Fruit and vegetable intake may influence mental well-being. The aim of this study was to assess longitudinal associations between fruit and vegetable intake and depressive symptoms among rural South Africans.This longitudinal community study enrolled 3,891 adults (≥ 40 years) from the “Health and Ageing in Africa: A Longitudinal Study of an INDEPTH Community in South Africa (HAALSI)”. Fruit and vegetable intake was assessed by self-report at wave 1, and depressive symptoms were assessed using the Center for Epidemiological Studies Depression Scale at wave 1 and 2. Outcomes were incident and persistent depressive symptoms at wave 2. Logistic regression was used to assess the associations between fruit and vegetable intake at wave 1 and incident, and persistent depressive symptoms.Results indicate that in the fully adjusted model for individuals with no depressive symptoms at baseline, we found no significant association between frequency of fruit or vegetables intake and incident depressive symptoms. We also found no significant association between frequency of fruit or vegetable intake and persistent depressive symptoms.Fruit and vegetable intake was not significantly associated with incident and persistent depressive symptoms. Major depressive disorders are a major public health burden globally, including in African countries . In SoutIn four longitudinal studies among middle-aged and older adults in Australia, Taiwan and USA, higher fruit and vegetable intakes decreased the odds of incident depression . In a loIn a further review among adults of 10 studies  found that in four studies higher fruit intake was associated with lower odds of depression, while six studies did not find a significant association, and among eight studies, three showed that high vegetable intake decreased depression, while five studies did not find significant associations . For exaMost cohort studies reviewed in the general adult and older adult population were conducted in high-income countries and none in Africa, which limits generalizability of findings to low- and middle-income countries, in particular in Africa, and it remains unclear if found associations hold in populations in Africa. Therefore, the aim of this study was to assess longitudinal associations between fruit and vegetable intake and depressive symptoms among an ageing population in South Africa.We analysed data from a longitudinal study (2 waves), “Health and Ageing in Africa: A Longitudinal Study of an INDEPTH Community in South Africa (HAALSI)” in Agincourt, South Africa. Sampling details have been published . The firThe study protocol was approved by the “University of the Witwatersrand Human Research Ethics Committee (ref. M141159), the Harvard T.H. Chan School of Public Health, Office of Human Research Administration (ref. C13–1608–02), and the Mpumalanga Provincial Research and Ethics Committee.” Participants provided written informed consent. All methods were carried out in accordance with relevant guidelines and regulations.Depressive symptoms (scores ≥ 3) were measured with the “Center for Epidemiological Studies-Depression Scale eight-item scale (CES-D 8) or the CES-D 20” , 20, intFruit and vegetable intake was sourced from the following items:“In a typical week, on how many days do you eat fruit? […vegetables?] (…days)” 2) “How many servings of fruit do you eat on a typical day? […vegetables] (on any one day) (…servings)”  .Social and demographic data: household wealth, age, country of birth, education level, sex, and marital status .Current tobacco use was defined as current smokeless and/or current tobacco smoking .Alcohol dependence was assessed with the 4-item CAGE scale ,  do you drink in a typical week?” “[USE SHOWCARDS OF POPULAR DRINKS LOCALLY.]” .2, normal weight: 18.5 to 24.9 kg/m2, overweight: 25 kg/m2 to 29.9 kg/m2, obesity: kg/m2 “) [Height and weight were measured, and Body Mass Index (BMI) calculated following World Health Organization (WHO) criteria (“underweight: < 18.5 kg/mkg/m2 “) .Hypertension was measured (averaging of the last two of three blood pressure readings) and defined based on National Committee criteria .Dyslipidaemia: taking medication for high cholesterol and/or “total cholesterol > 6.21 mmol/L, HDL-C < 1.19 mmol/L, LDL-C > 4.1 mmol/L, triglycerides > 2.25 mmol/L.” .Diabetes: taking glucose lowering medication and/or “fasting glucose (defined as > 8 h) > 7 mmol/L (126 mg/dL).” Physical activity  was measured with the “General Physical Activity Questionnaire (GPAQ)” , 25.p < 0.05 was considered as significant. “Inverse probability weights were applied to account for attrition and mortality at follow-up” [A total of 5,059 individuals (≥ 40 years) participated in wave 1, and of these 4,176 were followed at wave 2. Our analytic sample included participants who provided data on depressive symptoms at wave 2, giving a final sample of 3891. These sample restrictions were imposed as information of fruit and vegetable consumption and all other variables assessed in this analysis were obtained from wave 1. Descriptive statistics was used to describe baseline data. Chi-squared tests were used to