Alcaligenaceae and Sutterella were more abundant in individuals with higher DT scores at the family and genus levels (p = 0.017), while Streptococcaceae at the family level and Streptococcus at the genus level were prevalent in those with lower DT scores . This study illuminates the intricate interplay of stress, QOL, and gut microbiota in newly diagnosed breast cancer patients, offering valuable insights for potential interventions of biomarker or probiotics aimed at alleviating stress and enhancing QOL in this patient cohort.There is little research about the stress, quality of life (QOL) and gut microbiota in newly diagnosed breast cancer patients. In this study addressing the dearth of research on stress, quality of life (QOL), and gut microbiota in newly diagnosed breast cancer patients, 82 individuals were prospectively observed. Utilizing the Functional Assessment of Chronic Illness Therapy (FACT)-Breast questionnaire to assess health-related quality of life (HRQOL) and the Distress Thermometer (DT) to gauge distress levels, the findings revealed a mean FACT-B score of 104.5, underscoring HRQOL's varied impact. Significantly, 53.7% reported moderate to severe distress, with a mean DT score of 4.43. Further exploration uncovered compelling links between distress levels, FACT-B domains, and microbial composition. Notably,  Due to the early screening and improvement in the diagnosis and treatment, breast cancer patients’ survival outcomes including disease-free survival and overall survival improved progressively2. This improvement has also led to the need for more intensive management in terms of psychological issues such as quality of life (QOL), stress, anxiety and depression in breast cancer patients. The anxiety and depression negatively affect the quality of life and survival rates in breast cancer patients3.Female breast cancer has surpassed lung cancer as the most common diagnosed cancer, with an estimated 2.3 million new cases (11.7%), and with 68.5 thousand deaths6. The gut microbiota appears to influence breast cancer risk, response to treatment, and recurrence by affecting human health through metabolic, neural, and endocrine signaling, and immune activity7. The gut microbiota dysbiosis  may lead to the development of breast cancer through the crosstalk among microbiota and both endogenous hormones and estrogen-like compounds might synergize to provide protection from disease but also to increase the risk of developing hormone-related diseases11. Besides, diversity and specific microbiota were linked to chemotherapy response as well as prognosis in breast cancer patients12. Microbiota diversity was also predictive of side effects such as neurological symptoms, weight gain, and constipation. Emerging evidence indicates that gut microbiota affects the response to anticancer therapies by modulating the host immune system and gut microbiota involvement in trastuzumab efficacy represents the foundation for new therapeutic strategies aimed at manipulating commensal bacteria to improve response in trastuzumab-resistant breast cancer patients13. Fu et al. found that depletion of intratumor bacteria significantly reduced lung metastasis without affecting primary tumor growth, offering new methods for improved breast cancer management14. Furthermore, modulating microbiota by nutritional treatment with probiotics and prebiotics is as emerging and promising strategies for prevention and treatment of breast cancer in the future15.The human microbiota is the collection of microbes that inhabit various parts of the body, primarily the gut, skin, vagina, mouth, and among others. Each body site has a distinct microbiota and there is significant inter-individual variability in microbiomes, which can contribute to diseases such as metabolic disorders, inflammatory diseases, allergies, and cancer16. The gut microbiome plays a role between stress response, inflammation, and depression, and anxiety through the microbiome-gut-brain axis, which plays a key role in the regulation of brain function and behavior19. Recent study revealed gut bacteria composition may play a role in depression through the production of neurotransmitters, such as serotonin and glutamate20. A meta-analysis of 34 controlled clinical trials found that probiotics showed small but significant benefits for depression and anxiety, while prebiotics did not differ from placebo in their effects on depression or anxiety21. Additional randomized clinical trials with psychiatric samples are necessary fully to evaluate their therapeutic potential. There is little research about the quality of life22, stress, and gut microbiota in newly diagnosed breast cancer patients, so we designed this prospective study hoping to find potential probiotics for decreasing stress and improving quality of life in breast cancer patients.Many of the breast cancer patients experience fatigue, depression, and/or anxiety months to years after their breast cancer diagnosis with these symptoms being associated with greater disability and a poorer quality of life23 , Taipei, Taiwan (19MMHIS061e). The breast cancer patients with stage I-IV who were diagnosed by core biopsy and age greater than 20 years old were included. All patients were treated in MMH and provided written informed consent. Recurrent breast cancer patients or those patients with a history of mental illness were excluded. The patients were recruited as convenience samples. The quality of life was evaluated by FACT-Breast questionnaire23. The FACT-Breast questionnaire consists of a set of questions that measure different aspects of HRQOL such as physical, emotional, and social functioning, as well as overall well-being. The FACT-B consists of two parts, including the FACT-General (FACT-G) with 27 questions and the Breast Cancer Supplement (BCS) with 10 questions. It uses a 5-point scoring system, where 0 represents no at all and 4 represents a lot. The FACT-G includes four sub-scales: Physical Well-Being (PWB) with 7 questions, Social/Family Well-Being (SWB) with 7 questions, Emotional Well-Being (EWB) with 6 questions, and Functional Well-Being (FWB) with 7 questions. The BCS domain includes additional specific items about breast cancer: physical, psychological and aesthetical disorders due to cancer and therapies. The score of the FACT-B is the total of all life quality scores, with a higher score indicating higher satisfaction with life quality. The FACT-Breast questionnaire is widely used in research and clinical practice and has been shown to have good reliability and validity and the FACT-B in the Chinese version were confirmed26.The Functional Assessment of Chronic Illness Therapy (FACT)-Breast is a health-related quality of life (HRQOL) questionnaire specifically designed to assess the impact of breast cancer on an individual's daily functioning25 is a single-item, 11-point visual analogue scale, with respondents indicating how distressed they have felt over the past week (from “No Distress” to “Extreme Distress”). The most recent version of the NCCN practice guidelines for the management of distress recommends that a DT score of 5 or higher indicates moderate-to-severe distress. It is a simple, self-report, pencil-and-paper measure, using a thermometer format line to rate the level of distress and is an accurate, valid screening tool for depression, anxiety27.The Distress Thermometerrpm, 1 min. Wash filter by AW1, AW2 buffer. Elute DNA sample by 100 uL ATE buffer. DNA amount and quality was measured by nanodrop 2000 .Each fecal samples were collected before treatment when breast cancer confirmed by core biopsy and store in − 20 °C refrigerator before use. DNA extraction from fecal samples using QIAamp Fast DNA stool mini kit  followed by user’s manual. Briefly, 0.2 g sample in 1 mL InhibitEX buffer with glass beads, homogenized by precellys homogenizer  4500 beat per min, 2 min. Heat the suspension for 10 min at 70 °C centrifuge sample for 1 min to pellet stool particles. 25 uL proteinase K in a new 2 mL centrifuge tube add 600 uL supernatant from stool pellet. Then add 600 AL buffer and mix-well. Incubate at 70 °C for 10 min. Add 600 uL 100% ethanol and mix-well. Filtrate sample by QIAamp spin column 13,000 Variable regions of 16S rRNA are frequently used in phylogenetic classifications such as genus or species in diverse microbial populations. 2.5 μl (50 ng) of DNA was used to set up the first PCR with 0.2 μM V3 + V4 forward and reverse primers  and 12.5 μl 2X Kapa HiFi HotStart ReadyMix (KapaBiosystems) in 25 μl reactions. The PCR cycling conditions were 3 min at 95 °C, 25 cycles of 30 s at 95 °C, 30 s at 55 °C and 30 s at 72 °C, followed by 5 min at 72 °C. The amplified DNA was purified with Agencourt AMPure XP Reagent beads . The second PCR was set up to add indexes to the amplified DNA by adding 5 μl of purified DNA to 25 μl 2X Kapa HiFi HotStart ReadyMix , 5 μl Nextera XT Index 1 and 2 primers  in 50 μl reactions. The reaction was set at 3 min at 95 °C, 8 cycles of 30 s at 95 °C, 30 s at 55 °C and 30 s at 72 °C, followed by 5 min at 72 °C on an Applied Biosystems 2720 thermocycler , followed by another Agencourt AMPure XP Reagent beads purification .https://www.basespace.illumina.com). The classification was based on the NCBI database (https://www.ncbi.nlm.nih.gov). The output of the workflow was a classification of reads at several taxonomic levels: kingdom, phylum, class, order, family, genus, and species. Then the sequences were analyzed using the QIIME2 software package version 2017.10 (https://qiime2.org/). Potential chimeric sequences were removed using DADA228, followed by trimming 30 and 90 bases of the 3′ region of the forward and the reverse reads, respectively. Taxonomical classification was performed using Naive Bayes classifier trained on the Greengenes13.8 with a 99% threshold of OTU full-length sequences.We used qPCR (KAPA SYBR FAST qPCR Master Mix) to quantify each library using Roche LightCycler 480 system and pooled then equally to 4nM for illumina MiSeq NGS system . More than 80,000 reads with paired-end sequencing (2*300bp) were generated, and the metagenomics workflow classified organisms from the amplicon using a database of 16S rRNA data 30. Alpha-diversity is measured within a single sample using Shannon index with the QIIME software package version 2017.10 (https://qiime2.org/)31. For genera with a median relative abundance exceeding 1%, we conducted multiple regression analysis with adjustment for confounders to examine the association between FCR and the bacterial composition. We excluded from the analysis bacterial taxa that were not detected in 5% or more of the final participants. Furthermore, we calculated the skewness and kurtosis of the bacterial compositions and transformed the distribution of any bacterial compositions that did not assume a normal distribution using Box-Cox transformation. A p-value and T-test are calculated for each genus and species to assess statistical significance. Beta-diversity is measured between different samples using the Bray–Curtis index, and PCoA is used to visualize the results. PERMANOVA is used to test the significance of differences between samples, and F-value, r-squared, and p-value are reported. The taxonomy labels using QIIME. The stacked plot shows the percentage abundance (PA) of different genera and species in each sample, and the Top 20 genera and species are presented in separate graphs to highlight the most abundant taxa. Linear