs with minor allele frequency <0.01, SNPs imputed with poor accuracy (INFO <0.4) and SNPs with missing data proportion >0.05. The data were split into males and females. After these QC steps, 262 278 SNPs in 195 638 females and 267 937 SNPs in 168 055 males remained for analysis.10(p)>4.90 (p<1.26×10−5) or −log10(p)>4.91 (p<1.23×10–5) were regarded as significant following multiple testing correction in females and males, respectively. We then ran multiple association analyses between all SNPs in the region of interest with a number of different phenotypes linked to AF. We investigated AF (ICD-10 code ‘I48’), stroke (ICD-10 code ‘I64’), acute myocardial infarction (ICD-10 code ‘I21’), dementia (combined ICD-10 codes ‘F00–03’), asthma (ICD-10 code ‘J45’), anaemia (combined ICD-10 codes ‘D50−52’) and gastrointestinal (GI) disorders (combined ICD-10 codes ‘K50−59’). The association analyses were run for each phenotype in males and females separately. Fifteen principal components, array and age were included in the model as covariates. Finally, we used MAGMA2 gene-based analysis was used on the summary statistics from the association analyses, and unadjusted p values are presented.First, SNPs in the region of interest  were extracted: 3966 and 4060 SNPs remain in females and males, respectively; individual SNPs with −log4.91 p<1.×10–5 werhttp://doi.org/10.17035/d.2022.0230251614.UK Biobank data are available upon application to that resource.Of the 86 male Xp22.31 deletion carriers previously identified in UK Biobank, 9 had been diagnosed with AF. These nine individuals, were on average, significantly older than the remaining 77 , but the two groups did not differ with respect to self-reported blistering/desquamating disorder diagnoses (1 of 9 (11%) vs 3 of 77 (4%), Fisher’s exact test p=0.36). In the nine subjects with AF, the most common comorbid diagnoses were: GI problems  and respiratory conditions . Comparison of the most common diagnoses in the male deletion carrier AF versus non-AF group revealed that only the prevalence of ‘non-infective gastroenteritis and colitis’ differed significantly between groups, being more common in the former group (2 of 9 (22%) vs 1 of 77 (1%), p=0.028 Fisher’s exact test). Of the 312 female deletion carriers identified in the UK Biobank, just 3 had been diagnosed with AF. All three carriers diagnosed with AF displayed prominent cardiovascular abnormalities and had also been diagnosed with left ventricular failure. The other most common comorbidities in female carriers with AF were dyspnoea  and bone/joint conditions (rheumatoid arthritis in one patient and fracture in the second); one individual diagnosed with AF presented with acute renal failure and anaemia.2(1)=8.88, p=0.003).We recruited a total of 191 participants , although not all participants completed all aspects of the survey. The prevalence of self (or parent)-reported AHRs was similar across the three groups ; across the three groups, individuals affected by AHRs and those unaffected did not differ significantly with respect to age, country of residence and ethnicity, although individuals experiencing AHRs tended to be older on average . Across Of all the comorbid medical conditions assessed, only ‘gut problem’ was significantly more frequently self-reported in males with AHR than males without . ‘Heart murmur’ , anaemia  and asthma  were all significantly more common in female carriers reporting heart rhythm abnormalities than in those without. When adult/young male, and female deletion-carrying groups were all combined, four medical conditions were significantly more common in AHR than in non-AHR groups: ‘heart valve disease or malformation’ (OR: 10.6 (95% CI: 1.2 to 97.4), p=0.025), ‘anaemia’ (OR: 3.2 (95% CI: 1.1 to 8.7), p=0.027), ‘asthma’ (OR: 2.9 (95% CI: 1.3 to 6.4), p=0.010) and ‘gut problem’ (OR: 4.9 (95% CI: 1.7 to 14.3), p=0.004); of these, only ‘gut problem’ survived Benjamini-Hochberg False Discovery Rate correction (adjusted p=0.1) .In adult men, AF had been diagnosed by a medical professional in 60% of individuals with AHR, and tachycardia in 27%. Thirty per cent of female carriers with AHR had been diagnosed with AF, and 60% with tachycardia. In boys with XLI and AHR, 53% had been diagnosed with tachycardia, 40% with bradycardia and 20% with AF.Stress was cited in the top three precipitating factors for AHR across all three groups ; other precipitants were less-consistently identified but included ‘no obvious cause’ (men and boys with XLI), ‘exercise’  and ‘increased body temperature’ (boys with XLI) . There wA total of ≥80% of men with XLI  and fema−6 and 2.39×10−6, respectively), and this was with GI disorders in males (42 680 cases (25.4%) vs 125 375 controls (74.6%)). These SNPs are located closest to the PNPLA4 transcriptional start sitePNPLA4 in small intestine terminal ileum .STS  and AF in males (4556 cases (2.7%) vs 163 499 controls (97.3%), p=0.041) but not in females (2144 cases (1.1%) vs 193 494 controls (98.9%), p=0.793) (PNPLA4 and asthma (p=0.040) and anaemia (p=0.013) were noted in females only. Finally, gene-based analysis suggested significant associations between VCX3A, VCX, PNPLA4 and VCX2 genes and GI conditions in males only . Gene-based analysis results for medical conditions related to AF are presented in Across all conditions, only two closely linked individual SNPs  exceeded the threshold for significant association (p=2.24×1080 cases .4% vs 12p=0.793) . No signUsing the UK Biobank sample, we previously showed that approximately 10% of middle-aged men carrying Xp22.31 deletions had been diagnosed with AF, a prevalence around four times than that in the non-carrier male population.Our online survey suggested self-reported AHRs in 28%–35% of male and female carriers of Xp22.31 deletions, and, consistent with this, many affected carriers had received a cardiac arrhythmia-related diagnosis from a medical professional and reported a family history of cardiovascular issues. This prevalence figure is likely higher than the 10% figure reported previously as it includes multiple causes of AHR in addition to AF and may include subclinical and undiagnosed cases; on the one hand, true prevalence rates may be underestimated given that only perceptible and symptomatic AHRs can be self-reported, but on the other hand, reported rates may be somewhat overinflated due to response bias. While we acknowledge the limitations of self-reported data on AHRs , this assessment method allows interrogation of comparatively large samples and provides information on AHRs occurring at time points across the lifespan as opposed to temporally restricted clinical monitoring via, for example, ECG. While AHR prevalence rates across the three online samples appear broadly similar, we note that the expected pattern of data was obtained, that is, highest rate (and worst response to intervention) in older, hemizygous males and lower rates (and better intervention responses) in heterozygous females and younger males. The prevalence of AHRs in our deletion-carrying population appears elevated compared with that in non-carrier samples, for example, arrhythmia was self-reported in 13.3% of men and 21.9% of women aged 40–49 years in a large European general population sample previouslySo far as we could determine and bearing in mind lack of power, there was no clear relationship between heart rhythm abnormalities and the severity or presence of other features commonly associated with XLI  implying partially dissociable causes. Our data suggest that, as with idiopathic cardiac arrhythmias, the prevalence of AHR within the Xp22.31 deletion population increases with age. They also indicate that AHRs in Xp22.31 deletion carriers typically resolve quickly and respond well to intervention where this is required. The most commonly reported precipitant within this subpopulation was psychological and/or physiological stress.STS on male  risk of idiopathic AF. These genetic findings, in combination with: (a) our finding of high rates of AHR in female carriers not affected by XLI and boys unlikely to have been on long-term medication, and (b) medication not being reported as a significant contributor to AHR in males with XLI, provide further evidence against the idea that elevated rates of AHR in Xp22.31 deletion carriers are a secondary consequence of pharmacotherapy but instead support the idea of a biological predisposition to risk.Gene-based analyses suggested an aggregate effect of SNPs within STS is highly expressed in adult arterial vasculature and at a lower level in the atrial appendage.STS deficiency appears a strong functional candidate for AF risk in Xp22.31 deletion carriers given that PNPLA4 in asthma and anaemia risk in females only, and PNPLA4 in GI disorder risk in males only. PNPLA4 (previously known as GS2 and iPLA2eta) encodes an enzyme with triacylglycerol lipase and acylglycerol transacylase activities,PNPLA4 in female Xp22.31 deletion carriers may predispose to asthma/anaemia while in males loss of PNPLA4 may predispose to GI issues, and that these medical vulnerabilities may exacerbate any AHR risk incurred as a consequence of STS deficiency. Potentially, effective treatment of GI issues and atopic conditions/anaemia in male and female deletion carriers, respectively, could reduce AHR risk. We did not identify any genetic signatures within Xp22.31 associated with possible downstream consequences of AF including stroke, acute myocardial infarction and dementia; this may be because common variants within Xp22.31 contribute marginally towards AF risk, and AF, in turn, only contributes to the pathogenesis of a relatively small fraction of stroke, acute myocardial infarction and dementia cases.Despite the UK Biobank and online samples having different ascertainment biases and characteristics, they provided converging evidence that GI problems, asthma and anaemia are the comorbidities most closely associated with AHR in deletion carriers, with GI issues more prominent in male carriers with AHR, and asthma and anaemia more strongly comorbid in female deletion carriers. GI issues are increasingly being recognised as contributors to AF risk via multiple plausible pathways.Sts-deficient mice), in combination with more focused clinical analyses in Xp22.31 deletion carriers guided by the preliminary results presented here, should clarify the physiological, cellular and molecular mechanism(s) through which genetic variants at Xp22.31 affect risk of AF and other relevant medical conditions. In turn, this should lead to better-informed genetic counselling for deletion carriers.Work in relevant in vitro  and in vivo models  are strongly in favour of cardiac screening shortly after diagnosis to mitigate long-term health consequences associated with AHRs for them and their offspring. As such, the utility and viability of screening within these populations, and particularly in individuals with comorbid GI disorders, asthma or anaemia where clinical prognosis appears worse,