test for differences in proportions. Univariable and multivariable logistic regression was applied to assess the association between fruit and vegetable intake at wave 1 and incident and persistent depressive symptoms. Incident depressive symptoms were assessed among those without depressive symptoms at wave 1 and referred to new cases of depressive symptoms at wave 2. Persistent depressive symptoms were measured only among those who had depressive symptoms at wave 1 and were defined as having depressive symptoms at wave 2. The multivariable models were adjusted for age, sex, education, wealth and marital status, alcohol dependence, current tobacco use, frequency of cans/bottles/cups of sugary drink consumption, physical activity, body mass index, hypertension, diabetes and dyslipidaemia. llow-up” . StataSEN = 3,891) was 61.0 years (SD = 12.4 years), and 43.9% were males. The prevalence of depressive symptoms at baseline was 15.5% (N = 598), and at wave 2 there were 835 new cases with depressive symptoms (incident depressive symptoms). Of those who had depressive symptoms at wave 1, 148 (30.8%) continued to have depressive symptoms at wave 2 (persistent depressive symptoms).Baseline sample characteristics compared to fruit and vegetable consumption pattern are shown in Table Among those without depressive symptoms at wave 1, the prevalence of depressive symptoms at wave 2 (incident depressive symptoms) was higher among those with higher vegetable consumptions but did not differ with frequency of fruit consumption and frequency of fruit and vegetable consumption. Among those with depressive symptoms at wave 1, the prevalence of depressive symptoms at wave 2 (persistent depressive symptoms) did not differ with frequency of fruit and vegetable consumption, frequency of fruit consumption and frequency of vegetable consumption , which could have influenced depressive symptoms. Furthermore, dietary behaviour, including health risk behaviours may have changed between wave 1 and wave 2.Fruit and/or vegetable intake was not significantly associated with incident and persistent depressive symptoms among middle-aged and older South Africans.


Malignant peritoneal mesothelioma (MPM) is a rare disease characterized by atypical symptoms, difficult diagnosis, variable course and poor prognosis, and it develops mainly in elderly individuals. The authors aimed to identify the clinical-pathological characteristics, prognosis, and prognostic factors in elderly MPM patients.From the National Cancer Institute Surveillance Epidemiology End Results (SEER) database, 1492 patients with MPM from 1975 to 2016 were selected and divided into the elderly group (≥ 65) and the adult group (< 65). We compared the clinical-pathological characteristics and treatment methods of the elderly group (N = 665) and the adult group (N = 827). At the same time, we analysed specific selected clinicopathological parameters and prognostic factors for elderly MPM patients.P = 0.017) and white patients (P = 0.043) and lower proportions of insured patients (P < 0.001) married patients (P < 0.001), patients with peritoneal tumours (P = 0.006) and patients who underwent surgery (P < 0.001) and chemotherapy (P < 0.001). There was a significant difference in the differentiation grade between the two groups (P = 0.003). Elderly patients had a shorter median survival time than adult patients (6 months vs. 19 months). Uninsured (hazard ratio (HR): 5.187, P = 0.005), sarcomatoid type , poorly differentiated , distant metastasis , no cancer-directed surgery , and no chemotherapy  were independently associated with poorer prognosis in elderly MPM patients.Compared with the adult group, the elderly group had higher percentages of male patients  rate of elderly MPM patients was significantly lower than that of adult patients. Insurance status, histology type, differentiation grade, stage, surgery status, and chemotherapy status were all independent prognostic factors for elderly MPM patients. Malignant mesotheliomas (MM) are aggressive tumours arising from serous surfaces, including the pleura 65%-70%), peritoneum 30%), tunica vaginalis testis, and pericardium 1%-2%) [%-2% [1].0%, tunic%-70%, peHowever, there have been few studies on the clinical-pathological characteristics and prognostic factors of elderly patients. Therefore, in this study, we selected patients from the Surveillance, Epidemiology and End Results (SEER) database . With thA retrospective case series analysis was performed using the SEER database. The database records patients' basic information, diagnosis basis, stage and grade, treatment plan, follow-up time and cause of death, covering approximately 28% of cancer patients in the United States, providing valuable oncology data for medical researchers worldwide.Patients with pathologically confirmed MPM were enrolled from 1975 to 2016 using ICD-O-3 histology codes 9050-9053  combined with site codes 48.0, retroperitoneum; 48.1, specified parts of the peritoneum; 48.2, peritoneum not otherwise specified; and 48.8, overlapping lesion of retroperitoneum & peritoneum. Patients who had other malignancies or did not have