discriminant analysis (LDA) effect size (LEfSe) is a statistical method used to identify features that are differentially abundant between groups of samples. It calculates a p-value and Log LDA score for each feature and reports the results in a graphical format. The original sample pool is divided into Family, Genus, and Species categories, and features with a p-value < 0.05 and Log LDA score > 1.0 are considered significant.We presented bacterial compositions at the Family, Genus and Species levels and calculated the alpha, beta-diversity indices by MicrobiomeAnalyst . As in supplement Table Descriptive statistics for FACT-B different domain scores at diagnosis of breast cancer patients are shown in Table An initial DT was completed by all 82 patients. The mean score was 4.43 (range 0–10), with 53.7% (44/82) of the patients reporting moderate to severe distress (score 5 or above). Table To determine the risk factors of DT score, we compared the DT score (DT score of 5 or higher indicates moderate-to-severe distress) with FACT-B subscale scores, stage, treatment modality and lifestyle variants separately. There is no difference of DT score in FACT-B subscale scores Table , stage, The index of alpha- and beta-diversity of different study groups in genus and species levels are presented in supplement Table Alcaligenaceae (p = 0.017) at the family level and Sutterella (p = 0.017) at the genus level were found to be significantly more abundant in individuals with higher scores on the DT scale  at the family level and Streptococcus (p = 0.023) at the genus level were significantly more abundant in individuals with lower scores on the DT scale  and Ruminococcaceae (p = 0.025) at the family level, and Faecalibacterium (p = 0.014), Coprococcus (p = 0.046) at the genus level, and Obeum (p = 0.001), Prausnitzii (p = 0.014), and Plebeius (p = 0.0018) at the species level were significantly more abundant in breast cancer patients who reported having depression in the DT questionnaire. The relative abundance of Eubacterium (p = 0.019) at the family level and dolichum (p = 0.019) at the genus level were significantly higher in individuals who did not report having depression in the DT questionnaire.The top 5 bacterial taxa that are relatively abundant in different study groups were determined using LEfSe criteria, with a p value < 0.05 and log LDA score > 1. Table Alcaligenaceae (p = 0.022) at the family level and Sutterella (p = 0.022) at the genus level. In the SWB group, the relative abundance of Adlercreutzia (p = 0.005) was significantly higher in individuals with below-average scores. In the EWB group, the relative abundance of Carnobacteriaceae (p = 0.044) at the family level and Granulicatella (p = 0.044) at the genus level were significantly higher in individuals with above-average scores. Conversely, in the EWB below average group, there was a significantly higher relative abundance of Distasonis (p= 0.032) and V2 (p = 0.037) at the species level. The relative abundance of Prevotellaceae (p = 0.045) at the family level and Prevotella (p = 0.045) at the genus level, and Copri (p = 0.045) at the species level were significantly higher in individuals with above-average scores of FWB group patients. Conversely, in the BCS below average group, there was a significantly higher relative abundance of Lachnospiraceae (p = 0.033) and Pasteurellaceae (p = 0.037) at the family level, and Acidaminococcus (p = 0.018) and Haemophilus (p = 0.037) at the genus level, and Parainfluenzae (p = 0.037) and Catus (p = 0.041) at the species level. The FACT-G below average group had a significantly higher relative abundance of Clostridiaceae (p = 0.048) at the family level, and Acidaminococcus  scale had a significantly higher relative abundance of Clostridiaceae. Alcaligenaceae, a bacterial family within the gut microbiome, is implicated in a range of health and disease contexts. It shows associations with conditions such as inflammatory bowel diseases34, chronic kidney disease35, cholelithiasis36, thyroid cancer37, colorectal cancer38, esophageal squamous cell carcinoma39, and breast cancer40, suggesting potential roles in disease development or progression. The families Pseudomonadaceae, Sphingomonadaceae, Alcaligenaceae, Ruminococcaceae, and Clostridia were reported to be decreased in adjacent breast tissue compared with breast cancer tissue41. Additionally, the presence of Alcaligenaceae, a proinflammatory bacterial family, was found to be higher in depressed patients without anxiety compared to those with anxiety symptoms and the showed the proportion of Alcaligenaceae and Sutterella in the anxiety-negative depressed group was significantly higher than in the anxiety-positive group in first-episode depression of Chinese patients42. This suggests that the composition of the gut microbiota, including Alcaligenaceae, may influence the manifestation and severity of depression. However, more research is required to establish causality and understand the underlying mechanisms. Sutterella, belonging to Betaproteobacteria, are Gram-negative, non-spore-forming rods that grow in a microaerophilic atmosphere or under anaerobic conditions. Emerging research highlights the intricate relationship between the Sutterella and various aspects of health and disease, including irritable bowel disease, Crohn’s disease, autism spectrum disorder, Down syndrome and multiple sclerosis43, cancer therapy outcomes44, and sleep duration45. In the context of cancer therapy, particularly CAR-T cell therapy for hematologic malignancies, the presence of Sutterella in the gut microbiota has been associated with treatment responses and survival, emphasizing its potential role as a therapeutic target44. In contrast, studies related to autism spectrum disorder (ASD) have revealed differences in gut microbiota composition, including the presence of Sutterella, in children with ASD, suggesting a link between the microbiome and neurodevelopmental disorders. Conversely, lower abundance of Sutterella was been observed in people with depression46. Sutterella has demonstrated varying abundance levels in different psychiatric conditions, including lower levels in individuals with depression and higher levels in some children with autism47. This suggests its potential role in the intricate microbial-brain-gut axis. Additionally, Sutterella may possess immunomodulatory properties and contribute to Th-17 differentiation43. In a pilot study, lower relative abundance of Sutterella was consistently observed in adults with shorter sleep durations, suggesting a connection between this bacterium and sleep patterns45. Clostridiaceae plays a multifaceted role in health and disease. In menopause, it contributes to alterations in the gut microbiome, potentially affecting cardiometabolic health, with certain members like Clostridium lactatifermentans associated with protective effects against cardiovascular risk factors48. Additionally, Clostridiaceae, particularly Clostridium species, has been implicated in the gut microbiome of children with ASD, emphasizing its link to neurodevelopmental conditions49. The bidirectional relationship between stress, the hypothalamus-pituitary-adrenocortical axis, and the gut microbiome involves Clostridium species, potentially influencing overall well-being and distress50. Furthermore, emerging research suggests that Clostridiaceae bacteria may impact breast cancer outcomes by interacting with the immune system, highlighting their relevance in the context of cancer treatment and quality of life for survivors51.These findings suggest that the microbiome may play an important role in the development of distress and impacts of LOQ of newly diagnosed breast cancer patients.In this study, we observed significant differences in the abundance of certain bacterial families and genera in relation to the role of gut microbiota in distress and LOQ of newly diagnosed breast cancer patients. Specifically, Streptococcaceae was significantly (p = 0.028) more abundant in individuals with lower scores on the DT scale in this study. Accompanied by inflammation, Streptococcus mutans (S. mutans), an oral bacterium, invades endothelial cells (ECs) and disrupts their integrity, thereby promoting tumor cell extravasation and ultimately facilitating metastasis of breast cancer cells to the lungs52. The role of neurotransmitter imbalance, particularly insufficient levels of monoamine neurotransmitters like serotonin, dopamine, and norepinephrine, in contributing to emotional distress and depression. Serotonin, a key neurotransmitter in the brain-gut axis, is mainly synthesized in the gut by certain bacteria53. Various bacteria such as Streptococcus spp., Enterococcus spp., Escherichia spp., Lactobacillus plantarum, Klebsiella pneumoniae, and Morganella morganii were reported to have the ability to produce serotonin54. The high abundance of Streptococcaceae was observed in people with depression and the linkage between gut microbiota pattern and depression may be through the brain-gut microbiome axis46. One animal study revealed that a combination of living Bifidobacterium, Lactobacillus and Streptococcus may be used for treatment of anxiety55. But high abundance of Streptococcaceae was observed in people with depression46. It needs further study to define the role of Streptococcaceae in the distress and QOL in newly diagnosed breast cancer patients and evaluate its potential interventions of biomarker.The 56. The DT was developed as a simple tool to effectively screen for symptoms of distress and offers a user-friendly screening tool57, its limitations in addressing the multifaceted nature of different problems, such as pain, and distress in newly diagnosed breast cancer patients should be recognized, and supplementary assessments or tools may be necessary for a more in-depth understanding of this complex relationship. We would like to clarify that our study primarily aimed to explore the microbiome's potential links with depressive tendencies as measured by the DT scale, rather than to establish a direct causative relationship.The primary limitation of the Distress Thermometer (DT) in this study lies in its potential inadequacy for assessing the complex relationship between psychological distress and different problem list variables, such as pain, in newly diagnosed breast cancer patients. For example, while the study aims to screen for distress in this population using the DT scale, it faces challenges in capturing the nuances of pain experiences and their psychological impact. The DT's single-item nature remains subjective and may not sufficiently differentiate between different sources and origins of pain, making it less suitable for assessing pain-related distress comprehensively. Moreover, the study's diverse breast cancer patient population and the potential variations in distress of newly diagnosed cancer patients highlight the need for a more tailored and multidimensional assessment approach. Therefore, while the DT is recommended routine screening for distress in all cancer patients since1999 by the National Comprehensive Cancer Network (NCCN)In conclusion, this prospective study defines the relationships among QOL, stress and gut microbiota in newly diagnosed breast cancer patients and provides many useful information to find potential interventions of biomarker or probiotics for decreasing stress and for improving quality of life in breast cancer patients.Supplementary Information.