Availability of the dopamine and noradrenaline precursor tyrosine is critical for normal functioning, and deficit in tyrosine transport across cell membrane and the blood-brain barrier has been reported in bipolar disorder and schizophrenia. Clozapine and lithium are two psychoactive agents used to treat psychosis, mood disorders and suicidal behavior, but their mechanism of action remains largely unknown.To characterize immediate and delayed differences in tyrosine uptake between healthy controls (HC) and bipolar patients (BP) and see if these differences could be normalized by either clozapine, lithium or both. A second objective was to see if clozapine and lithium have additive, antagonistic or synergistic effects in this.Fibroblasts from five HC and five BP were incubated for 5 min or 6 h with clozapine, lithium, or combination of both. Radioactive labelled tyrosine was used to quantify tyrosine membrane transport.There was significantly reduced tyrosine uptake at baseline in BP compared to HC, a deficit that grew with increasing incubation time. Clozapine selectively increased tyrosine uptake in BP and abolished the deficit seen under baseline conditions, while lithium had no such effect. Combination treatment with clozapine and lithium was less effective than when clozapine was used alone.There was significant deficit in tyrosine transport in BP compared to HC that was reversed by clozapine but not lithium. Clozapine was more effective when used alone than when added together with lithium. Potential clinical implications of this will be discussed.The online version contains supplementary material available at 10.1007/s00213-023-06397-5. The cells were stored in a Biobank at the Neuropsychiatric Research Laboratory, School of Medical Sciences, Örebro University. Ethical approval was obtained from the Ethics Committee at the Karolinska Institutet, Stockholm, Sweden (Rnr: 01-205) and Swedish Ethical Review Authority .n = 5) and BP-I (n = 5) between 7 and 16 passages were used in the present study  containing minimum essential medium (MEM) supplemented with 10% FBS, 2 mM L-glutamine, Amino-Max and 100 mg/mL penicillin, and streptomycin . Radiolabeled [14C] L-tyrosine with the specific activity 482.0 mCi/mmol  and 51.8 mCi/mmol  was used to quantify tyrosine uptake in cells seeded in 24 multi-well plates . All solutions were made with phosphate buffered saline (PBS)  with pH held at 7.35–7.40.Fibroblast cell lines from HC   was dissolved in 1.0 ml dimethyl sulfoxide  to a stock solution of 50 mmol/L and stepwise diluted to final concentrations of 0.5–1.5 μmol/L in phosphate buffer (PBS); final concentration of DMSO was under 0.01%. Lithium sulfate  was diluted to a final concentration of 1.0 mmol/L in PBS.t-test and one-way ANOVA or nonparametric test with independent samples Mann-Whitney U or independent samples Kruskal-Wallis test were used as found appropriate. Where significant interaction between group and medication was found, post-hoc test was performed to determine differences. SPSS (IBM v28) was used for all statistical analysis and p-value ≤ 0.05 after Bonferroni correction for multiple comparison was considered significant. All significant values shown are for two-sided tests.Tyrosine uptake was compared between HC and BP in the absence and presence of the psychoactive drugs clozapine and lithium. Both within-group (lithium/clozapine vs. control conditions in the absence of psychoactive drugs) and between-group  pairwise comparisons were conducted. Kolmogorov-Smirnov test showed data was not always normally distributed, thus parametric statistics with independent sample Ten fibroblast cell lines were randomly selected from a biobank, 5 healthy controls (HC), and 5 patients with bipolar disorder (BP). Mean age, gender distribution, and number of passages for both HC and BP is shown in Table n=6 at each incubation time). Repeated measures ANOVA showed significant main effect for condition for the 5-min , but not for the 30-min (p = 0.39) or 60-min (p = 0.16) incubation times. Bonferroni corrected pairwise comparison for the 5-min experiments showed significant clozapine effect at 1.0 μmol/L, which decreased tyrosine uptake by roughly 10% . Although there was no main effect for condition for the 60-min incubation time, post-hoc pairwise comparison showed significant effect of clozapine at 1.0 μmol/L  and 1.5 μmol/L , which again did not survive Bonferroni correction for multiple comparison.Lithium which has a narrow therapeutic window 0.5–1.2 mmol/L) was used at a concentration of 1 mmol/L, which is at the upper end of its therapeutic dose interval. To determine optimal dose for clozapine within its wider therapeutic range, preliminary experiments were conducted in a single cell line from a healthy donor incubated with saline or 0.5, 1.0, and 1.5 μmol/L clozapine for 5, 30, or 60 min (.2 mmol/Ln = 24 for HC and n = 20 for BP) or 6 h (n = 28 for both HC and BP). At 5-min incubation time, BP had 15.5% lower tyrosine uptake compared to HC . The difference in tyrosine uptake between HC and BP grew with increasing incubation time and there was roughly 31.2% lower tyrosine uptake in BP compared to HC at 6-h incubation time . The difference at 6-h, but not that at 5-min incubation time, survived Bonferroni correction , p = 0.016) and 6-h , p < 0.001) incubation times (Table N=117), p = 0.035) but not HC , p = 0.95). Bonferroni adjusted post-hoc within-group analysis in BP showed significant effect for clozapine compared to baseline (p = 0.026), but not for lithium (p = 0.24) or combination of clozapine and lithium (p = 0.18) (Table N=92), p = 0.83) nor in BP , p = 0.39), although there was a similar trend with clozapine enhancing tyrosine uptake in BP and reversing the difference between HC and BP seen under baseline conditions.To study immediate and sustained effects of psychoactive drugs on tyrosine uptake in the two groups (HC vs. BP) under four different conditions , therapeutic doses of 1.0 μmol/L clozapine, 1.0 mmol/L lithium, or a combination of both at the above concentrations was added to the incubation medium for 5 min or 6 h. Independent samples Kruskal Wallis test showed significant main effect for group (HC vs. BP) in both the 5-min  remained significant for lithium (p = 0.002) and combination of clozapine and lithium (p = 0.00046), but not for clozapine (p = 0.084) at 6-h incubation (Fig. Between-group pairwise post-hoc analysis of HC and BP under the different experimental conditions is shown in Table ion Fig. . In otheClozapine and lithium have unique place in the treatment of psychotic and affective disorders. Both drugs are first-line options for treatment-resistant schizophrenia and bipolar disorder, respectively. Clozapine is also known to have mood-stabilizing effects (Kapczinski et al. Tyrosine is a precursor of dopamine and noradrenaline, whose conversion to L-DOPA by tyrosine hydroxylase is the rate-limiting step in the synthesis of the two signal substances. Dopamine and noradrenaline are known to have widespread cognitive and affective functions (Berridge and Arnsten Fibroblasts cultured from patients with different diagnosis have previously been used to study pathological mechanisms of psychiatric disorders (Batalla et al. Contrary to our expectation, we saw no additive or synergistic effects of clozapine and lithium on tyrosine uptake when the two drugs were added together. To the contrary, clozapine was less effective in modifying tyrosine transport when added together with lithium than when added alone. One possible explanation for this could be that the two drugs competed for binding at some putative common site of action, and clozapine in the presence of lithium could not bind well enough to reach the required threshold for action. Irrespective of possible mechanisms behind this, we believe this finding may have important implications in the clinical setting where polypharmacy is routinely utilized. Combination treatments might under certain circumstances lead to less optimal outcome, not just by increasing risk for adverse events or through pharmacokinetic interactions, but also through pharmacodynamic interactions even when the medications do not have any known common mechanism of action.Tyrosine transport across the blood brain barrier occurs through