active follow-up since the time of MPM diagnosis were excluded from our study cohort. Based on these criteria, our study cohort consisted of a total of 1,492 MPM patients.Demographic variables of interest to our study included gender, race, age, marital status, and insurance status; clinical data of interest included the status of surgery, radiotherapy, chemotherapy, and survival time (from MPM diagnosis until cause-specific death (CSD), as of December 31, 2016, in months); pathological characteristics of interest included histology type, differentiation grade, and tumour stage.For further analysis, some demographics and clinical factors were classified based on SEER database record and our clinical experiences: for insurance status, patients with “Any Medicaid”, “Insured”, and “Insured/no specifics” were regarded as the “Insured” group, for marital status, “Separated”, “Divorced”, “Single”, and “Widowed” were clustered as the “Unmarried” group, differentiated grade was defined as “Well differentiated”, “Moderately differentiated”, “Poorly differentiated”, “Undifferentiated” or “Unknown”, tumour staging was defined as “Localized”, “Regional”, “Distant”, or “Unknown”, and histological type was defined as “Epithelioid”, “Sarcomatoid”, “Biphasic” or “Unknown”, radiotherapy and chemotherapy status were defined as “Yes” or “No or unknown”, for surgery status, the database did not contain details of MPM surgery before 1987, and the classification of surgery has also changed over time since 1987, so surgery status was classified as “Cancer-directed surgery done”, “No cancer-directed surgery” or “Unknown” groups in order to obtain long-term data.P values were two-sided, and P < 0.05 was considered to be statistically significant.Statistical analyses were performed using SPSS software (version 26.0). The study subjects were stratified by age into two groups: the adult group (< 65) and the elderly group (≥ 65). Using Student’s t test and the chi-square (χ2) test, selected clinical-pathological and treatment characteristics were compared between the adult and elderly groups as well as between the males and females in the elderly group. The CSS rate was determined by the Kaplan–Meier method, and the differences in CSS rates were determined by the 2-sided log-rank test. Cox proportional hazards analysis was performed to assess the independent risk factors for CSD in the elderly group. All P = 0.017) and white patients  and lower proportions of insured patients , married patients  and patients with peritoneum tumours . The differentiation grade in elderly patients was relatively worse than that in adult patients (P = 0.003), the ratios of well-differentiated patients (4.96% < 10.16%) and moderately differentiated patients (1.65% < 2.06%) were lower than those in adult patients, and the ratio of poorly differentiated patients was higher than that in adult patients (7.22% > 5.68%). Surgery  and chemotherapy  incidence was less common in elderly patients than in adult patients.From 1975 to 2016, a total of 1492 MPM patients were identified from the SEER registry, and there were 665 individuals in the elderly group (≥ 65). The baseline characteristics of the selected patients are summarized in Table P = 0.002). There was a higher percentage of males than females who underwent surgery .There were 391 (58.80%) males and 274 (41.20%) females in the elderly group. For the elderly MPM patients, the comparison of selected clinical-pathological characteristics between males and females is shown in Table P < 0.001, Fig. As of December 31, 2016, a total of 549 patients in the elderly group (N = 665) had died. The median survival time was six months, and the 1-, 3- and 5-year CSS rates in the adult and elderly patients are shown in Table P < 0.001). The patients who had surgery for MPM had a higher 5-CSS rate than those who did not (19.14% vs. 8.76%).The median survival time and the 1-, 3- and 5-year CSS rates in the different groups of elderly MPM patients are shown in Table P = 0.006, Fig. P = 0.001, Fig. P = 0.046, Fig. P < 0.001, Fig. P < 0.001, Fig. P < 0.001, Fig. P < 0.001, Fig. P < 0.001, Fig. Factors that were associated with the CSS of elderly patients included gender . Notably, elderly MPM patients with the biphasic and sarcomatoid types had a higher risk of CSD than those with the epithelioid type . The poorly differentiated and undifferentiated patients had a lower CSS rate  than well differentiated patients. Distant metastasis was a risk factor for poor prognosis . The no surgery group had 1.733 times the CSD risk of the surgery group. Moreover, patients in the no chemotherapy or unknown group had a 53.2% higher risk than those who underwent chemotherapy.These associated variables were included in the multivariate analysis. Cox proportional hazards analysis showed that insurance status . A study published in 2009  concludeAt present, there is no mature TNM staging system for MPM. Yan et al. proposed a TNM staging system for diffuse malignant peritoneal mesothelioma in 2010 . The SEEFor treatment, the effect of radiotherapy for MPM patients is not clear. Silja A.S. Salo et al.  showed tIn this study, only 33.38% of all elderly MPM patients underwent surgery, consistent with Anish Thomas’ report in 2015 (32%) . SurgicaP = 0. 