Whereas dilated cardiomyopathy  was less common, hypertrophic cardiomyopathy  and Brugada syndrome  were present more often. During 1-year-follow-up, mortality  and the rate of major adverse cardiac or cerebrovascular events  were lower in patients with syncope and inducible VA. Among patients with inducible VA, post-procedural adverse events including rehospitalization  did not differ between those with vs. without syncope. Taken together, patients with syncope and inducible VA have better clinical outcomes than patients with a secondary preventive defibrillator indication, but comparable outcomes to patients without syncope, which underlines the relevance of VA inducibility, potentially irrespective of a syncope.History of syncope is an independent predictor for sudden cardiac death. Programmed stimulation may be considered for risk stratification, but data remain sparse among different populations. Here, we analyzed the prognostic value of inducible ventricular arrhythmia (VA) regarding clinical outcome in patients with syncope undergoing defibrillator implantation. Among 4196 patients enrolled in the prospective, multi-center German Device Registry, patients with syncope and inducible VA  vs. those with a secondary preventive indication , defined as previously documented sustained ventricular tachycardia or ventricular fibrillation, serving as a control group were studied regarding demographics, device implantation and post-procedural adverse events. Patients with syncope and inducible VA  presented less frequently with congestive heart failure (15.1% vs. 29.1%;  Programmed ventricular stimulation (PVS) is indicated for risk stratification of SCD in post-infarct patients in whom syncope remains unexplained after non-invasive evaluation. It may further be considered for selected populations including patients with dilated or arrhythmogenic right ventricular cardiomyopathy2. In this context, it has been confirmed that patients with coronary artery disease, left ventricular dysfunction and inducible sustained ventricular arrhythmia (VA) during PVS have a higher risk of SCD and a higher overall mortality than those without inducible VA3. For other patient populations data remain controversial.The implantable cardioverter-defibrillator (ICD) is the mainstay of therapy for the primary and secondary prevention of sudden cardiac death (SCD)4. However, patients with syncope and inducible VA during PVS, comprising a small group of all patients undergoing defibrillator implantation, are not well represented in clinical trials and data on the prognostic value of inducible VA regarding clinical outcome during the first time period after ICD implantation in patients with syncope are sparse5. Therefore, the purpose of this study was to assess patient characteristics, data on device implantation and post-procedural adverse events in patients with syncope and inducible VA, derived from a prospective, multi-center database, compared to those with a secondary preventive indication, defined as previously documented occurrence of either sustained ventricular tachycardia or ventricular fibrillation, serving as a well-studied control group with respect to ICD implantation as a gold standard for reducing the rate of mortality and major adverse cardiac or cerebrovascular events (MACCE). Considering the limited indications for PVS, in an additional analysis of all patients with inducible VA, those with vs. without previous syncope were compared for patient characteristics and clinical outcomes after defibrillator implantation.The history of syncope is an independent predictor for SCD as well as appropriate device discharge and may be helpful for identification of patients at increased risk6.The German Device Registry is a prospective, multi-center non-profit database of ICD implantations with or without cardiac resynchronization therapy (CRT) under supervision of the “Stiftung Institut für Herzinfarktforschung” . A total of 50 participating centers enrolled patients between March 2007 and April 2010. The ethics committee of the Rhineland‐Palatinate State Medical Council  approved the study, which was conducted in accordance with the provisions of the Declaration of Helsinki, all relevant guidelines and regulations. Patients gave written informed consent for procedure and registry participation. All data were entered into an internet-based, secure electronic database and transmitted to the IHF using a secure socket layer for encryption. The registry captured baseline data on patients’ demographics, device indication, implantation procedure and complicationsThe present study population includes patients undergoing ICD implantation or replacement divided into patients with a history of at least one syncope and inducible VA, defined as inducible sustained monomorphic ventricular tachycardia or ventricular fibrillation, during PVS vs. patients with a secondary preventive indication, defined as previously documented occurrence of either sustained ventricular tachycardia or ventricular fibrillation, serving as a well-studied control group. PVS was performed in line with the given ESC guidelines as interpreted at the operator’s discretion. In an additional analysis of patients with inducible VA, those with vs. without previous syncope were compared.6.Follow-up contacts were scheduled 1 year after device implantation or replacement by telephone and performed by IHF representatives. The telephone interview included questions on arrhythmias, cardiac events, clinical symptoms, medication, complications, hospitalizations and quality of life. In case of an ineffective call, further information was gathered from other caring physicians or civil registration offices. Data on mortality and ICD shocks were additionally obtained from available records and device interrogationsTo put our presented findings from a large prospective, multi-center database into context, we performed a systematic literature review on prospective studies assessing patients with previous syncope and inducible VA during PVS. The literature search was conducted using the terms “programmed ventricular stimulation AND syncope” OR “inducible ventricular tachycardia AND syncope” OR “ventricular stimulation AND syncope” OR “VT induction AND syncope” in PubMed/MEDLINE. We supplemented our database searches with manual searches of the reference lists of published studies. Potentially eligible studies were assessed independently by 2 investigators (A.K.K. and C.M.). Studies were included if they (1) assessed patients with syncope and inducible VA; and (2) reported demographics and/or data on ICD implantation and/or outcomes (after ICD implantation) in this population. The following exclusion criteria were applied: (1) Case reports and case series; (2) studies published in abstract form only; (3) studies of which the abstract was not available in English; (4) studies investigating arrhythmias other than inducible VA; (5) studies only including patients with documented ion channel diseases. The initial review included all prospective and retrospective studies with or without a control group and was subsequently restricted to prospective studies only.8.In addition, we reviewed classes of recommendations and levels of evidence for ICD implantation in patients with syncope and inducible VA according to international guidelinesU test, the chi-square test or the Fisher’s exact test, respectively. Survival analysis was estimated using the Kaplan–Meier method and compared with the log-rank test. A p value < 0.05 was considered statistically significant. All analyses were performed at the Biometrics Department of the IHF.Continuous variables are presented as mean ± SD or median with interquartile ranges (25th–75th percentile). Categorical variables are expressed as absolute numbers with percentage of patients. Statistical differences between groups were compared using the Mann–Whitney p < 0.001) and any underlying structural heart disease  than patients with a secondary preventive indication. Whereas dilated cardiomyopathy  was less common in patients with syncope and inducible VA, hypertrophic cardiomyopathy  and Brugada syndrome  were present more frequently  with a lower percentage of patients assigned to left ventricular ejection fraction ≤ 30%  and New York Heart Association class III–IV . Intraventricular conduction disorders were found less frequently in patients with previous syncope and inducible VA . Previous cardiopulmonary resuscitation was reported in 55.4% of patients undergoing secondary preventive defibrillator implantation. The rate of previous sudden cardiac death in the family history did not differ between groups . Demographic data of patients undergoing first-time defibrillator implantation are shown in Supplementary Table Out of 4196 patients enrolled in the German Device Registry, 285 (6.8%) had a history of syncope or presyncope and inducible VA during PVS, 1885 patients (45.2%) had a secondary preventive defibrillator indication with documented ventricular tachycardia in 51.8% and ventricular fibrillation in 45.8% of patients. Demographic data at baseline are shown in Table p = 0.005). Of all procedures, 77.5% were first-time implants, with fewer previous implants in the cohort of patients with syncope and inducible VA . Previous implants were mainly ICDs without  or with CRT . Pacemakers were implanted in 4.3% and 4.8% of patients, respectively (p = 0.71).Device implantation was performed more often urgently in patients with syncope and inducible VA than in patients with a secondary preventive indication , and dual-chamber ICDs more often than in patients with a secondary preventive indication . ICDs with CRT were implanted in 10.5% vs. 13.9%, respectively (p = 0.12).During the index implantation procedure, single-chamber ICDs were chosen less frequently in patients with a history of syncope and inducible VA during PVS  as well as complications  during hospital stay did not differ between groups (Table p < 0.001) and digitalis  , 1-year-mortality  and the rate of MACCE  were lower in patients with syncope and inducible VA compared to patients with a secondary preventive indication , aldosterone antagonists  and diuretics  were applied less often in patients with a history of syncope and inducible VA, dronedarone , but not class III antiarrhythmic drugs in general , was applied more often (Table During a median follow-up of 16.4 IQR 12.8–24.3] months vs. 17.5 [IQR 13.3–25.0] months (.8–24.3 mp = 0.017) and prior myocardial infarction  than those without syncope  patients with a history of at least one syncope were compared to 86 (2.1%) without previous syncope, who underwent PVS mainly for diagnostic evaluation after remote myocardial infarction with symptoms such as palpitations suggestive of VA. Patients with syncope presented less often with coronary artery disease . In patients who were included for device revision, the first implantation was conducted 5 years [IQR 4–7] before study enrollment in both groups (p = 0.44). Previous implants in patients with vs. without syncope were mainly ICDs without  or with CRT . Pacemakers were implanted in 4.3% and 3.5% of patients, respectively (p = 0.25).Of all procedures, 76.4% were first-time implants, with a higher percentage in the cohort of patients with syncope , respectively. Single-chamber ICDs were chosen in 55.7% vs. 48.8% (p = 0.51), dual-chamber ICDs in 44.7% vs. 37.2% (p = 0.22). Rates of revision of the implanted or replaced defibrillator  and complications  during hospital stay did not differ between groups.The choice of the firstly implanted or revised device did not differ between groups, with an ICD without and with CRT implanted in 89.5% vs. 86.0% and 10.5% vs. 14.0% of patients (p = 0.004), there were no differences between the cohorts in terms of 1-year-mortality , MACCE , recurrent syncope , ventricular tachycardia storm , ICD shocks , rehospitalization  and improvement in quality of life  . Demographic data and outcomes of patients with inducible VA with vs. without syncope undergoing first-time defibrillator implantation are shown in Supplementary Tables During a median follow-up of 15.0 months IQR 12.8–18.5] vs. 17.7 months [IQR 13.4–22.8]  present less often with most cardiac comorbidities and (2) have lower rates of 1-year-mortality and MACCE than patients with a secondary preventive indication, and 3) have similar rates of post-procedural adverse events compared to patients undergoing defibrillator implantation for inducible VA without previous syncope  and rehospitalization (27.6% vs. 21.7%) were present in a comparable number of patients during follow-up, without any differences in the rate of 1-year-mortality and MACCE between both groups. Low rates of SCD and recurrent syncope are concomitant with a high positive predictive value supporting the use of defibrillators in patients with inducible VA5. Consequently, patients with inducible VA, who are not well represented in trials so far but may be at risk to be underdiagnosed due to fewer cardiac comorbidities, experience a relevant number of post-procedural adverse events not differing between those with vs. without syncope, which supports the indication for defibrillator implantation in this population. Besides one registry investigating the context in which PVS is used in ICD recipients, that also included patients with previous syncope and inducible ventricular tachycardia but did not focus on these patients in detail9, our here presented study is—to the best of our knowledge—the largest prospective one reporting demographics, data on device implantation and post-procedural adverse events during the first time period after ICD implantation in this patient population. As it is further one of only a few multi-center studies and consists of a large group of patients with different underlying structural heart diseases, our study enables insights into a more representative patient sample, increases generalizability of findings and helps to put this relatively rare patient population in relation to a well-studied control group. Importantly, so far, literature and guidelines are heterogeneous, partly controversial8, and data are still somehow limited to mainly single-center and retrospective studies consisting of only relatively small numbers of patients with syncope and inducible VA . However, the here investigated study cohort corresponds to the currently available guidelines still representing a small part of all patients undergoing ICD implantation, which underlines the clinical relevance of the presented data even after development of heart failure medication and ICD programming. In the future with the updated ESC guideline recommendation, patients not being inducible during PVS might serve as a valuable control group but have not been included in the here investigated registry. Nonetheless, the German Device Registry enables insights into a large, prospective real-world cohort undergoing defibrillator implantation for syncope and inducible VA, which has not been described before.The present study has some limitations. In a registry-based cohort, potential confounding factors and a reporting bias have to be considered, as follow-up was only centralized at 1 year post-implantation and performed during clinical routine before. Device implantations and post-procedure care were left to each participating center and are therefore heterogeneous in nature. The follow-up was limited to 1 year after ICD implantation. However, our study aimed to concentrate on post-procedural events during the first time period after ICD implantation, which has only been rarely described, paving the way for subsequent trials including a long-term follow-up. Whether our here presented findings are transferable to a long-term follow-up remains speculative but is supported by the landmark MUSTT dataPatients with syncope and inducible VA undergoing defibrillator implantation, who only represent a small part of all patients undergoing device implantation, present less often with most cardiac comorbidities and have lower rates of 1-year-mortality and MACCE than patients with a secondary preventive indication. Numbers of post-procedural adverse events are comparable in patients with inducible VA with vs. without syncope, which underlines the relevance of VA inducibility, potentially irrespective of the history of syncope.Supplementary Tables.