the LAT1 and LAT2 transporters, both of which are expressed in fibroblasts. Thus, fibroblast cell lines collected from healthy donors and patients with bipolar disorder can be used as surrogate models for the study of tyrosine uptake in the CNS. Tyrosine supplementation has also been shown to have cognition enhancing effects under stressful and demanding conditions when dopaminergic neurons are actively releasing transmitter substance (Jongkees et al. The mechanism of action of clozapine as mood stabilizer is not known, while its antipsychotic effects at least in part is assumed to be related to dopamine D2 blockade (Lieberman et al. Although clozapine is known to have mood stabilizing effects, its main area of use is in schizophrenia and related psychotic disorders. Before we can draw any conclusion pertaining to clozapine’s antipsychotic mechanism, these experiments need to be replicated in fibroblasts from patients with schizophrenia. Furthermore, although fibroblasts express similar amino acid transporter proteins as those found in the blood-brain barrier and neuronal membranes, these may differ in function so the findings here cannot simply be extrapolated to also apply to neuronal tissue. Another limitation that needs to be mentioned here is that there was significant age difference between HC and BP in our study, which may have contributed to the differences observed in tyrosine uptake between HC and BP.Tyrosine uptake was reduced in BP compared to HC under baseline conditions, a deficit that was normalized by clozapine but not lithium. Clozapine selectively enhanced tyrosine uptake in BP but slightly reduced it in HC, indicating that it had little effect on tyrosine transport under physiological conditions, but restored function under pathological conditions. Not only was there no additive or synergistic effect in this, but clozapine was no longer as effective when added together with lithium, indicating that medications that may initially have been effective when used alone may no longer be effective when used together with other medications, which we believe may have implications for polypharmacy in clinical settings. Here, it should be remembered, however, that in clinical practice clozapine and lithium are being increasingly used together, in some cases to mitigate the known side effect of clozapine in causing neutropenia, and in other cases to optimize treatment of patients with severe self-harm, and it is not generally believed that lithium and clozapine are clinically more effective when used alone than when combined together.ESM 1


Glioblastoma (GBM) has a poor prognosis after standard treatment. Recently, metformin has been shown to have an antitumor effect on glioma cells. We performed the first randomized prospective phase II clinical trial to investigate the clinical efficacy and safety of metformin in patients with recurrent or refractory GBM treated with low-dose temozolomide.2, daily)] or an experimental group [metformin  plus low-dose temozolomide]. The primary endpoint was progression-free survival (PFS). Secondary endpoints were overall survival (OS), disease control rate, overall response rate, health-related quality of life, and safety.Included patients were randomly assigned to a control group [placebo plus low-dose temozolomide (50 mg/mp = 0.679). The median OS was 17.22 months (95% CI 12.19–21.68 months) in the experimental group and 7.69 months (95% CI 5.16–22.67 months) in the control group, showing no significant difference by the log-rank test . The overall response rate and disease control rate were 9.3% and 46.5% in the control group and 5.3% and 47.4% in the experimental group, respectively.Among the 92 patients screened, 81 were randomly assigned to the control group (43 patients) or the experimental group (38 patients). Although the control group showed a longer median PFS, the difference between the two groups was not statistically significant (2.66 versus 2.3 months, Although the metformin plus temozolomide regimen was well tolerated, it did not confer a clinical benefit in patients with recurrent or refractory GBM.Trial registration NCT03243851, registered August 4, 2017.The online version contains supplementary material available at 10.1007/s12672-023-00678-3. Glioblastoma (GBM) is the most common tumor of primary malignancy in the brain and spine. Current treatment options for GBM include surgical resection, radiotherapy, and concomitant and/or adjuvant chemotherapy. However, GBM is a fatal disease with a poor prognosis . AlthougMetformin  is a biguanide antidiabetic medication. It has been suggested as the first-line medication for patients with type 2 diabetes mellitus (DM). It can inhibit mitochondrial complex I, lower cellular adenosine triphosphate (ATP) levels, cause adenosine monophosphate (AMP)-activated protein accumulation, and disrupt downstream cAMP-protein kinase A (PKA) signaling, resulting in inhibition of glycolysis in the liver . Lower sThe antitumor effect of metformin on glioma cells has been suggested by in vitro and in vivo murine models , 15. HowThis study was planned as a prospective, multicenter, phase 2 trial by the Korean Neuro-Oncology Group of the Korean Society for Neuro-Oncology (KSNO). Patients were randomized into two groups, a control group and an experimental group, with a double-blind process by a central agency. The trial was registered with ClinicalTrials.gov (NCT03243851). It was conducted at twelve tertiary hospitals in the Republic of Korea.9/L, absolute neutrophil count > 1500 per mm3 without transfusion or granulocyte colony-stimulating factor, platelet count ≥ 100,000 per µL), liver function ; and renal function test results (serum creatinine ≤ 1.5 mg/dL). Assessment of O6-methylguanine-DNA methyltransferase (MGMT) methylation status at first surgery was also needed. The exclusion criteria were pregnancy or breastfeeding; leptomeningeal metastasis; diabetes; hypersensitivity or intolerance to metformin; patients who were currently taking thiazolidinediones, sulfonylureas, metformin, and insulin regardless of the reason; congestive heart failure, unstable angina or incomplete arrhythmia, uncontrolled hypertension despite optimal medical management; myocardial infarction within 6 months before the start of drug administration; cerebrovascular accident; or uncontrolled infection.GBM patients with recurrence or progression after surgery, radiotherapy, chemotherapy, and/or targeted therapy were eligible for inclusion. Additional inclusion criteria were as follows: age of at least 19 years; Karnofsky performance score (KPS) ≥ 60; disease progression on MRI as defined by the Response Assessment in Neuro-Oncology (RANO) criteria  at leastScreened patients were enrolled according to a randomization sequence created by an independent statistician using the PROC PLAN procedure of SAS 9.4 with a 1:1 allocation using block randomization. This allocation was implemented by the central agency. Each enrolled patient was assigned an encrypted trial number. Data analysts and outcome assessors were blinded. Statisticians remained blinded to treatment allocation until all data had been analyzed to minimize bias.2) daily. To manage toxicity, prespecified dose modifications were planned in the protocol. Patients requiring discontinuation and dose reduction of metformin could have their dose raised again at the discretion of the investigator once toxicity had been resolved to baseline levels. Treatment was discontinued if toxicity did not reverse after 4 weeks of discontinuation.Patients randomly assigned to the control (placebo plus low-dose temozolomide) or experimental (metformin plus low-dose temozolomide) group were administered medication daily. Placebo or metformin was given at 1000 mg/day for the first week, at 1500 mg/day for the second week, and at 2000 mg/day from the third week until death, disease progression, unacceptable toxicity, or withdrawal of consent. Simultaneously, patients were administered low-dose temozolomide . Responses were assessed by each institutional investigator according to the RANO criteria . The MGMThe primary endpoint of the study was progression-free survival (PFS) time, which was defined as the time from randomization to tumor progression based on the RANO criteria or to death. The secondary endpoint was overall survival (OS), which was the time from randomization to death due to any cause. The proportion of patients in whom disease control was achieved, including stable disease, partial response, and complete response, and the proportion with a good response, including partial or complete response, were also evaluated as secondary endpoints. Additionally, the health-related QoL and safety of patients were assessed as secondary endpoints. Radiological and clinical findings were evaluated by each institutional investigator and supervised by the central agency.The primary endpoint of PFS was used to determine the sample size for the study based on the following assumptions: a two-sided log-rank test at the 0.1 level of significance, 80% power to detect a hazard ratio (HR) for the experimental group versus the control group of 0.58 corresponding to 25% control group and 45% experimental group at 6 months for PFS. With an anticipated accrual period of 18 months, a follow-up of 6 months, and a 10% dropout, we calculated that at least 54 patients per group  were needed. Sample size calculations were performed using PASS 13 software . The intention-to-treat (ITT) population comprised all randomized patients by their assigned treatment arms, whereas the per-protocol (PP) population was composed of ITT patients who had received