049). However, some studies have shown that systemic chemotherapy has no positive effect on the prognosis of MPM patients [Chemotherapy is often combined with surgery to treat MPM, which can be delivered in the form of heated intraperitoneal chemotherapy (HIPEC). Nagata Y et al.  found thpatients . In thisAt present, cytoreductive surgery (CRS) combined with HIPEC as the first-line treatment of MPM has been proven to improve the prognosis of MPM patients –37. A syHowever, several limitations in our study should be considered. First, the SEER database does not clearly distinguish patients who did not receive chemotherapy or radiotherapy and those who did not know whether they had received these treatments, and we could not determine the effect of chemotherapy or radiotherapy on elderly MPM patients more precisely. Second, there are no specific chemotherapy regimens in SEER, so the influence of different chemotherapy regimens on prognosis cannot be studied. Third, for the variables of histology type and differentiation grade, the majority of patients were in the unknown group, which affected the accuracy of our results.In


Background: The combination of immune checkpoint inhibitors (ICIs) and chemotherapy (CT) is a new strategy to explore cancer treatment in recent years, and it is also practiced in triple-negative breast cancer (TNBC). However, several published randomized controlled trials (RCTs) reported heterogeneous results. We conducted this meta-analysis to yield insights into the efficacy and safety of the combination of ICIs and CT for TNBC patients in both the adjuvant and neoadjuvant settings.Method: EMBASE, PUBMED, Cochrane, and www.clinicaltrials.gov databases were searched to determine potential eligible studies from the inception to 20 May 2022. Published RCTs on PD-1/PD-L1 ICIs combined with CT for TNBC patients were included.Result: This meta-analysis included six double-blind RCTs comprising 4,081 TNBC patients treated with PD-1 or PD-L1 ICIs plus CT or placebo plus CT. The combination strategy benefited a better pathologic complete response (pCR) by 29%  and a better progression-free survival (PFS)  in the neoadjuvant and the adjuvant settings, respectively, especially in PD-L1-positive population . The safety profiles were generally tolerable in both settings but the combination treatment will increase the risk of severe adverse events in the adjuvant setting . Additionally, the combination will increase the risk of any-grade hypothyroidism, hyperthyroidism, pneumonia, and rash in the adjuvant setting, and the risk of any-grade hypothyroidism, hyperthyroidism, infusion-related reactions, and severe cutaneous reactions in the neoadjuvant setting.Conclusion: This meta-analysis demonstrated a significant pCR benefit and confirms the PFS benefit with PD-1/PD-L1 ICIs plus CT in TNBC patients with tolerable safety events in both neoadjuvant and adjuvant settings. Triple-negative breast cancer (TNBC) stands as a particularly aggressive type of cancer with a relatively poor prognosis. TNBC indicates the presence of cancerous cells that screen negative for three molecular constituents of breast cancer: estrogen receptors (ERs), progesterone receptors (PRs), and sufficient levels of human epidermal growth factor (HER2) protein . Due to One molecular attribute that is often indicative of cancer, is the ability of cells to evade immune response through various pathways such as the PD-1/PDL-1 pathway. Programmed death protein 1 (PD-1) is an inhibitory cell surface receptor that binds to programmed death-ligand 1 (PD-L1), and activates a downstream signaling cascade that inhibits T-cell activation, and represses the immune system’s ability to initiate any inflammatory response . When PDThe efficacy and use of immuno-monotherapy for TNBC patients have not yet been defined . HoweverA meta-analysis together with a systematic review was performed to enroll RCT studies concerning the combination strategy of PD1/PD-L1 ICIs plus CT compared with CT monotherapy in TNBC patients applied whether as adjuvant therapy or neoadjuvant therapy. The meta-analysis was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines [20].www.clinicaltrials.gov databases to enroll potential eligible studies from the inception to 20 May 2022. We performed the searching using the following strategy: [“nivolumab” OR “pembrolizumab” OR “atezolizumab” OR “avelumab” OR “BMS-936559″ OR “durvalumab” OR (“pd l1 inhibitors” OR “pd l1 inhibitors” OR “pd l1 inhibitor” OR “pd l1 inhibitor” OR “programmed death ligand 1 inhibitors” OR “programmed death ligand 1 inhibitors” OR “pd 1 inhibitors” OR “pd 1 inhibitors” OR “pd 1 inhibitor” OR “inhibitor pd 1″ OR “pd 1 inhibitor” OR “programmed cell death protein 1 inhibitor” OR “programmed cell death protein 1 inhibitors” OR “pd 1 pd l1 blockade” OR “blockade pd 1 pd l1″ OR “pd 1 pd l1 blockade” OR “Immune Checkpoint Inhibitors")] AND {[(“ER-Negative” AND “PR-Negative” AND “HER2-Negative”) AND “breast neoplasms”] OR {[(“eye rep” OR “expert rev mol med” OR “educ res” OR “econ rec” OR “ER”) AND (“Negative” OR “negatively” OR “negatives” OR “negativities” OR “negativity”) AND (“psychopathol rev” OR “pharmacol rep” OR “pharmacognosy res” OR “partis rev” OR “PR”) AND (“Negative” OR “negatively” OR “negatives” OR “negativities” OR “negativity”) AND “HER2″] AND “negative breast neoplasms”} OR “triple negative breast cancer” OR “breast cancer triple negative” OR “breast cancers triple negative” OR “triple negative breast cancers” OR “triple negative breast neoplasm” OR [(“breast neoplasms” OR (“Breast” AND “Neoplasms”) OR “breast neoplasms” OR (“Breast” AND “Neoplasm”) OR “breast neoplasm”] AND “Triple-Negative”) OR “breast neoplasms triple negative” OR “triple negative breast neoplasm” OR “Triple Negative Breast Neoplasms” OR “er negative pr negative her2 negative breast cancer” OR “er negative pr negative her2 negative breast cancer” OR “triple negative breast cancer” OR “Triple Negative Breast Neoplasms”}. Available Medical Subject Headings (MeSH) in our strategy were searched by MeSH. The relevant references and reviews were either retrieved to acquire potential studies. Abstracts of conferences posted before 20 May 2022, were also reviewed.We searched EMBASE, PUBMED, Cochrane, and The following criteria should be considered and met for the enrolled eligible studies: 1) RCTs concerning the combination therapy of ICIs and CT compared with CT monotherapy; 2) should be applied whether as adjuvant therapy or neoadjuvant therapy; 3) in TNBC patients; 4) RCTs with efficacy outcomes assessed by a hazard ratio (HR) and confidence intervals (CI); 5) adverse events (AEs) and response rate (assessed by the WHO criteria) were analyzed and released.Exclusion criteria are as follows: 1) non-RCT studies such as single-arm trials and retrospective studies; 2) studies that did not apply placebo plus CT as the strategy of the control group; 3) studies that did not use a PD-1/PD-L1 ICIs plus CT treatment in the experiment group; 4) ongoing clinical trials without released results at the time of the literature search; 5) reviews, systematic reviews, basic research, case reports, meta-analysis, letters, editorials, and expert opinions; 6) unpublished or duplicated studies.The titles and abstracts of all screened studies were reviewed by two authors, namely, YQ and RJ independently. The full texts were subsequently assessed for potentially eligible studies. A standardized piloted form was applied to retrieve information from the enrolled studies.The following variables were retrieved: name of the studies, first author, publication year, study design, endpoint, blinding status, study phase, lines of treatment, median follow-up time, population characteristics, study sample size, experimental group sample size, control group sample size, ICIs used in combination with CT as an experimental arm, CT regimen used as a control arm, biomarker and PD-L1-positive definition assays, intention-to-treat population, PD-L1 status subgroups, safety population sample size; ECOG performance status, median age, female percentage, race percentage, number of metastatic sites, previous neoadjuvant/adjuvant CT, stage IV at initial diagnosis, frequency of pCR events in neoadjuvant studies, HR with associated 95% CI for PFS and OS, frequency of AEs of any grade reported, grade ≥3 AEs, and severe adverse events (SAEs) for the experimental group. Adverse events are graded by 1–5, and recorded according to Version 4 of the Common Terminology Criteria for Adverse Events of the National Cancer Institute, CTC for AE version. The discrepancies were discussed thoroughly and well-solved.The risk of bias was discussed and assessed according to the Cochrane Collaboration’s Risk of Bias tool by two independent investigators. We assessed each study’s risk of bias according to exclusion criteria, study design, and observation period considerations.The primary endpoints are as follows: 1) pCR rate in neoadjuvant therapy studies, defined as pathological complete response with no cancer cells existing in the pathological examination of cancer patients after treatment; 2) PFS in adjuvant therapy studies, which was defined as the time from the date of randomization to the date of the first record of disease progression (according to RECIST 1.1) or any-cause death. The secondary efficacy endpoint was OS, which was defined as the time from randomization to any-cause death. Safety endpoints are 1) percentage of AEs of any grade, 2) grade ≥3 AEs, and 3) severe AEs.PFS and OS were analyzed by calculating HR with 95% CI to summarize the efficacy. For the pCR rates and safety assessment, RR with 95% CI was determined to obtain an overall estimation. HR < 1 indicates a protective effect with ICIs plus CT, while an RR > 1 indicates a higher possibility of adverse events for patients treated with the combination therapy.2 statistics were performed to assess heterogeneity between included studies. The meta-analysis was performed using the fixed-effects model only if the I2 value was less than 50%, otherwise, the random-effects model will be selected. Otherwise, the random-effects model