The acquisition of geriatric-friendly resources is an important part of adapting emergency department (ED) care to the needs of vulnerable older patients. The aim of this study was to explore the availability of geriatric-friendly protocols, equipment and physical environment criteria in EDs and to identify related improvement opportunities.The head nurse of 63 EDs in Flanders and Brussels Capital Region was invited to complete a survey in collaboration with the chief physician of the ED. The questionnaire was inspired by the American College of Emergency Physicians Geriatric ED Accreditation Program and explored the availability, relevance and feasibility of geriatric-friendly protocols, equipment and physical environment. Descriptive analyses were performed. A region-wide improvement opportunity was defined as a resource that was never to occasionally (0–50%) available on Flemish EDs and was scored (rather or very) relevant by at least 75% of respondents.A total of 32 questionnaires were analysed. The response rate was 50.8%. All surveyed resources were available in at least one ED. Eighteen out of 52 resources (34.6%) were available in more than half of EDs. Ten region-wide improvement opportunities were identified. These comprised seven protocols and three physical environment characteristics: 1) a geriatric approach initiated from physical triage, 2) elder abuse, 3) discharge to residential facility, 4) frequent geriatric pathologies, 5) access to geriatric specific follow-up clinics, 6) medication reconciliation, 7) minimising ‘nihil per os’ designation, 8) a large-face, analogue clock in each patient room, 9) raised toilet seats and 10) non-slip floors.Currently available resources supporting optimal ED care for older patients in Flanders are very heterogeneous. Researchers, clinicians and policy makers need to define which geriatric-friendly protocols, equipment and physical environment criteria should become region-wide minimum operational standards. Findings of this study are relevant to facilitate the development process of this endeavour.The online version contains supplementary material available at 10.1186/s12877-023-03994-z. Older adults presenting to the emergency department (ED) are characterized by interacting multi-domain problems and outcomes that are generally poorer compared to those of their younger counterparts –4. As coIn Belgium, the ED population is growing and aging. The total number of admissions to Belgian EDs increased by 23% between 2010 and 2019 , while over the same period of time the relative growth in the number of ED patients over 65 and over 75 years old was 38.5% and 30.7%, respectively. In 2019, the proportion of ED patients over 65 and 75 years old was 20% and 12%, respectively [The aim of this study was to explore the availability of geriatric-friendly protocols, equipment and physical environment criteria in EDs and to identify related improvement opportunities.A cross-sectional survey was performed in Belgian EDs with Dutch as working language. This concerned a total of 63 EDs, which were all located in Flanders (i.e. the northern part of Belgium), except for one. One ED in the bilingual Brussels Capital Region (French and Dutch) was eligible, as well. As the questionnaire was only available in Dutch, EDs with French as working language were not approached for study participation. These non-eligible EDs were situated in the southern part of Belgium  and in the bilingual Brussels Capital Region. This manuscript was reported using the Consensus-Based Checklist for Reporting of Survey Studies (CROSS)  gave feedback on the readability, comprehensibility and completeness of a draft version developed by the junior authors . Subsequently, in the second phase, the board of the Flemish Emergency Nurses Association assessed the readability, comprehensibility and completeness of the questionnaire. The final questionnaire is available as Additional file Data were collected from 9 January 2020 until 13 March 2020 using an online platform, Qualtrics . After iRegion-wide improvement opportunities’ were defined as resources that were never to occasionally (0–50%) available, while being scored (rather or very) relevant by at least 75% of respondents. These region-wide improvement opportunities were classified as ‘difficult to achieve’ and ‘easy to achieve’ if at least half of respondents scored the operational standard ‘not (or rather not) feasible’ and ‘(rather or very) feasible’, respectively.Statistical Package for the Social Sciences (version 25) and Excel 2016 were used to calculate frequencies, modes, medians, means, standard deviations, quartiles and interquartile ranges (IQR), as appropriate. Questionnaires with a missing data rate of 50% or more were excluded from analysis. Availability of standards was classified into five categories: never (0%), seldom (1–25%), occasionally (26–50%), often (51%-75%) and very often (76–100%). ‘n = 32) had a male/female ratio of 69:31, a median age of 49 years old (IQR = 44–56) and a median seniority in the current job of 12 years (IQR = 5–15). Chief ED physicians (n = 32) had a male/female ratio of 56:44, a median age of 48 years old (IQR = 42–55) and a median seniority in the current job of 7 years (IQR = 7–13).Qualtrics registered 61 questionnaires. Of these, four overlapped with other registrations. For example, two head nurses of one ED started completing the questionnaire separately, and in three other cases, the head nurse interrupted completion of the questionnaire and began all over in a new questionnaire. These incomplete and initial registrations were excluded. Therefore, the number of different responses received was 57. As one respondent withdrew consent for study participation after questionnaire completion and as 24 questionnaires had a missing data rate of 50% or more, the final data analyses were based on 32 questionnaires, which implies a response rate of 50.8% (≈32/63). The final data analyses represented 32 EDs, located in three university and 28 non-university hospitals. Head nurses  reported having between 1 to 9 protocols. Nine EDs (i.e. 28.1%) had between 10 to 19 protocols. Table Out of 27 surveyed geriatric-friendly protocols, one ED 3.1%) reported to have none, while another (3.1%) had 22. The majority of respondents . Often available protocols focused on physical restraint use , fall risk assessment , access to patient transport services , pain management , and urinary catheter use . Five easy to achieve region-wide improvement opportunities were also identified. These included i) a protocol with criteria for access to a geriatric approach starting from physical triage, ii) an elder abuse protocol, iii) a protocol to facilitate discharge to a residential facility, iv) protocols for work-up and initial treatment of frequent geriatric pathologies and v) a protocol for access to geriatric-specific follow-up clinics. Two protocols were identified as difficult to achieve region-wide improvement opportunities. These focused on medication reconciliation and minimisation of ‘nihil per os’ status including better access to appropriate drinks and food.One protocol was very often available in Flemish EDs. This comprised a protocol to inform a patient’s general practitioner after ED discharge  EDs reported to have 1 to 4, while 17 (53.1%) had 5 to 9 geriatric-friendly equipment. One (3.1%) ED had 11 elements. Table n = 27/32; 84.4%), ii) blanket warmer , iii) bedside commodes  and iv) low beds or high-low beds . The only equipment element that was often available were reclining arm chairs . Each type of walking aid  was available in only one (3.1%) or two (6.3%) EDs. One ED had both a cane and walker available. Non-slip socks were available in 12 (37.5%) EDs. No region-wide improvement opportunity was identified.Four equipment elements were very often available in Flemish EDs: i) pressure-ulcer reducing mattresses and pillows  reported to have 1 to 4, while the other half had 5 to 9 geriatric-friendly physical environment criteria. Table Out of 11 surveyed physical environment criteria, half of EDs  and ii) wheel-chair accessible toilets . Five elements were often available: i) easy access to food and drink , ii) adequate hand rails in sanitary facilities , iii) high quality signage and way-finding , iv) enhanced lightning  and v) efforts at noise reduction . Two easy to achieve region-wide improvement opportunities were identified. These included having a large-face and analogue clock in each patient room and availability of raised toilet seats. Availability of non-slip floors was the only identified difficult to achieve region-wide improvement opportunity.Two physical environment criteria were very often available on Flemish EDs: i) seating for visitors (i.e. at least two seats per room)  , 14. In n= 22/32; 68.8%). Knowing this, it is remarkable that all surveyed walking aids were only available in one or two EDs. Even more, the majority of respondents scored walking aids as not or rather not relevant, while their purpose should be preventing falls during ED stay and avoiding unnecessary admissions among patients with balance difficulties . As availability of mobility aids is for these reasons one of the few minimum requirements to obtain a geriatric ED accreditation label, it is clear that purchasing walking aids and organizing trainings on how to use them is an important improvement opportunity for Flemish EDs [To guarantee a reflection of high quality geriatric emergency care, operational standards should be defined with an interdisciplinary panel . This might avoid obtaining operational standards with care-related inconsistencies, which are also present in the results of this study. For example, in the current survey, almost 70% of the respondents reported having a standardized fall assessment guideline with follow-up possibilities  EDs at the moment of the survey . This might have introduced selection bias .Additional file 2. Questionnaire.Additional file 3. Availability, relevance and feasibility of few available protocols that were not identified as ‘region-wide improvement opportunities’.Additional file 4. Differences between excluded and included emergency departments.