at least one dose of study therapy without any major protocol deviations. We first performed primary efficacy analysis on the ITT population for primary and secondary endpoints. Sensitivity analyses were performed for the PP population. The PP population was also used for safety analyses.The primary end point for PFS was defined as the time from randomization to progression or death, whichever occurred first. Censoring rules for PFS followed those described in the 2007 FDA guidance on clinical trial endpoints. OS was defined as the duration between the date of randomization and death or censoring at the date of the last follow-up. We estimated PFS and OS using the Kaplan–Meier method with the median survival duration summarized. The log-rank test was used to compare the control and experimental groups. The pointwise 95% confidence interval (CI) of the overall survival rate or progression-free survival rate for 6, 12, and 24 months was calculated with Greenwood’s formula using complementary log-log transformation. Hazard ratios (HRs) and 95% confidence intervals (CIs) for PFS and OS were estimated using the Cox proportional hazard model. Proportional hazards assumptions were confirmed by Schoenfeld residuals and Supremum tests.Response rates were compared between groups using the chi-square test or Fisher’s exact test when appropriate. We evaluated and tabulated the number of patients with at least one reported adverse event by the group. The effects of treatments on QoL were analyzed by generalized estimating equation (GEE) models with an unstructured correlation structure, normal distribution, and identity link function. A GEE model included treatment, time, and the interaction term between treatment and time. The results were regarded as statistically significant when the p value was less than 0.05. All statistical analyses were performed using SAS ver. 9.4 .A total of 12 tertiary hospitals in the Republic of Korea participated in this study, and ethical approval was received from each institutional review board before enrollment began. All patients provided written informed consent in accordance with national guidelines. Patients were not compensated for their participation.p = 0.045)  or an experimental group (n = 38). Among the 43 patients assigned to the control group, the study was interrupted for 7 patients because of withdrawal of consent (n = 5) or medical problems (n = 2). The remaining 36 patients in the control group were treated with the scheduled protocol. Eleven patients completed six cycles of intervention, whereas 25 patients did not complete treatment due to disease progression (n = 20), side effects (n = 3), cerebral infarction (n = 1), or denial of treatment (n = 1). Among the 38 patients assigned to the experimental group, the study was stopped for 6 patients because of withdrawal of consent (n = 2) or deterioration of the patient’s condition (n = 4) before the clinical trial. The remaining 32 patients in the experimental group followed the treatment schedule. Twenty-eight patients received up to 2000 mg/day of metformin. Ten patients completed the intervention, whereas 22 patients did not complete treatment because of disease progression (n = 21) or side effects (n = 1) . PFS at 6 and 12 months was 32% (95% CI 0.18–0.47) and 24% (95% CI 0.11–0.40) in the control group and 29% (95% CI 0.15–0.45) and 15% (95% CI 0.04–0.32) in the experimental group, respectively. OS tended to be improved in the experimental group compared with the control group. The median OS was 17.22 months (95% CI 12.19–21.68 months) in the experimental group and 7.69 months (95% CI 5.16–22.67 months) in the control group, showing no significant difference by the log-rank test . OS at 6 and 12 months was 83% (95% CI 0.64–0.93) and 75% (95% CI 0.55–0.88) in the experimental group and 60% (95% CI 0.42–0.74) and 46% (95% CI 0.28–0.62) in the control group, respectively  of 43 in the control group and 28 (73.7%) of 38 in the experimental group. The median PFS time was 2.66 months (95% CI 1.74–4.40 months) in the control group and 2.3 months (95% CI 0.67–1.87 months) in the experimental group. The PFS time between the two groups was not significantly different by the log-rank test  in the control group. Physical function, role function, and emotional function were similar between the two groups except that the control group showed better results at a visit time of 12 weeks. Cognitive function and social function were also similar between the two groups. In symptom scales, fatigue, nausea/vomiting, pain, insomnia, appetite loss, constipation, diarrhea, and financial impact were similar. Dyspnea was higher in the experimental group at 12 weeks. For the BN20 symptom scales, future uncertainty, communication deficit, seizure, drowsiness, hair loss, itchy skin, and weakness of legs were similar. Visual disorder and motor dysfunction were higher in the experimental group at 8 and 12 weeks, respectively. Headache was higher in the control group at 8 weeks. Bladder control was higher in the control group at 16 weeks. The results of the QoL questionnaires according to the time flow are summarized in Supplementary Table 1.In vitro and experimental in vivo studies have demonstrated the antitumor effect of metformin on glioma cells, including human and rat glioma cells. The most proposed mechanism involved in this effect of metformin is the activation of AMPK. AMPK is a metabolic tumor suppressor that maintains cell metabolism and growth at appropriate levels. It can also activate the response to energy stress in the microenvironment of tumors. AMPK can act on mTOR, p53, and fatty acid synthase, resulting in suppression of cell proliferation and growth . Two antMetformin also exhibits a cytotoxic effect on glioma stem cells mediated by inhibition of the AKT pathway , 27. In TMZ plus metformin shows a synergistic effect on glioma cells in vitro. Compared with TMZ or metformin monotherapy, this combination enhances AMPK phosphorylation and inhibits mTOR phosphorylation, AKT phosphorylation, and p53 expression , 33. SynThe therapeutic effect of metformin in patients with malignant gliomas has been reported but remains controversial. A retrospective investigation was performed to determine the effects of diabetes mellitus (DM), corticosteroid therapy, and metformin therapy on progression and survival in 276 primary glioblastoma patients . DespiteA phase Ib clinical trial, the only clinical prospective study to the best of our knowledge, was conducted with metformin and chloroquine, an antimalarial drug with a putative anticancer function, for IDH-1-mutated solid cancers. This study included 12 cholangiocarcinoma, 2 glioma, and 3 chondrosarcoma patients in total. The combinational treatment was performed with a median duration of 43 days under a well-tolerated state, but it did not show a clinical benefit in this trial . SimilarOur study is the first clinical study designed as a prospective, double-blind, randomized trial to investigate the clinical significance of metformin in patients with recurrent or progressive GBM. This study could not identify any survival benefit of metformin on PFS or OS in the included patients. Metformin had no survival advantage at the 3 time points . The tendency of OS prolongation might be attributable to postprogression therapy at each institution. However, treatment with metformin at a maximal dosage of 2000 mg/day was safe and tolerated with minimal toxicity and well-preserved QoL in patients with GBM.This study included a relatively limited number of patients with recurrent GBM. To minimize bias of patients by major protocol deviations, such as withdrawal of consent, this study was analyzed with an ITT method, including all randomized patients. However, 7 (16.3%) and 6 (15.8%) patients in the control and experimental groups, respectively, were interrupted before the treatment. It is possible that the difference in survival outcomes between the two groups has not been confirmed due to a decrease in the number of patients participating in clinical studies. Because the clinically recommended maximum dosage of metformin is 2000 mg/day, this study was designed with 1000 mg/day as the starting dose and 2000 mg/day as the maximum dose. However, it is also possible that this 2000 mg/day dosage of metformin is still insufficient to show a positive survival outcome. In future studies, it will be necessary to confirm the difference in survival outcomes based on metformin dosages including a dosage of over 2000 mg/day or based on a novel delivery system. Additionally, metformin can inhibit insulin-mediated tumor growth by lowering systemic insulin levels. However, systemic insulin levels were not measured in this study, so the survival outcome depending on insulin level by metformin was not assessed.Although metformin is well tolerated, our study did not show that metformin confers a benefit in survival outcomes or radiological response in patients with recurrent or refractory GBM.Supplementary Material 1