was selected. The Egger’s test and funnel plot were conducted to examine the potential publication. All analyses were performed using R statistical software version 3.6.2 (R packages metafor and meta).The Q test and IWe identified 692 potential articles, 286 studies of which were excluded due to duplications. We screened the titles and abstracts of the remaining 406 articles, 383 of which were again removed according to our inclusion or exclusion criteria. An additional of 17 studies were excluded because they did not contain our data of interest. Ultimately, six studies were pooled for our meta-analysis. The study selection diagram is shown in The study design and baseline information on the involved clinical trials are demonstrated in n = 2,400), all three studies are phase III clinical trials, with atezolizumab used in IMpassion 130 and IMpassion 131  and pembrolizumab used in KEYNOTE-355  as immunotherapy agents. Nab-paclitaxel, paclitaxel, and gemcitabine–carboplatin were, respectively, used as CT regimens in different studies. In neoadjuvant settings , IMpassion 031 using atezolizumab  and KEYNOTE-522 using pembrolizumab  are phase III clinical trials, and GeparNuevo using durvalumab  is a phase II study. Among these 4,081 patients, 2,577 patients were PD-L1+ , the pooled evaluation in the ITT population demonstrated a benefit for the ICIs plus CT group with no heterogeneity was found  (n = 984) in the combination of ICIs and CT regimens   . Further2 = 13%) .n = 2,400). The pooled OS evaluation demonstrated no benefit of ICIs plus CT with heterogeneity in both the ITT population  (2 = 79%)   and the 2 = 79%) .n = 1,553). The pooled evaluation demonstrated no benefit of ICIs plus CT with no heterogeneity in both the ITT population  (2 = 0%) (The pooled time to deterioration (TTD) in global health status/health-related quality of life (GHS/HRQoL) was analyzed in IMpassion 131 and IMpassion 130 (I2 = 0%)  and the (2 = 0%) .n = 1,681). The pooled evaluation in the ITT population demonstrated that a significant pCR was in favor of the ICIs plus CT group with no heterogeneity found   .n = 2,400). The estimation demonstrated that the combination of ICIs and CT arm was associated with a higher incidence of AEs in any grade with no heterogeneity , and a higher incidence of AEs in severe grades with no heterogeneity . However, the estimation in the random-effects model showed that no significant variation was found between the experimental arm and control arm in the incidence of AEs more than grade III  and any-reason death   patients were included in the safety analysis. The pooled overall AEs in the adjuvant setting was analyzed in IMpassion 130, IMpassion 131, and KEYNOTE-355 (2 = 68%) .2 = 0%), more than grade III AEs  and the SAEs   .2 = 0%), hypothyroidism , pneumonitis , and rash  with no heterogeneity , hypothyroidism , infusion-related reactions , and severe cutaneous reactions  with no heterogeneity (2 = 0%) for combination strategy . Our study aimed to push the dense fog aside and clarify the efficacy and safety of the combination of PD-1/PD-L1 ICIs and chemotherapy applied to adjuvant and neoadjuvant treatments.Our findings confirmed that neoadjuvant ICIs plus CT combination improves the pCR rate of early-stage TNBC patients in the ITT group by 29%, and we also verified that adjuvant combination therapy benefits the PFS in unresectable locally advanced or metastatic TNBC in both ITT and PD-L1-positive groups. Driven by the PD-L1 status, this PFS benefit enables a 29% relative risk of progressive reduction in the PD-L1-positive population. However, no benefit was found in terms of OS and TTD in both ITT and PD-L1-positive populations under the adjuvant setting, and no benefit for any-reason death in the PD-L1-positive population was found.In terms of the safety of this combination strategy, though the general profile was consistent with the published side effects of the regimen in individual studies, discrepancies were shown between adjuvant and neoadjuvant settings. There is no doubt that the idiosyncratic AEs can occur in the experimental group due to the use of immunosuppressants. However, our analysis shows that the side effects of this combination therapy are overall safe and tolerable.In general, the combination strategy did not increase the risk of any AEs in the neoadjuvant setting. Although the experimental group had more adverse reactions at any grade in the adjuvant setting, there was no difference concerning the AEs above grade III. In detail, in addition to increasing the risk of hypothyroidism, hyperthyroidism, pneumonia, and rash at any grade in the adjuvant setting, there is an increasing risk of hypothyroidism, hyperthyroidism, infusion-related reactions, and severe cutaneous reactions at any grade in the neoadjuvant setting, as well as the risk of severe cutaneous reactions above grade III in both settings, there was no difference existing in other adverse events. Hypothyroidism is widely acknowledged as the most common immune-related endocrine toxicity, which occurs in approximately 30%–40% of anti-PD-1/PD-L1-treated patients . AdditioSpecifically, it