Although the incidence of late-onset colorectal cancer (LOCRC) has decreased, the incidence of early-onset colorectal cancer (EOCRC) is still rising dramatically. Heterogeneity in the genomic, biological, and clinicopathological characteristics between EOCRC and LOCRC has been revealed. Therefore, the previous prognostic models based on the total CRC patient population might not be suitable for EOCRC patients. Here, we constructed a prognostic classifier to enhance the precision of individualized treatment and management of EOCRC patients.EOCRC expression data were downloaded from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases. The regulatory pathways were explored by gene set enrichment analysis (GSEA). The prognostic model was developed by univariate Cox-LASSO-multivariate Cox regression analyses of GEO samples. TCGA samples were used to verify the model. The expression and mutation profiles and immune landscape of the high-risk and low-risk cohorts were analyzed and compared. Finally, the expression and prognostic value of the model genes were verified by immunohistochemistry and qRT‒PCR analysis.MCM2, INHBA, CGREF1, and KLF9 was constructed. The risk score was an independent predictor of overall survival. The area under the curve values of the classifier for 1-, 3-, and 5-year survival were 0.856, 0.893, and 0.826, respectively, in the training set and 0.749, 0.858, and 0.865, respectively, in the validation set. Impaired DNA damage repair capability (p < 0.05) and frequent PIK3CA mutations (p < 0.05) were found in the high-risk cohort. CD8 T cells (p < 0.05), activated memory CD4 T cells (p < 0.01), and activated dendritic cells (p < 0.05) were clustered in the low-risk group. Finally, we verified the expression of MCM2, INHBA, CGREF1, and KLF9. Their prognostic value was closely related to age.The cell cycle was identified as the most significantly enriched oncological signature of EOCRC. Then, a 4-gene prognostic signature comprising In this study, a robust prognostic classifier for EOCRC was established and validated. The findings may provide a reference for individualized treatment and medical decision-making for patients with EOCRC.The online version contains supplementary material available at 10.1186/s12885-023-11075-y. P < 0.0001), reflecting whole genome hypomethylation and chromosomal instability [Colorectal cancer (CRC) is a very common malignant tumor worldwide . The ovetability , 12. MSItability . Oxidatitability . Yanlei tability  identifitability  constructability , 4, 17.The cell cycle refers to the entire process that a continuously dividing cell undergoes from the end of one mitosis process to the end of the next. Maintaining the integrity of the genome is crucial for chromosome separation and cell proliferation . The celThe prognosis of EOCRC is still controversial. Patients with EOCRC often receive more radical treatment, but unfortunately, not all patients experience a benefit –30. ThusThus, we processed and analyzed the transcriptome data of early-onset colorectal cancer from the GEO and TCGA databases and combined these data with clinical and pathological factors to build a precise classifier to predict the overall survival (OS) of EOCRC patients. This was followed by experimental verification. In conclusion, our study may provide novel insights regarding clinical prognosis assessment and potential individualized treatment for patients with EOCRC.CRC patients in the Department of Gastrointestinal Surgery, Guangdong Provincial People's Hospital from January 2017 to March 2018 were enrolled to analyze the clinical features of EOCRC. Patients with solitary primary colorectal carcinoma without a history of cancer who did not receive any preoperative neoadjuvant therapy followed by radical resection and who had complete clinical and survival data were our target population. Information on sex, age, TNM stage, and tumor location was obtained from the patient's medical records. This study complied with the Helsinki Declaration and was approved by the author's organization. All experiments followed relevant regulations. All enrolled CRC patients provided written informed consent, and the collection of the clinical sample was approved by the Institutional Review Board of Guangdong Provincial People's Hospital under grant number GDREC2019504H (R2).https://www.ncbi.nlm.nih.gov/geo/). Moreover, the RNA-Seq and corresponding clinical information were downloaded from the TCGA website .The gene microarray and clinical data of EOCRC were downloaded from the Gene Expression Omnibus (GEO) (GEO Series (GSE) 41,258 (31 EOCRC samples versus 10 control samples), GSE87211 (19 EOCRC samples versus 14 control samples), and TCGA (69 EOCRC samples versus 51 control samples) data were used to find the differentially expressed genes between EOCRC and normal cohorts. Patients had to meet the following criteria: (1) both the age of patients whose tumor and nontumor samples were used should be < 50 years for the GEO; and (2) tumor samples should be from patients < 50 years at diagnosis and all normal samples should be from patients < 50 years for the TCGA.Moreover, data for 202 EOCRC patients from GSE41258 (31 patients), GSE39582 (66 patients), GSE17536 (20 patients), GSE17537 (9 patients), GSE12945 (7 patients), and TCGA (69 patients) datasets, were obtained for survival research; the included patients met the following criteria: (1) tumor patients who were diagnosed when they were < 50 years of age; (2) patients with available survival information; and (3) patients with survival time > 30 days. The clinical information of samples from the GEO and TCGA datasets is presented in Supplementary Table In addition, mutation data for 63 patients were obtained from the TCGA database.p value < 0.05, discovery rate (FDR) q-value < 0.25, and normalized enrichment score (NES) > 1 were regarded as significantly enriched.GSEA was applied to investigate the clusters of significant differentially expressed genes between EOCRC and normal tissues in GSE41258. Pathways with a false nominal http://software.broadinstitute.org/gsea/msigdb). Then, a novel complete cell cycle gene set consisting of 2579 unique genes was generated after assembling and removing duplications.All cell cycle-related genes were screened from the molecular signatures database (MSigDB)  between EOCRC and normal samples in the GSE41258, GSE87211, and TCGA datasets were identified using the “DESeq2” package in R . |Log2FCSince the data are derived from different cohorts, the RNA expression data of GSE41258, GSE39582, GSE17536, GSE17537, GSE12945, and TCGA were log10-transformed and normalized using the “limma” R package  and werep < 0.05). Then, to prevent model overfitting, the least absolute shrinkage and selection operator (LASSO) algorithm [GEO samples were used as the training group on account of their larger sample size. First, survival-related cell cycle genes were found by univariate Cox analysis (lgorithm  was emplp < 0.05). Next, a nomogram was constructed. Finally, the calibration curve was utilized to assess the nomogram. As an external validation group, the TCGA samples were assigned to the high-risk group and low-risk group on the basis of the same median risk score of the training group. The Kaplan–Meier curve, ROC curve, and calibration curve were also plotted to validate the model.According to the median risk score calculated by the signature, samples were divided into high-risk and low-risk groups. We compared the prognosis between the high- and low-risk cohorts with the packages "survival" and "survminer", and a significant p value was obtained. Kaplan–Meier survival curves and receiver operating characteristic (ROC) curves for OS were plotted to test the applicability of the classifier. The independent clinicopathological factors affecting OS were found by multivariate Cox analysis   could idThe "maftools" package  was applhttp://gepia.cancer-pku.cn) was used to validate the expression levels of the model genes between colorectal carcinoma and normal tissues. GEPIA data come from the TCGA and GTEx databases, including 275 colon cancer samples and 349 normal colon samples, 92 rectal cancer samples and 318 normal rectal samples.GEPIA  qualified tissue specimen quality; (2) complete follow-up data; (3) sporadic CRC. The exclusion criteria were as follows: (1) missing or poor-quality tissue specimens (2) incomplete follow-up data. The Institutional Review Committee of Guangdong Provincial People's Hospital approved the collection of tissue samples and clinical data. All included patients signed informed consent documents.The cancerous tissue was dewaxed in xylene solution for 15 min. Then, a series of graded ethanol solutions  were applied for 5 min each. Then, antigen repair was carried out at a high temperature and high pressure for 5 min. Peroxidase inhibitor was applied at 37 °C for 20 min. The primary antibody was incubated at 4 °C overnight. The secondary antibody was added and incubated at room temperature for 1 h. DAB chromogenic solution was developed under an electron microscope, hematoxylin was stained for 5 min, and ethanol hydrochloride was differentiated for 1 min. Resin and cover glass were used to seal the film and photographed under an electron microscope. The staining scores were as follows:—for negative, + for weakly positive, +  + for moderately positive, and +  + for strongly positive. Staining scores as—or + was considered to represent low expression, and staining scored as +  + or +  +  + was considered to represent