This study aimed to examine the effects of mindfulness-based stress reduction (MBSR) in patients with acute myocardial infarction (AMI) after primary percutaneous coronary intervention (PPCI).A retrospective study was conducted with data collected from AMI patients who underwent successful PPCI. The study included 61 cases that received 8-week MBSR intervention (MBSR group) and 61 cases that received weekly health education (control group) over the same period. Outcome measures, including hemodynamic parameters, psychosocial characteristics , health-related quality of life , and major adverse cardiovascular events (MACE), were assessed at baseline (T1), post-intervention (T2), 1 month after the post-intervention (T3) and 3 months after the post-intervention (T4).Compared to the control group, the MBSR group showed improvements in blood pressure, specifically in systolic blood pressure (SBP) at T4, and diastolic blood pressure (DBP) at T3 and T4, and mean arterial blood pressure (MABP) at T3 and T4. Additionally, the MBSR group had lower scores of anxiety and perceived stress  and higher scores of perceived social support (PSSS) after the intervention. Furthermore, the MBSR group had higher scores on the SAQ-7 at all measurement points. The control group had a significantly higher total MACE rate compared to the MBSR group (26.23% vs. 9.84%).This study provides support for the potential benefits of MBSR as an adjunctive treatment for AMI patients undergoing PPCI. Negative emotions such as depression, anxiety, and stress, are commonly experienced by cardiac patients following a myocardial infarction (MI), contributing to poorer mental health and increased risk for adverse outcomes , 2. PrimMindfulness, an popular psychological intervention worldwide originally designed in the 1970s by Jon Kabat-Zinn for chronic pain patients , involveOver the past decade, several studies have investigated the effectiveness of the MBSR program in cardiac patients. Lundgren O et al. examined the feasibility of MBSR for CAD patients with depressive symptoms and found positive changes in depressive symptoms and mastery scores . AnotherTherefore, this study aimed to fill the research gap by conducting a robust evaluation of the effectiveness of MBSR in AMI patients after PPCI, providing valuable insights into the potential benefits of MBSR in this specific population. The study utilized a retrospective design and compares two groups: an MBSR group receiving 8 weekly sessions of 2.5 h each and a 1-day retreat, and a control group receiving weekly health education. Various outcome measures, including hemodynamic parameters, psychosocial characteristics, health-related quality of life (HRQoL), and major adverse cardiovascular events, are assessed at four time points. This study’s innovative approach and focus on the specific population of AMI patients after PPCI contribute to the existing literature and are highly relevant to post-AMI cardiac care.The study was approved by the ethics committee of our hospital, and the informed consent was waived due to the retrospective nature of the study.2, attendance of mindfulness training or a stress reduction course  within the past two years, presence of psychosis, dementia or communication disturbances, and a history of alcoholism or other substance abuse.This retrospective analysis collected data between June 2019 and June 2022 on AMI patients who underwent successful PPCI, were moved to an internal medicine ward, and then discharged from our hospital. AMI was defined as acute symptoms of ischemia persisting for more than 30 min within 24 h from the onset of symptoms, with elevations in cardiac biomarker values (preferably cardiac troponin) and eventual detection of coronary artery disease by emergency coronary angiography . The incThe study included 61 cases that received 8-week MBSR intervention (MBSR group) and 61 cases that received weekly health education (control group). Following a previously standard protocol , the MBSImpedance cardiography was used to measure hemodynamic parameters, including heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial blood pressure (MABP).The perceived Stress Scale (PSS) and Perceived Social Support Scale (PSSS) were used to assess the degree of perceived stress and perceived social support from family, friends and significant others, respectively. The 14-item PSS questionnaire examined stress levels using a 4-point scale , with a total score ranging from 0 to 56 . The 12-HRQoL was measured using the 7-item Seattle Angina Questionnaire (SAQ-7), which includes three domains: physical limitation, angina frequency, and quality of life . Higher Major adverse cardiovascular events (MACE) during the follow-up period were defined as heart failure (HF) requiring hospitalization, recurrent MI, repeat revascularization, and cardiac death. Hospitalization and death due to non-cardiac cause was not considered events.post hoc sample size calculation was performed using G*Power 3.1.9.2 software to determine the appropriate sample size for a t-test comparing the means of two independent groups. The calculation was based on the variable SBP at T4. The MBSR group consisted of 61 participants, with a mean SBP of 129.6 mmHg and a standard deviation (SD) of 11.61. The control group also comprised 61 participants, with a mean SBP of 135.9 mmHg and a SD of 12.83. The effect size (d) was determined to be 0.514. With an α error (significance level) set at 0.05, a post hoc sample size calculation was conducted, yielding a power of 80.53%.A P value of less than 0.05 considered statistically significant.All statistical analyses were performed using Prism . Continuous data were summarized as means ± standard deviation (SD) or median ± interquartile range (IQR) after testing for normality using the Shapiro-Wilk test. Between-group comparisons were conducted using t-test or Mann-Whitney test, depending on the distribution of the data. Categorical variables were presented as n (%) and compared using chi-square or Fisher’s exact test. Two-tailed tests were performed for each comparison, with a P = 0.244), age (P = 0.395), marital status (P = 0.788) and level of education (P = 0.320). Additionally, there were no significant differences in comorbidities, clinical presentation, lesion characteristics, culprit vessel, procedural characteristics, in-hospital adverse events, medication use at discharge, and length of hospital stay between the MBSR group and control group .A total of 122 patients participated in this study, with a mean age of 58.69 ± 14.80 years (ranging from 18 to 72 years). The majority of the patients were male (68.03%), married (91.80%), and had a high school education or higher (68.85%). Table P > 0.05). However, the MBSR group showed decreased blood pressure compared to the control group at specific time points: SBP at T4 (P = 0.005), DBP at T3 (P = 0.018) and T4 (P < 0.001), and MABP at T3 (P = 0.003) and T4 (P < 0.001).As shown in Table P > 0.05). However, significant differences were observed between the MBSR and control groups at T2, T3, and T4, with lower scores of HADS and PSS and a higher score of PSSS in the MBSR group compared to the control group .Figure P > 0.05). However, significant improvements in physical limitation, angina frequency, and quality of life were observed in the MBSR group at T2-T4 compared to the control group . However, the total MACE rate was significantly higher in the control group as compared to the MBSR group .The occurrence of MACE was evaluated Table . During A growing body of research evidence suggested that MBSR has measurable and long-lasting benefits in medical and psychological well-being in various conditions , 23. ConThe ability of patients to track and cope with stress was also found to increase with greater awareness of their emotions and bodily sensations . Our stuSAQ-19 was designed to be a more sensitive measure of recent changes in angina, and SAQ-7 was to assess patients’ satisfaction with the care they received for their coronary disease, which was more appropriate for most applications as reported by Thomas M et al. . The MBSA major limitation of our study was its retrospective observational design instead of a randomized controlled trial. Furthermore, despite observing differences in blood pressure between the MBSR and control groups during the 3-month follow-up, it is crucial to acknowledge several limitations in our study. Firstly, the 3-month follow-up period may not fully capture the long-term effects of MBSR on blood pressure. It is possible that the observed differences might diminish or disappear beyond this time frame, as most AMI patients without complications tend to return to baseline or normal life and have stable hemodynamics after 3 months. Future studies with longer follow-up durations are needed to provide more comprehensive insights into the sustained effects of MBSR on blood pressure. Secondly, individual variations in response to MBSR could have influenced the observed differences in blood pressure. Some patients may have experienced more pronounced improvements, while others may not have shown significant changes. The influence of these individual differences should be considered when interpreting the results. It would be valuable to explore potential factors that may contribute to differential responses to MBSR, such as baseline characteristics or psychological factors. Lastly, it is essential to note that our study focused on a specific population of AMI patients without complications. The generalizability of our findings to other populations or individuals with different clinical profiles should be approached with caution. Future research should aim to replicate these findings in larger and more diverse samples to determine the broader applicability of MBSR in different patient populations.In summary, this study provides evidence that MBSR can be a beneficial adjunctive treatment in AMI patients undergoing PPCI, as it shows positive effects on alleviating negative emotion and perceived stress, increasing perceived social support, and reducing MACE. However, it is important to recognize that these findings are specific to the studied population and caution should be exercised in extrapolating the results to all cardiac patients.