should be noted that in the adjuvant setting, the combined strategy produced a 33% statistically significant relative increase in the risk of severe AEs but not grades more than three AEs and death events. This may result from the fact that although the overall number of high-grade AEs in the two groups is roughly the same, the AEs caused by the combination are concentrated in more serious events but not death. In terms of that in the neoadjuvant setting, the analysis cannot be performed due to a lack of relative data. Therefore, based on current evidence, the safety of combination therapy is generally mild and well-tolerated, but we can never be careless about serious events, especially in the adjuvant setting, which needs more in-depth research and discussion to reduce and control them.n = 9], but with the longest resolution time . Therefore, in a neoadjuvant setting, rash symptoms should be paid more attention to at the initiation of the durvalumab and should be given more time to recover.The side effect profiles of the neoadjuvant therapy group and adjuvant therapy group were almost the same, but there were still some differences. For example, there were more dermatitis and infusion-related reactions in the neoadjuvant treatment group, while there was a high rate of pneumonia in the adjuvant treatment group. One thing that should be clarified is that the analysis was not performed due to insufficient infusion-related reaction data in the adjuvant treatment group. According to the existing evidence, it can be speculated that the difference in pneumonia may be related to the baseline characteristics of the population to some extent: most patients in the neoadjuvant treatment group are in the early stage, with low ECCG scores and no lung metastasis, while patients in the adjuvant treatment group are in the advanced stage, with high ECOG scores, and nearly half of the patients have lung metastasis. The intervention of ICIs may be more likely to affect this part of the population. Additionally, compared with patients in other clinical trials, patients in the GeparNuevo have a higher risk of rash, which may be because GeparNuevo is the only study using durvalumab as an ICI, and rash/dermatitis was reported to be one of the most common adverse events among patients receiving durvalumab . AdditioYunhai Li et, al. and Ji Qiao et, al. conducted meta-analyses regarding the efficacy and safety of the combination of ICIs and chemotherapy among TNBC patients , which rThis meta-analysis’s limitations are as follows. First, we did not perform analysis stratified by age, CT regimen, ICI regimens, ECOG, number of metastatic sites, and so on. Second, PD-L1 was assessed in different cells in these studies: PD-L1 was assessed in immune cells in IMpassion 131, IMpassion 130, and IMpassion 031, whereas PD-L1 was assessed in iTILs  in KEYNOTE-355, KEYNOTE-522, and GeparNuevo. In addition, the PD-L1 assessment techniques were different: IMpassion 131 and IMpassion 130 were assessed by VENTANA PD-L1 (SP142) immunohistochemical testing, while KEYNOTE 355, IMpassion 031, and KEYNOTE 522 were assessed using the IHC 22C3 pharmDx assay and characterized by the CPS, and the GeparNuevo used the Ventana SP263 antibody to assess. Therefore, we should proceed with extra caution when interpreting the findings of PD-L1-positive populations in these studies. Third, since the adverse events were not homogenous among these six studies, the safety analysis was performed only for those adverse events reported in at least two studies. Fourth, our analysis is based on only three studies each in adjuvant and neoadjuvant settings, which could be inadequate for visual or statistical examination of publication bias. Fifth, due to the lack of OS data in the KEYNOTE 355 study, the pooled OS analysis in the adjuvant setting only included IMpassion 131 and IMpassion 130. Sixth, due to the extremely low incidence rate of some of the detailed AEs in the neoadjuvant setting, the pooled evaluation should be carefully interpreted. In addition, this study may be subjected to any errors and biases from the original investigators, thus the meta-analysis findings are generalizable only to patients eligible for these clinical trials.Our meta-analysis demonstrated that a combination of ICIs and CT can effectively improve the pCR rate in early-stage TNBC patients in the neoadjuvant setting, and it also benefits a better PFS in untreated, unresectable, locally advanced, or metastatic TNBC patients in the adjuvant setting. The PD-L1-positive status is regarded as a driven factor for better efficacy. The safety profiles of the experimental arm are generally good and tolerable compared with the control arm, but it is still essential for clinical physicians to pay close attention to those severe adverse events. To summarize, based on the current evidence concerning efficacy and safety data, the combination of ICIs and CT regimen can be recommended in early-stage TNBC patients in the adjuvant setting and untreated unresectable locally advanced or metastatic TNBC patients in the adjuvant setting, especially in PD-L1-positive population.