high expression. Antibodies and dilutions were as follows: INH3A , CGREF1 , KLF9 , and MCM2 .Six EOCRC and six LOCRC samples were randomly selected from samples obtained from CRC patients who underwent radical surgery at Guangdong Provincial People's Hospital from January 2017 to March 2018. All enrolled patients underwent radical surgery, and the postoperative pathology was adenocarcinoma. The Institutional Review Committee of Guangdong Provincial People's Hospital approved the collection of tissue samples and clinical data. All included patients signed informed consent documents.Total RNA was extracted using the RaPure Total RNA Kit , and cDNA synthesis was performed using the HiFiScript cDNA Synthesis Kit , after which Hieff® qPCR SYBR Green Master Mix (No Rox) was used for qPCR (Yeasen Biotechnology (Shanghai) Co., Ltd.). The relative identified mRNA expression levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and calculated with the 2–ΔΔCT method. The BLAST tool was applied to verify the specificity of the designed primers, the sequences of which are shown in Supplementary Table https://www.r-project.org/) and GraphPad Prism version 8.4.3 . Categorical variables were analyzed with the chi-square or Wilcoxon rank-sum test. Continuous variables were analyzed with Student's t test. Kaplan‒Meier analysis estimated the cumulative OS. The log-rank test was used to compare the survival curves. OS referred to the time from tumor resection to patient death. Factors associated with OS were calculated by the Cox proportional hazards model and indicated by hazard ratio (HR) and 95% confidence interval (CI). The time-dependent receiver operating characteristic curve was constructed and the area under the curve (AUC) was determined by the "timeROC" R package. The calibration curve was plotted by the "rms" package. Mutations in the high- and low-risk groups were assessed by the Fisher test in the "maftools" R package. The p values of for DEG analysis, GSEA, and GSVA were adjusted based on multiple testing corrections. Unless otherwise stipulated, p < 0.05 was considered to indicate statistical significance.Statistical analyses were conducted using R version 4.0.2 (p < 0.05).In all, 273 CRC patients (EOCRC = 42 samples and LOCRC = 231 samples) who accepted curative surgery from January 2017 to March 2018 at our hospital were included in the analysis of the clinical characteristics   were found. Following stepwise multivariate Cox proportional hazards regression analysis, a 4-cell cycle gene prognostic signature was established.We removed the batch effect of the GSE41258, GSE39582, GSE17536, GSE17537, GSE12945, and TCGA datasets  was used as the external validation set and had the same risk formula and cutoff point as the GEO cohort. The high-risk group had markedly poorer outcomes  and tumor stage  were confirmed as independent predictors by univariate and multivariate Cox regression analyses. Then, a nomogram based on the risk score and tumor stage was constructed and assessed , while patients in the TCGA cohort showed no significant difference in TNM stage (p = 0.298).The clinicopathological features between the identified high-risk and low-risk cohorts were compared, and patients in the high-risk group in the GEO cohort had advanced TNM stage , TTN (50%), and KRAS (43%), while those in the low-risk group were APC (83%), TP53 (69%) and TTN (43%). PIK3CA mutations were more frequent in the high-risk group than in the low-risk group .As shown in the mutation waterfall plot Figs. B, C, thep < 0.05), activated memory CD4 T cells (p < 0.01) and activated dendritic cells (p < 0.05) were significantly enriched in the low-risk cohort, while eosinophils (p < 0.05) were enriched in the high-risk group.CIBERSORT revealed the immune landscape of the two groups. As shown in Fig. KLF9  and CGREF1  had significantly better OS than those who had lower expression. However, the expression of KLF9  and INHBA  was not related to OS. Interestingly, as shown in Table p = 0.003), although these differences were not present in the LOCRC cohort . Similarly, higher expression of INHBA also indicated poor OS in young CRC patients  but not in LOCRC patients   and in LOCRC   and in LOCRC   to explore the clinical significance of the 4 genes. In our study, the expression of KLF9, MCM2, INHBA, and CGREF1 was significantly correlated with TNM clinical stage and tumor differentiation, excluding tumor location, age, and sex (Table p = 0.026) was expressed to a lesser extent in EOCRC, while MCM2 (p = 0.004) and INHBA (p < 0.001) were more highly expressed in EOCRC than in LOCRC. CGREF1 (p = 0.002) expression was not significantly different  to localize to the nucleus in eukaryotic cells is necessary for helicase activity in DNA replication . High exferation –54. Prevferation . MCM2 maferation , 56. A pKrüppel-like factor 9 (KLF9) is a member of the SP/KLF family of DNA-binding transcriptional regulators , which cInhibin βA (INHBA) is a member of the transforming growth factor-β superfamily. This protein has been found to be overexpressed and to promote cell proliferation, invasion, and metastasis in many cancer types –68. Highp53 and inhibits cell proliferation [CGREF1 has not yet been fully explored, and further research is warranted.Cell growth regulator with EF-hand domain 1 (CGREF1) is regulated by feration , 71. MecThese four genes are all closely related to tumor prognosis, as they regulate tumorigenesis and tumor development through the cell cycle pathway. In our study, the expression of these genes was determined to be related to tumor pathological stage and differentiation and was an independent prognostic factor in early-onset colorectal cancer patients. Notably, we found that KLF9 and INHBA were associated with prognosis in EOCRC but not in LOCRC, while the expression differences in KLF9, MCM2, and INHBA between the tumor and normal groups were more obvious in EOCRC than in LOCRC. The prognostic value of MCM2, KLF9, INHBA, and CGREF1 was closely related to age. Therefore, we inferred that these genes may play more important roles in the prognosis of young CRC patients.PIK3CA mutations, which were present in 15% of metastatic CRC cases [PIK3CA mutations than their older counterparts [PIK3CA mutations was common in EOCRC patients. PIK3CA mutations are usually considered to be closely related to advanced tumor stage and poor survival [We explored the reasons for the difference in prognosis between the high-risk and low-risk groups identified by our classifier. The results of the GSVA demonstrated that some DNA damage repair pathways that benefit from tumor inhibition, such as mismatch repair, base excision, and homologous recombination, were downregulated in the high-risk group. This might lead to rapid deterioration of the quickly proliferating EOCRC. Moreover, we speculated that defects in DNA damage repair may have a greater impact on the prognostic risk in EOCRC, which exhibits rapid growth with a high rate of cell proliferation. Another important finding of our research was that RC cases , 73, werRC cases , 75. EOCterparts . Moreovesurvival . Therefosurvival , 79. Densurvival . Young psurvival . The lowsurvival , EOCRC psurvival . It coulAlthough the cell cycle-based signature was shown to be an effective independent prognostic factor, some limitations should still be acknowledged. Due to the limited data on early-onset colorectal cancer, our study included only 202 samples. Therefore, our results should be verified by multicenter prospective studies with larger sample sizes. In addition, further studies in vitro and in vivo, such as those involving patient-derived organoids and patient-derived tumor xenografts, are warranted to verify our 4-gene classifier in the near future.MCM2, INHBA, CGREF1, and KLF9 play critical roles in EOCRC progression. This signature may be used as an important supplement to achieve individualized tumor treatment by optimizing prognosis evaluation.Due to the poor prognosis of EOCRC patients, effective predictive indicators are urgently needed. However, the heterogeneity of EOCRC limits the applicability of existing models. Thus, a novel prognostic classifier based on cell cycle profiles in EOCRC was developed and validated in our study. We also found that Additional file 1:Supplementary Figure 1. (A)HALLMARK and GO analysis of GSEA indentified cell cycle-associated genesets as the most enriched oncological signature of EOCRC cohort in GSE 41258 comparedwith the normal  cohort.  Before(B) and after(C) batchprocessing of GEO sets. (D) GSVAanalysis indicating downregulation of 9 pathway in high-risk group compare withlow-risk group based on GO set. (E)Normal and colorectal cancer samples could be distinguish definitely by PCAanalysis accroding to the 4 cell cycle- related hub genes. Abbreviations: NES, normalized enrichment score; FDR, false discovery rate; PCA, principal component analysis; READ, rectal cancer;COAD, colon cancer; GSEA, Gene set variation analysisand gene set enrichment analysis; GSVA, Gene Set VariationAnalysis; EOCRC, early-onset colorectal cancer. Supplementary Table 1. Baselinecharacteristics of early-onset colorectal cancer. patients in the GEO and TCGAcohorts. Abbreviations: GEO, GeneExpression Omnibus database; TCGA, The Cancer Genome Atlas  database. Supplementary Table 2. 98 commondifferentially expressed cell-cycle genes of early-onset colorectal cancer. Supplementary Table 3. Unicox genes of early-onset colorectal cancer. Supplementary Table 4. The sequences  of  qPCR primers.