Staphylococcus aureus (S. aureus), is becoming a leading public health concern demanding effective therapeutics. Metabolite modulation can improve the efficacy of existing antibiotics and facilitate the development of effective therapeutics. However, it remained unexplored for drug-resistant S. aureus (gentamicin and methicillin-resistant), primarily due to the dearth of optimal metabolite extraction protocols including a protocol for AMR-associated metabolites. Therefore, in this investigation, we have compared the performance of the two most widely used methods, i.e., freeze-thaw cycle (FTC) and sonication cycle (SC), alone and in combination (FTC + SC), and identified the optimal method for this purpose. A total of 116, 119, and 99 metabolites were identified using the FTC, SC, and FTC + SC methods, respectively, leading to the identification of 163 metabolites cumulatively. Out of 163, 69 metabolites were found to be associated with AMR in published literature consisting of the highest number of metabolites identified by FTC (57) followed by SC (54) and FTC + SC (40). Thus, the performances of FTC and SC methods were comparable with no additional benefits of combining both. Moreover, each method showed biasness toward specific metabolite(s) or class of metabolites, suggesting that the choice of metabolite extraction method shall be decided based on the metabolites of interest in the investigation.Emerging antimicrobial resistance (AMR) among Gram-positive pathogens, specifically in  This reto treat . Tremendto treat .S. aureus infections are spread through direct contact with an infected wound or contaminated hands and can be fatal if remains untreated  and the centers for disease control and prevention (CDC) have placed MRSA under a list of serious threat causing drug-resistant pathogens . MRSA alntreated . Furtherreatment .S. aureus, has become critically important. This can be achieved using various new-generation biological tools, such as genomic, transcriptomic, proteomic, and metabolomics. However, metabolomics allows the identification and quantification of metabolites that are the end product of any genomic- and proteomic-based biological activity of an organism at a given point of time and hence provide a characteristic chemical fingerprint of a specific cellular process , including the very first step of extracting AMR-associated metabolites.Therefore, understanding the molecular changes driving antibiotic resistance among Gram-positive bacteria, specifically  process . Recentlhia coli . A centrnfection . Further effects . A decreibiotics . However. aureus . This maS. aureus because the method of metabolite extraction dictates the possible range of metabolites that can be detected in the sample and thus has a large impact on the potential outcome(s) of the metabolomic studies. Most of the metabolomic investigations employ FTC or SC-based methods for extracting bacterial metabolites; however, their efficacy for extraction of AMR-associated metabolite from Gram-positive bacteria has not been investigated yet , a highly sensitive and advanced mass-spectrometer was used to potentially increase the metabolomic coverage. Finally, the list of identified metabolites was subjected to an intensive literature search for finding the potential association of identified metabolites with AMR. Altogether, this information was utilized to identify the optimal metabolite extraction method for investigating AMR-associated metabolites.Therefore, the goal of this study was to determine the optimal method for extraction of AMR-associated metabolites from ated yet . In this2.2.1.S. aureus (ATCC®33592) was inoculated in 5 ml Mueller Hinton Broth (MHB), followed by incubation for approximately 16 h at 37°C at 220 rpm. The secondary culture was inoculated (1% from primary culture) in fresh MHB and incubated till the exponential growth phase  was achieved. Approximately 107 cells (OD-based measurement) were harvested using centrifugation and washing with LC–MS grade cold water and kept at −80°C. The cell pellets were generated for 6 independent biological replicates. Since the cellular architecture and composition of member pathogens among a species are highly expected to be similar, a single strain of S. aureus was used for the study.The primary culture of 2.2.For metabolite extraction, the stored cell pellet was thawed for 10 min on ice, followed by resuspension in 500 μl of mass spectrometry (MS) grade chilled methanol (Sigma). To evaluate the metabolites extraction efficiency of three extraction methods, i.e., FTC, SC, FTC + SC, 13C-labeled L-valine (40 ng/ml), an internal standard, was added to each sample before metabolite extraction . For the2.3.The standard workflow for metabolite separation and measurement using ESI-LC–MS/MS was as followed. An aliquot (100 μl) of the sample was vacuum dried and the pellet was resuspended in 3:17, methanol: water mixture (25ul), followed by vortexing for 15 min, and 10 min centrifugation at 11000 rpm at 4°C. Data acquisition was done on C18 Reversed Phase HPLC Columns (C18) and Hydrophilic interaction LC (HILIC) columns with positive and negative mode separately. The collected metabolites were separated using C18 (HSS T3) and HILIC (XBridge BEH Amide) columns on UPLC ultimate 3,000 maintained at 40°C and 35°C temperature, respectively. A gradient of mobile phase A consisting of water +0.1% formic acid and mobile phase B (methanol + 0.1% formic acid) was used as a mobile phase for the C18 column. For the HILIC column, 20 mM ammonium acetate in the water of pH 9.0 (mobile phase A) and 100% acetonitrile (mobile phase B) were used for separation. For separation in the reverse phase, a gradient of 1% mobile phase B to 99% mobile phase B over 14 min (flow rate of 0.3 ml/min) was set and for the HILIC column, 85% mobile phase B to 10% B over 16 min (flow rate of 0.35 ml/min) was used. 5ul sample was injected into the column and for data acquisition. The Orbitrap Fusion Tribrid Mass Spectrometer (Thermo-Scientific) equipped with heated electrospray ionization (HESI) source was used for processing the sample using the following settings: 4000 positive mode spray voltage, 35,000 V for negative mode, 60–900 m/z mass range, AGC (Automatic gain control) was targeted at 100,000 ions. For data acquisition, 120,000 resolutions in MS1 mode and 30,000 resolutions in data-dependent MS2 scan mode were used. For MS, 50 ms was used as the maximum injection time while for MS/MS, an AGC target of 20,000 ions and a maximum injection time of 60 ms was used.2.4.The untargeted workflow of Progenesis QI software for metabolomics from Water Corporation was used at default settings for acquiring the data and its analysis. The MetaScope plugin of Progenesis QI metabolites was used for matching the mass, fragmented ions pattern and retention time of identified compounds with a list of 950 metabolites from our in-house library followed by an online spectral library search to confirm the identified metabolites. Peak detected in ≥4 replicates  with intensity ≥100  receives an identification by spectral match. Further, ±1 min and 5 ppm retention time error for MS and MS/MS with fragmentation pattern match were also considered as a criterion for identifying metabolites .p (FDR) cutoff of ≤0.05. 