The objectives of this study were to assess the changes in the left myocardial function after chemotherapy for childhood lymphoma and observe the predictive or monitor value for cancer treatment-related cardiac dysfunction (CTRCD) by speckle-tracking echocardiography.A total of 23 children with histopathological diagnoses of lymphoma were included, with age-matched normal controls. Comparative analysis of clinical serological tests and left heart strain parameters in children with lymphoma, including left ventricular global longitudinal strain (LVGLS); global myocardial work (GMW) indices, which include global work index (GWI), global constructive work (GCW), global wasted work, and global work efficiency; and the LS of subendocardial, middle, and subepicardial layer myocardium during left ventricular systole were measured: left atrial strain of reservoir phase (LASr), left atrial strain of conduit phase (LAScd), and left atrial strain of contraction phase (LASct).p < 0.05), and the degree of decrease also showed a regular pattern of epicardial layer < middle layer < subendocardial layer while the difference was not significant (p > 0.05). After chemotherapy, maximum flow rate in early mitral relaxation/left atrial systolic maximum flow rate (E/A) and left atrial volume index of each group were in the normal range, and the values of LASr, LAScd, and LASct slightly increased in the second cycle and decreased significantly in the fourth cycle after chemotherapy, reaching the lowest level; LASr and LAScd were positively correlated with GLS.One-way ANOVA showed that GLS, GWI, GCW, LASr, and LAScd were closely associated with CTRCD and multivariate logistic regression analysis showed that GLS was the most sensitive predictor for detecting patients at lofty risk of anthracycline-related cardiotoxicity. Both before and after chemotherapy, GLS in the left ventricle showed a pattern of basal segment < middle segment < apical segment and subepicardial < middle < subendocardial layer (LVGLS is a more sensitive and earlier indicator to predict CTRCD compared with conventional echocardiography-related parameters and serological markers, and GLS of each myocardial layer showed a certain regularity. Left atrial strain can be used for early monitoring of cardiotoxicity in children with lymphoma after chemotherapy. Anthracyclines (ANTs) are first-line drugs for lymphoma chemotherapy, and antitumor mechanisms primarily include the inhibition of DNA and RNA synthesis in cells and inhibition of topoisomerase II activity, which are toxic to cardiomyocytes while inhibiting tumor cell growth and being progressive, irreversible, and dose-dependent . ChemothWe quantitatively evaluated the functional changes of LV and LA after ANT chemotherapy in children with lymphoma and explored the clinical application value of conventional echocardiography, serological makers, and ultrasound strain parameters to early detect the chemotherapy-induced cardiac dysfunction and to provide a new rationale for the clinical monitoring of the cardiotoxicity of ANTs.From January 2019 to March 2020, 28 children with lymphoma-confirmed histopathological diagnosis were first selected at Children's Hospital of Chongqing Medical University, who completed at least two courses of chemotherapy with normal LVEF (>53%) and sinus rhythm. The exclusion criteria included congenital heart disease, myocarditis, severe arrhythmia, cardiac function impairment due to anemia and nephrotic syndrome, and poor image quality due to excessive obesity and severe pneumonia. Therefore, we excluded 5 patients , and a total of 23 subjects were included in the final analysis; the flowchart of this cohort shown in The age, height, weight, blood pressure, heart rate, and chemotherapy regimen of every child were recorded, and the child's body surface area and the cumulative amount of Adriamycin after every two courses of treatment were measured. Venous blood samples were taken from each kid before chemotherapy and within 24 h after every two chemotherapies to measure the serum levels of creatine kinase isoenzymes MB (CK-MB) and troponin I ultra (TnI-Ultra) before and after chemotherapy.The same physician performed echocardiograms before chemotherapy and within 24 h after every two courses of chemotherapy using a GE system  and M5S transducer (3.5 MHz) as per current guidelines , 15. PosThe images were obtained in DICOM format, and the left atrial myocardial motion was depicted in the apical four-chamber view and apical two-chamber view with the matching software EchoPAC 204 to calculate the left atrial end-diastolic dimension (LAEDd), left atrial end-diastolic volume (LAEDV), left atrial end-systolic volume (LAESV), and the biplane left atrial ejection fraction (LAEF) and left atrial volume index (LAVI); in the mode of Auto-EF, the apical two-chamber and four-chamber end-systolic left ventricular intimal surfaces were depicted, and LVEF was calculated by the biplane Simpson method; the crest mitral valve orifice blood flow rate was measured by spectral Doppler in the apical four-chamber view to calculate E/A.In auto function imaging (AFI), the corresponding standard view of three-chamber, four-chamber, and two-chamber heart  was selected. The software can automatically track the LV membrane surface to form a range of interest through the electrocardiogram; manually depict or finetune if necessary; calculate the GLS of LV in APLAX, 4CH, and 2CH; and the software automatically calculates the average value GLPSAvg (LVGLS) . At the The left atrial global strain was automatically tracked at a finishing time of diastole , left atrial strain of reservoir phase (LASr), left atrial strain of conduit phase (LAScd), left atrial strain of contraction phase (LASct) at 4CH and 2CH were calculated and record it respectively, and their mean values BiSr, BiScd, and BiSct, recorded as LASr, LAScd, LASct . All part test, Single factor variance analysis, and repeated measures analysis of variance (ANOVA); non-normally distributed data are expressed as the median ± interquartile range and between groups were analyzed with the Wilcoxon signed rank test. Correlation analysis was performed using Pearson correlation analysis, and diagnostic efficacy was assessed using binary logistic regression analysis and receiver operating characteristic (ROC) curve. p < 0.05 was considered statistically significant.Statistical analysis was performed using SPSS 25.0 software. LVGLS, LAScd, and Sct were negative values, and absolute values were taken for a description in this paper; normally distributed data and approximately normally distributed data are expressed as the mean ± standard deviation (SD); differences between groups were analyzed with independent-sample n = 23) consisted of 20 males and 3 females with a mean age of 9.34 ± 3.59 years (range 5–15 years). They all completed at least two chemotherapy courses (cumulative ANT dose 50 mg/m2): 19 patients completed four chemotherapy courses (cumulative ANT dose 100 mg/m2), 12 patients completed six chemotherapy courses (cumulative ANT dose 150 mg/m2), and 8 patients completed eight chemotherapy courses (cumulative ANT dose 230 mg/m2). Twenty-three children developed varying degrees of nausea, vomiting, and myelosuppression after chemotherapy only within different chemotherapy cycles, which resolved spontaneously after the end of chemotherapy without other discomforts such as palpitations or dizziness and had no significant abnormalities in myocardial markers or electrocardiograms. In addition, only 1 of the 23 patients died of severe infection within 1 year after the end of chemotherapy. The control group consisted of 23 patients, 20 males and 3 females, with an average age of 9.42 ± 3.15 years . There were no obvious differences in age, weight, height, and gender distribution between the control and study groups (p > 0.05). The diastolic blood pressure (DBP) was significantly lower than healthy controls (p < 0.05). There was no statistical significance in body surface area (SA), heart rate (HR), and systolic blood pressure (SBP) between 23 children with lymphoma before chemotherapy and 2, 4, and 6–8 times after chemotherapy and the control group (p > 0.05) (The study population ( > 0.05) . During  > 0.05) .p > 0.05). After chemotherapy, the data of LVEF determined by the Simpson method decreased in the normal range compared with that before chemotherapy (p > 0.05); LAVI before chemotherapy in children with lymphoma were lower when compared with the normal group   .p < 0.05), while GCW, LASr, LAScd, and LASct were not significantly different between the two groups (p > 0.05). Compared with that before chemotherapy, the mean values of GLS in children with lymphoma after chemotherapy at cycles 2, 4, 6, and 8 showed a decreasing trend at cycles 2 and 4 . However, they fluctuated at cycles 6 and 8 (p > 0.05) (p > 0.05).LVGLS, GWI, GWW, and GWE were significantly lower than healthy controls ( > 0.05) . There wp < 0.05) whether before chemotherapy or after chemotherapy; and the degree of decreased also showed revealed a regular pattern of epicardial layer < middle layer < subendocardial layer while the difference was not significant (p > 0.05)  .p > 0.05). Correlation analysis showed that LASr was weak positively correlated with LAVI, LVEF, GLS, and LASct ; LAScd was weak positively correlated with E/A, LAEF, and LAVI , moderate positively correlated with LVEF and GLS , and strong positively correlation with LASr ; LASct was weak positively correlated with LAVI , moderate positively correlated with LASr, and there was no significant association with E/A, LAEF, LVEF, GLS, and LAScd (r = −0.138 to 0.229)  had LASr lower than 35%. LASr, LAScd, and LASct were slightly increased after the second cycle of chemotherapy but decreased after the fourth cycle of chemotherapy, and the overall mean value was below that of the healthy control group, and slightly increased again in the sixth and eighth cycles after chemotherapy. The overall mean value exceeded that before chemotherapy but was lower than that in the second cycle of chemotherapy ; at the o 0.229) .B = −0.965, Exp (B) = 0.381, p < 0.05]. When the absolute value of LVGLS was < 17.67, the sensitivity for predicting CTRCD was 0.765, the specificity was 0.917, and the area under the curve (AUC) = 0.857 , but the minimum age of this study was 50 years, which was different from our study subjects in age. Unfortunately, we lacked the criteria for the diagnosis of LAVI abnormalities in children, so the predictive value of LAVI in children may be underestimated.The left atrial changes are asymmetric and difficult to measure quantitatively, and the left atrial size is influenced by many factors, which leads to unreliable results such as LAESV. We discovered that LAVI before chemotherapy in lymphoma children was clearly lower than those in the control group, LAVI predicts diastolic dysfunction by reflecting the magnitude of LV filling pressure, and monitors LV diastolic function more accurately than E/A and LAD . Followi2 in breast cancer patients, and serum Hs-cTnT concentrations after anthracycline chemotherapy showed a gradually increasing trend over four chemotherapy stages, while only one of our children had anthracycline concentrations reaching 350 mg/m2, mostly less than 100 mg/m2. This could also explain why serum indicators in our investigation did not indicate any changes  and the European Association of Cardiovascular Imaging (EACVI) recommended LVEF criteria were not met, but a GLS decrease of >15% from baseline can be considered subclinical CTRCD , 46, 47.LVGLS is a more sensitive and earlier indicator to predict CTRCD compared with conventional echocardiography-related parameters and serological markers. GLS of each myocardial layer showed a certain regularity. Left atrial strain can be used for early monitoring of cardiotoxicity in children with lymphoma after chemotherapy.