2-D PCA was performed displaying 95% confidence regions to observe the inner clusters and find the apparent outliers. Hierarchical clustering heat maps were prepared using t-test/one-way ANOVA and Euclidean distance measure and Ward clustering method.For data analysis, the list of identified metabolites in HILIC and C18 (both positive and negative mode) was combined, and duplicates were removed . MetaboAMetabolomics Workbench.For quantitation, the peak intensity average of replicates for each metabolite extraction method  was considered and used to identify differences among the extraction method. Chemical class-based analysis and functional categorization of metabolites were done using 2.5.A majority of antibiotics work by targeting cellular processes like (1) cell wall synthesis, (2) cell metabolism, (3) nucleotide biosynthesis and/or (4) protein synthesis. So, it is evident that establishing and identifying metabolites associated with these processes can help us in understanding the metabolic fingerprint and mechanism of antibiotic resistance. Therefore, a literature search to identify the role of each metabolite in nucleotide synthesis, protein synthesis, cell wall biosynthesis, cell proliferation  and cell death  was performed using Google Scholar and PubMed and the resulting information was utilized to fetch the AMR-associated metabolites and followed by data interpretation.3.3.1.S. aureus using three methods, FTC, SC, and their combination FTC + SC, were separated using UPLC ultimate 3,000 (C18and HILIC column), detected and quantified using an Orbitrap Fusion Mass Spectrometer and identified using the untargeted workflow of Progenesis QI software for metabolomics (Water Corporation). The list of identified non-polar and polar metabolites from the C18 and HILIC columns, respectively, was merged to get a collated list of metabolites extracted from each method-FTC, SC, and FTC + SC  predominantly enabled the identification of different chemical classes of amino acids followed by saturated fatty acids and dicarboxylic acid  and identified using ESI-LC–MS/MS , a highly sensitive and advanced mass-spectrometer to potentially achieve increased metabolomic coverage. Further, we have used 2 different Liquid Chromatography (LC) columns, i.e., HILIC and C18 which are specific for separating compounds with different physico-chemical properties were also used. Generally, HILIC columns are very well known for separating polar amino acids, organic acids, sugars, phosphorylated sugars, nucleobases, nucleotides, phosphorylated metabolites, hydrophilic vitamins, and coenzymes  and 107 (450 M) metabolites were identified using cold methanol and vortexing (vigorously for ~1 min) based metabolite extraction method with HPLC coupled with a TSQ Quantiva Triple Quadrupole mass spectrometer in (methicillin-resistant S. aureus) MRSA and (methicillin sensitive S. aureus) MSSA strains, respectively, after exposure to a sublethal dose of antibiotic   . Howeverloxacin) . Anotherloxacin) . Althougloxacin) . Among tloxacin)  (Ammons loxacin) . Similarloxacin) . The pos4.2.S. aureus and Gram-positive pathogens; however, the metabolite of interest shall be key in choosing the method for metabolite extraction.Of the identified 163 metabolites, 69 (42.0%) were observed to be associated with AMR in published literature . Among t4.3.S. aureus were identified using SC (119 metabolites) and subsequently by FTC (116 metabolites) and FTC + SC (99 metabolites). Whereas for K. pneumoniae, the maximum number of metabolites were identified by the FTC (151) method followed by FTC + SC (132 metabolites) and SC . A similar pattern was observed in AMR-associated metabolites. This supports that the metabolomic architecture and AMR-associated metabolites among S. aureus (a Gram-positive pathogen) may be different than the K. pneumoniae (a Gram-negative pathogen). This also suggests that the optimal method for extracting metabolites from Gram-positive pathogens may be different than the method for Gram-negative pathogens.In this investigation, the maximum number of metabolites from 5.S. aureus is becoming a global health concern and urgently demands strategies to control it. Understanding the metabolomic footprint of antibiotic-resistant/sensitive pathogens has been shown to improve our understanding of the emergence/spread of AMR superbugs. However, very few efforts have been made in this direction possible due to the lack of appropriate methods of metabolite extraction.The increasing emergence of AMR among Gram-positive pathogens such as S. aureus (gentamicin and methicillin-resistant) using a highly sensitive and advanced HPLC-coupled mass-spectrometer (ESI-LC–MS/MS). The SC and FTC methods were observed to identify a comparable number of total metabolites as well as AMR-associated metabolites and hence may be utilized for investigating the metabolome of S. aureus or other Gram-positive bacteria after further validation. FTC + SC gave a lower yield of metabolites possible due to the degradation of already extracted metabolites by FTC during sonication. The methods of metabolite extraction were also observed to have biasness toward specific metabolite(s) or class of metabolites  and hence can potentially impact the overall finding of the metabolomics-based studies. Therefore, the method of metabolite extraction shall be primarily chosen based on the metabolites of interest in the investigation. Altogether, our data can help in designing/planning pathway specific/directed metabolomics studies which could improve understanding of the emergence/spread of AMR superbugs and ultimately contribute to improving the efficacy of existing antimicrobial therapies.Therefore, in this investigation, we have compared the performance of the two most common methods, i.e., FTC and SC alone and in combination (FTC + SC), for extracting metabolites from The datasets presented in this study can be found in online repositories (MetaboLights study identifier-MTBLS7338). The names of the repository/repositories and accession number(s) can be found in the article/NK conceived, designed, and coordinated the study. SK and MK generated the study material. AK and YK performed most of the experiments. RS, LT, AK SK, YK, and NK performed the data analysis. LT, RS, and SR wrote the manuscript. NK and YK supervised the manuscript writing and revisions. All authors contributed to the article and approved the submitted version.This work was supported by grants awarded to NK from the Indian Council of Medical Research Govt. of India (Grant No. AMR/Adhoc/233/2020-ECD-II) and Translational Health Science and Technology Institute Faridabad, India. RS received Senior Research Fellowship from the Council of Scientific and Industrial Research, Govt. of India, and SS and LT received Junior Research Fellowship from the Department of Biotechnology, Govt. of India.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Accumulating evidence indicates that type II cystatin (CST) genes play a pivotal role in several tumor pathological processes, thereby affecting all stages of tumorigenesis and tumor development. However, the prognostic and predictive value of type II CST genes in GC has not yet been investigated.The present study evaluated the expression and prognostic value of type II CST genes in GC by using The Cancer Genome Atlas (TCGA) database and the Kaplan–Meier plotter (KM plotter) online database. The type II CST genes related to the prognosis of GC were then screened out. We then validated the expression and prognostic value of these genes by immunohistochemistry. We also used Database for Annotation, Visualization, and Integrated Discovery (DAVID), Gene Multiple Association Network Integration Algorithm (GeneMANIA), Search Tool for the Retrieval of Interacting Genes/Proteins (STRING), nomogram, genome-wide co-expression analysis, and other bioinformatics tools to analyze the value of type II CST genes in GC and the underlying mechanism.P = 0.015). Multivariate Cox regression analysis confirmed that the high expression level of CST4 was an independent prognostic risk factor for OS.The data from the TCGA database and the KM plotter online database showed that high expression of CST2 and CST4 was associated with the overall survival (OS) of patients with GC. The immunohistochemical expression analysis showed that patients with high expression of CST4 in GC tissues have a shorter OS than those with low expression of CST4 (HR = 1.85,95%CI: 1.13–3.03, Our findings suggest that CST4 could serve as a tumor marker that affects the prognosis of GC and could be considered as a potential therapeutic target for GC.The online version contains supplementary material available at 10.1186/s12885-023-11550-6. Globally, gastric cancer (GC) is the fifth most common malignant tumor in new incidence and the fourth leading cause of cancer-related death worldwide . The incThe cystatin (CST) superfamily are a kind of thiol proteinase inhibitors (TPIs) that are widely present in human tissues and body fluids and function as competitive and reversible inhibitors of cysteine proteases. The CST superfamily is divided into three subfamilies according to their three-dimensional molecular structure and biochemical function : (1) typhttps://portal.gdc.cancer.gov) database and the Kaplan–Meier plotter (KM plotter) online database. We also used immunohistochemistry to validate the expression of type II CST genes in GC tissues and their prognostic value. Finally, various bioinformatics tools were used to determine the potential function and mechanism of type II CST genes.To determine the prognostic value and potential function