Sarcopenia is highly prevalent in elderly individuals and has a significant adverse effect on their physical health and quality of life, but the mechanisms remain unclear. Studies have indicated that transcription factors (TFs) and the immune microenvironment play a vital role in skeletal muscle atrophy.RNA-seq data of 40 muscle samples were downloaded from the GEO database. Then, differentially expressed genes (DEGs), TFs(DETFs), pathways(DEPs), and the expression of immune gene sets were identified with limma, edgeR, GO, KEGG, ORA, GSVA, and ssGSEA. Furthermore, the results above were integrated into coexpression analysis by Pearson correlation analysis (PCA). Significant coexpression patterns were used to construct the immune-related transcriptional regulatory network by Cytoscape and potential medicine targeting the network was screened by Connectivity Map. Finally, the regulatory mechanisms and RNA expression of DEGs and DETFs were identified by multiple online databases and RT‒qPCR.p < 0.05), 4 DETFs , 304 DEPs (enrichment scores (ES) > 1 or <  − 1, p < 0.05), and 1208 differentially expressed immune genes sets (DEIGSs) (p < 0.01). Based on the results of PCA (correlation coefficient (CC) > 0.4 or <  − 0.4, p < 0.01), we then structured an immune-related network with 4 DETFs, 9 final DEGs, 11 final DEPs, and 6 final DEIGSs. Combining the results of online databases and in vitro experiments, we found that PAX5-SERPINA5-PI3K/Akt  was a potential transcriptional regulation axis, and B cells  may play a vital role in this signal transduction. Finally, the compound of trichostatin A  might be a potential medicine for sarcopenia based on the PubChem database and the result of the literature review.We screened 808 DEGs (log2 fold change (FC) > 1 or <  − 1, We first identified immune-related transcriptional regulatory network with high-throughput RNA-seq data in sarcopenia. We hypothesized that PAX5-SERPIAN5-PI3K/Akt axis is a potential mechanism in sarcopenia and that B cells may play a vital role in this signal transduction. In addition, trichostatin A might be a potential medicine for sarcopenia.The online version contains supplementary material available at 10.1186/s12877-023-04152-1. Sarcopenia contributes to the accelerated loss of muscle mass and function with ageing and associated with increased risk of falls, postoperative complications, frailty, and mortality , 2. To dTranscription factors (TFs) can specifically interact with the cis-acting elements of eukaryotic genes and control the rate of genetic information from DNA to mRNA . A NumbeHere, we constructed an inclusive immune related transcriptional regulatory network in sarcopenia, which included the differentially expressed TFs, downstream genes, pathways, and immune-related events and cells. In this study, we first obtained the RNA-seq profiles from the Gene Expression Omnibus (GEO) database and obtained the differentially expressed genes (DEGs) by the limma package. Then, the differentially expressed TFs (DETFs) were identified by the edgeR package. We next explored the differentially expressed pathways (DEPs) with overrepresentation analysis (ORA) and Gene Set Variation Analysis (GSVA). Single-sample gene set enrichment analysis (ssGSEA) was also performed to identify the differentially expressed immune gene sets (DEIGSs) in sarcopenia. After that, we proposed a systematic and innovative regulatory network with an immune signature in sarcopenia by using Pearson correlation analysis(PCA) and Cytoscape. Finally, both online verification  and in vitro experiments were conducted to confirm our findings. Moreover, Connectivity Map (C-Map) was exploited to screen possible compounds targeting this network and develop potential drugs for sarcopenia.https://www.ncbi.nlm.nih.gov/geo/), which included 20 cases with sarcopenia and 20 age-matched and sex-matched healthy controls. Sarcopenia was defined based on harmonized consensus clinical definitions of the 2019 Asian Working Group for Sarcopenia Consensus (AWGS)  and Var[Y] represent the variance of two variables. The two variables were considered positively correlated (1 ≥ r > 0), uncorrelated .For the identification of final DEGs, the amount of gene expression for DETFs and DEGs was extracted from RNA-seq and input into the formula. The final DEGs were defined as those with an absolute value of PCC > 0.4 and p < 0.0106. The final DEIGSs were further identified by backtracking the corresponding DEIGSs of the final DEGs.For the identification of final DEIGSs, first, ssGSEA was performed by the R package GSVA to assess the ES of immune cells and pathways for every single sample, and the quantity of 29 immune gene sets in the sarcopenia group (SP) and nonsarcopenia group (NSP) was evaluated by the CIBER-SORT algorithm. Then, PCA was used to calculate the correlation between immune gene sets and DEGs by evaluating the ES and the amount of gene expression. The DEIGSs were defined as having absolute values of PCC > 0.4 and https://portals.broadinstitute.org/cmap/). The key medicines were identified with p < 0.01, and information of inhibitor compounds, such as chemical structural formula, biologic function, and clinical applications, was downloaded from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/).Then, network was built by Cytoscape (version 3.7.1) based on the PCA results. Finally, the sarcopenia-related hypothesis built on the bioinformatics was displayed by a signalling diagram. The potential inhibitors of the network were screened by C-Map (build 02) (https://www.ncbi.nlm.nih.gov/gene). Then, the target DETF binding site distribution in the target promoter regions was detected with the UCSC database(http://www.genome.ucsc.edu/). Finally, the JASPAR database(https://jaspar.genereg.net/) was used to predict the binding sites sequence and the relative score between the DETFs and final DEGs. The scan relative profile score threshold in the JASPAR database was set as 80%.For annotation of the final DEGs and DETFs, three algorithms  were used to repredict the transcriptional regulatory pattern among them. First, the promoter sequence regions of the final DEGs were obtained from the NCBI Gene database(2) based on the 2019 AWGS [In total, 24 Chinese male participants aged 69–84 years with or without sarcopenia were recruited from a prospective cohort study approved by the Shanghai Tenth People’s Hospital Affiliated with Tongji University School of Medicine. In brief, patients who were diagnosed with low-energy hip fracture and planned to undergo hip replacement surgery within 3 days after hip fracture were eligible for participation in the study between October 2021 and December 2021. The diagnosis of sarcopenia was defined as low muscle strength (dominant hand grip < 28 kg) and low appendicular skeletal muscle mass  equipment InbodyS10  in a supine position. DSM-BIA was performed using a tetrapolar 8-point electrode system following standard protocols . Lean boThe isokinetic strength of the participant’s dominant hand was measured using an electronic hand dynamometer  on the day of admission. The maximal value of three consecutive tests was recorded. Muscle strength was defined as the highest muscular force output (peak torque) in kilograms.3 muscle specimen was removed using sharp dissection. Then, small bleeding vessels were carefully coagulated by high-frequency electrosurgical equipment and the operation continued in routine fashion. No complications occurred from the biopsy procedure. After removing excess fat and connective tissue, the biopsy specimens were flushed with cold PBS to rinse the blood. The muscle specimens were immediately frozen in liquid nitrogen and stored at -80 °C until further analysis.Biopsy specimens were obtained at the beginning of hip replacement surgery under general anaesthesia. The edge of the tensor fascia femoris was exposed and a 1 cmn = 6 per group). For ultrastructural analyses, the fixation and Epon embedding of ultrathin sections were performed according to standard procedures, and the morphology of the biopsy specimens was observed using a transmission electron microscope  at an acceleration voltage of 80 kV. Three randomly selected fields within a 32.6 μm2 field of view for each sample were analysed to assess the ultrastructure of myofibrils, sarcomeres, lipid droplets, and mitochondria.Haematoxylin and eosin (H&E) staining and transmission electron microscopy (TEM) observation of the tensor fascia femoris muscle were performed following standard protocols. For H&E staining, biopsy specimens were fixed in 4% paraformaldehyde, embedded in paraffin, and then cut into sections. After staining with H&E, the sections were visualized under a light microscope system . Images of muscle section were taken randomly from sample, and the average cross-section areas (CSA) of muscle fibre was determined by manually encircling each cross-section of at least 100 fibres using standard imaging software ImageJ  , and the RNA quantity was determined by NanoDrop2000 . All RNA samples were homogeneous and passed quality control with 260/280 nm ratio > 1.8. Then cDNA of each sample was synthesized using 1.0 µg of RNA and the PrimeScript RT Reagent Kit  according to the manufacturer’s instructions. The qPCR was performed by QuantStudioTM Dx real-time PCR instrument  using TB Green Premix ExTaq II  according to the manufacturer’s instructions. The relative gene expression was calculated using the ΔΔCt method, and GAPDH was used as the internal control. The primers used in this q-PCR reactions were listed in Additional file p < 0.05 was considered statistically significant. All bioinformatics statistical analysis was performed using R version 3.5.3 .For descriptive statistics, the continuous variables with a normal distribution were expressed as the mean ± standard deviation (SD), while the median (range) was used for an abnormal distribution. Classified variables were expressed by counts and percentages. Only two-tailed |log2FC|> 1 and p < 0.05 between SP and NSP were defined as DEGs, and we identified 808 DEGs (207 downregulated and 601 upregulated) , “cornified envelope” for CC  and “structural constituent of skin epidermis” for MF  were the most significant GO items  was the most significant item  in C2, and “GO_ANTIMICROBIAL_HUMORAL_RESPONSE”  in C5, were also significantly upregulated in sarcopenia , mast cells , Th2 cells , TIL , B cells  and type II IFN response . Finally, the coanalysis for DETFs, final DEGs, final DEPs and final DEIGSs was performed, “trichostatin A” , “vorinostat” , and “thioridazine”  had the most potential as inhibitors of sarcopenia according to the results of C-Map. Based on the PubChem database, detailed information of thioridazine  of PAX5, TFAP2C and TP73 were located in the promoters of SERPINA5, ACTG2 and DSP respectively, and the results are illustrated in Fig. As a chronic common disease among the elderly population (present in 11–50% of people aged > 80 years) , sarcopeIn this study, we primary screened 808 DEGs, 4 DETFs, 304 DEPs and 1208 differential expressed immune cell- or immune response-related events. Based on the results of PCA, we then structured an immune-related transcription factor regulatory network with 4 DETFs, 9 final DEGs, 11 final DEPs, 5 immune cells and 1 immune reaction. Combining the identification results of online databases and in vitro experiments, we speculated that PAX5-SERPINA5-PI3K/Akt axis was the most potential pathogenic pathway and that B cells may play a vital role in signal transduction. Finally, according to the results of the literature review and C-Map, we hypothesized that trichostatin A might be a potential medicine for sarcopenia.TFs can direct genome expression and alter the activity of the signalling pathway by recognizing specific DNA sequences and controlling chromatin transcription to form RNA . With thPAX5, also known as a paired box 5, was the only downregulated transcription factor among the 4 DETFs in our study. PAX5 encodes a member of the PAX family and is a vital regulator of cell development, tissue differentiation and immune function . PreviouAs one of the extracellular alpha-1-antitrypsin clades of serpin, SERPINA5 can inhibit several plasma serine proteases and plays diverse roles in host defence, protein haemostasis, and lipid transport in multiple organs . PreviouSeveral recent studies have shown that immune cells play an important role in the development and progression of sarcopenia . HoweverSeveral limitations in our study should be noted. First, the muscle RNA-seq data in our analysis were collected from public databases, and much clinical information was incomplete, such as basic illness and medication use, which may cause potential bias and even errors. Second, the network demonstrated in our study was based on bioinformatics analysis and multidimensional correlation rather than biological mechanism research. Therefore, more cell and animal experiments still need to be performed to support our findings. In the future, we will conduct functional experiments to verify the PAX5-SERPINA5-PI3K/Akt axis in vivo and in vitro.In conclusion, our study was the first to predict the immune-related transcriptional regulatory network with high-throughput RNA-seq data in sarcopenia. We supposed that PAX5-SERPIAN5-PI3K/Akt axis is a potential mechanism in sarcopenia, and B cells may also play a vital role in this signal transduction. In addition, trichostatin A might be a potential medicine for sarcopenia.Additional file 1. Primers of DETFs and final DEGs.Additional file 2. Results of DEGs.Additional file 3. Functional enrichment analysis.Additional file 4. Results of ORA.Additional file 5. Results of DEPs.Additional file 6. Results of ssGSEA.Additional file 7. Results of DEIGSs.Additional file 8. Results of co-analysis.Additional file 9. Results of C-Map database.Additional file 10. Clinical characterization of patients.Additional file 11. The representative images of TEM.