of type II CST genes in GC, we analyzed the expression and prognostic value of type II CST genes by using The Cancer Genome Atlas  was used to analyze Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment of type II CST genes [http://www.genemania.org/; accessed May 20, 2019) was used to predict the function of type II CST genes [https://string-db.org/; accessed May 20, 2019) was used to detect protein–protein interaction (PPI) networks [The Database for Annotation, Visualization, and Integrated Discovery . The TCGA database included 407 patients diagnosed with GC, including 375 GC tissues and 32 adjacent normal tissues. After excluding cases with missing follow-up data, 351 patients with GC were included in our present analysis. We used the TCGA database to perform paired t-test on the mRNA expression levels of type II CST genes in GC and adjacent normal tissues, and scatter plots and receiver operating characteristic (ROC) curves were then plotted. For survival analysis, patients with GC were divided into two groups on the basis of the median expression level of type II CST genes: high expression group and low expression group. The mRNA expression of type II CST genes and clinical information in the TCGA database were used for the univariate survival analysis and the multivariate Cox regression model survival analysis.We downloaded mRNA expression data and the corresponding clinical information related to GC from the TCGA  was used to analyze the relationship between the mRNA expression of type II CST genes and the OS of patients with GC. The KM plotter online database was established using the gene expression data and survival information of 875 GC patients; these data were downloaded from the Gene Expression Omnibus (GEO)  [P were analyzed.The KM plotter online database (SE62254) . The KM https://CRAN.R-project.org/package=rms), along with its dependencies in the R platform, and visualized using gplot [A nomogram was constructed, and the prognostic risk between type II CST genes and the OS grade in patients with GC was assessed. The possible utility of type II CST genes to predict clinical rank was also assessed. According to prognostic clinical indicators and Cox regression model survival analysis, related factors and type II CST gene expression levels were included in the risk model. The scores for each factor were counted, and the 1-, 3-, 5-, and 10-year survival rates were calculated . The nomng gplot .The stratified analysis method was used to evaluate the prognostic value of type II CST genes in different stratifications in the TCGA and the KM plotter online databases.GC sections with complete clinicopathological and prognostic data were collected from the Affiliated Hospital of Guangxi Medical University. The sections were placed in 65◦C oven for 2 h, dewaxed with xylene, hydrated with a graded series of ethanol, and repaired with antigen repair buffer by EDTA method. Endogenous antigens were blocked with 3% hydrogen peroxide. The sections were incubated with CST2 antibody  and CST4 antibody  overnight at 4◦C. The sections were heated for 30 min to room temperature and incubated with secondary antibody for 20 min. The staining result was visualized using DAB color developing solution. The sections were stained with Hematoxylin, differentiated in 1% hydrochloric acid in alcohol, and dehydrated. Thereafter, the sections were naturally dried in a fume hood, transparentized by xylene, and sealed with neutral resin. The negative control used PBS instead of primary antibody, and the positive section was served as positive control. Immunohistochemistry was performed to determine the expression of type II CST genes in GC and adjacent normal tissues. Paired t-test was used to analyze the difference in the expression level between GC and adjacent normal tissues, and scatter plots and ROC curves were drawn. The X-tile software was used to determine the optimal cutoff value. X-tile, a statistical software developed by Yale University with a single function, can determine the cut-off point of continuous variables and facilitate the drawing of the Kaplan–Meier curve . Finally P < 0.05. These co-expressed genes were then used to perform GO and KEGG functional enrichment analyses by using DAVID v6.8 [Pearson’s correlation analysis was performed between type II CST prognostic genes and TCGA genome expression profile data, and the co-expressed genes were screened out. Pearson’s correlation coefficient was calculated by the cor function on the R 3.4.4 platform. The difference was considered to be significant at |r|> 0.6,VID v6.8 –24.Statistical analysis was performed using SPSS v.25.0 software  and GraphPad Prism software  to generate vertical scatterplots and survival curves. Nomograms and correlations were also generated using R software .The biological function of type II CST genes was assessed by DAVID, and biological process (BP), cellular component (CC), and molecular functionality (MF) were obtained from GO and KEGG pathway analyses Fig. A and B. P < 0.05, Fig. P < 0.001, Fig. P < 0.001, Fig. P = 0.018, HR = 1.48, 95% CI = 1.07–2.06; P = 0.018, HR = 1.48, 95% CI = 1.07–2.06; P = 0.010, HR = 1.53, 95% CI = 1.10–2.13; P = 0.030, HR = 1.43, 95% CI = 1.03–1.99, respectively).From the vertical scatter plot of type II CST gene expression levels in the TCGA, it was observed that the expression levels of CST1, CST2, CST3, and CST4 were significantly different in GC tissues and adjacent normal tissues , tumor stage, cancer status, residual tumor, chemotherapy, and targeted therapy. The results showed that age, tumor stage, cancer status, residual tumor, chemotherapy, and targeted therapy were associated with OS . Consistent with the TCGA analysis, the expression of CST2, CST4, and CST5 was significantly associated with worse prognosis . The ROC curve of the expression levels of CST2 and CST4 is shown in Fig. P < 0.05). Figure P = 0.010, HR = 1.78, 95% CI = 1.13–2.81, Fig. P = 0.015, HR = 1.76, 95% CI = 1.76–2.78) (Table (P > 0.05, Table Figure To determine the underlying mechanism by which CST4 plays a role in GC, we performed a genome-wide co-expression analysis. The regulatory network of CST4 and its co-expressed related genes in GC tissues from the TCGA cohort are shown in Supplemental Fig. SThe results of GO analysis of CST4 and its co-expressed genes of GC in the TCGA showed that it enriched in focal adhesion, negative regulation of cell proliferation, fascia adherens, cell-matrix adhesion, positive regulation of cell-substrate adhesion, negative regulation of cell migration, etc. Table . SimilarCancer development is a multifactorial long-term interaction process, and normal cells undergo multiple steps to transform into malignant cells . Many stThe maintenance of the balance between CST and cysteine proteases is crucial to the normal function of biological systems. Once the balance is disrupted, it will cause cell death, leading to tumorigenesis of malignancies . CSTs weThe combination of serum CST4 and DR-70 was reported to further enhance the diagnosis of early colorectal cancer . Recent Wang et al.  indicateThe mechanism by which CST4 plays a role in the occurrence and development of GC has not yet been elucidated. Zhang et al.  found thThe present study has one potential limitation. We used clinical specimens to validate the results obtained by bioinformatics, which largely confirmed the differential expression of CST4 and its significance in the survival and prognosis of patients with GC. However, we were unable to conduct in vivo and in vitro experiments to further confirm the mechanism by which CST4 affects GC occurrence and development. This aspect will be the focus of our future studies.In conclusion, this study systematically investigated the relationship between the expression of type II CST genes and OS in patients with GC. As the findings suggest, high expression of CST4 in GC is correlated with a poor prognosis. Therefore, CST4 may be a novel prognostic and predictive indicator for GC.Additional file 1: Supplemental Table 1. Demographic and clinical data for 351 GC patients.Additional file 2: Supplemental Figure S1. Pearson correlation analysis of correlation between TCGA genome-wide expression profile data and CST4.Additional file 3： Supplemental Data S1. CST4 and its co-expressed related genes in GC tissues from the TCGA cohort.Additional file 4: Supplemental Data S2. GO term enrichments of co-expressed genes of CST4 in gastric cancer.