BACKGROUND: Frailty is associated with adverse health outcomes, and vitamin D (VD) deficiency may be a risk factor. We aimed to identify frailty trajectories and examine the mediating effect of physical activity (PA) on the association between VD deficiency and frailty trajectories. METHODS: We included 2997 participants aged 60 to 85 years from ELSA. VD was measured using serum 25-hydroxyvitamin D [25(OH)D] . Frailty was assessed by a 60-item frailty index, and PA was measured on the basis of total energy expenditure. Frailty trajectories were identified using group-based trajectory modeling, and the mediation effect of PA was tested using causal mediation analysis. RESULTS: Three distinct frailty trajectories emerged: “Non-frail” (66.48%), “Pre-frail to frail” (25.67%) and “Frail to severely frail” (7.85%). VD deficiency was associated with the “Pre-frail to frail”  and “Frail to severely frail” trajectories . PA only mediated 48.4% (95% CI: 17.1%–270.8%) of the association between VD deficiency and the “Pre-frail to frail” trajectory. CONCLUSIONS: Vitamin D deficiency is associated with the onset and worsening of frailty in older adults, and reduced PA may mediate its impact on the transition from pre-frailty to frailty. Frailty is a clinical state characterized by reduced physiological function and reduced stress resistance in older adults, leading to adverse events including hospitalization, falls and death . A recenPrevious studies on VD deficiency and frailty have predominantly focused on the binary frailty status. For instance, a cross-sectional study among individuals aged ≥65 years showed that those with 25(OH)D < 50 nmol/L had a 130% increased risk of frailty . As a coUtilizing data collected from the English Longitudinal Study of Ageing (ELSA), the current study aimed to (1) identify distinct frailty trajectories over 8 years among adults aged from 60 to 85 years in the UK, (2) examine the association between VD deficiency and frailty trajectories and (3) test the hypothesis that VD deficiency bears an indirect effect on frailty trajectories through low physical activity.This analysis used data from the English Longitudinal Study of Ageing (ELSA). ELSA is a nationally representative longitudinal survey of people aged 50 and over in England. The first survey was conducted in 2002 with participants from the Health Survey for England (HSE) samples in 1998, 1999 and 2001. Participants in ELSA were followed up every two years with a computer-assisted personal interview plus a self-administered questionnaire. The detailed study design of ELSA has been reported elsewhere . We usedicipants . The ELSThe serum 25(OH)D levels were determined by the DiaSorin Liaison immunoassay performed on a fasting blood sample taken at the Royal Victoria Infirmary  in wave 6′s nurse visits. In this study, 25(OH)D concentration was classified into three categories with reference to the Institute of Medicine (IOM) criteria: sufficient ( nmol/L) .PA was measured by asking participants how often they engaged in vigorous, moderate and light activities. Each question had four options: never, one to three times a month, once a week and more than once a week. Participants were given specific examples of different PAs; for example, home repairs and laundry fell under vigorous PA, car washing and dancing fell under moderate PA, and cycling and swimming fell under light PA . We estiWe used the frailty index to quantify the level of frailty. Based on the methodology of Searle et al. , we used2 ≤ BMI < 25 kg/m2; overweight: 25 kg/m2 ≤ BMI < 30 kg/m2; obese: BMI ≥ 30 kg/m2). Since the blood 25(OH)D level is subject to seasonal influences, we further adjusted for season [Socio-demographic, anthropometric and health behavior factors from wave 6 were included as potential confounders. Socio-demographic variables included sex, education , marital status , employment (employed or unemployed) and annual income (in quintiles). Health behaviors included smoking , alcohol intake  and BMI  to identify distinct frailty trajectories with age. GBTM clusters individuals with similar trajectories into latent trajectory groups . In thisWe conducted multinomial logistic regression to examine the association among different 25(OH)D levels, PA in wave 8 and frailty trajectories. For each association analysis, 4 models were fitted consecutively: Model 1 was the unadjusted model; Model 2 adjusted for basic demographic factors, including sex, education, marital status, employment, wealth, smoking, alcohol intake and BMI; Model 3 further adjusted for VD supplement use and season; and Model 4 further adjusted for PA in wave 6.Causal mediation analysis was used to quantify the contribution of PA to the association between different 25(OH)D levels and frailty trajectories . Causal  follows :δ¯i(t)≡ p <0.05 was regarded as statistically significant.Data cleaning and analysis were conducted in R version 4.0.4 . GBTM was performed in SAS  using PROC TRAJ. Mediation analysis was performed using the R package “mediation”, and The sample included 2997 individuals with an average age of 68.6 years. Most participants were female (55.5%), were married (68.9%), were unemployed (79.5%), had no formal education (37.5%), were never smokers (89.5%), were overweight (45.1%), drank a few times per week (45.2%) and engaged in moderate PA (49.5%). The prevalence of VD insufficiency and deficiency was 32.1% and 19.6%, respectively .We identified three latent frailty trajectories for individuals who were 60 to 85 years of age . The firIn unadjusted multivariable logistic regression, participants with insufficient serum VD levels were more likely to follow the “Pre-frail to frail” trajectory and the “Frail to severely frail” trajectory  of the association. However, we did not observe any significant ACME on the association between VD insufficiency and the “Pre-frail to frail” trajectory. In the causal mediation analysis of the risk of being in the “Frail to severely frail” trajectory group versus the “Non-frail” group, PA did not show a significant ACME.In the causal mediation analysis of the risk of being in the “Pre-frail to frail” trajectory group versus the “Non-frail” group, PA showed a significant ACME on VD deficiency , which mediated 48.4% . In addition, PA showed a significant mediation effect on the association of VD deficiency with the “Frail to severely frail” trajectory among older adults who lived alone , had hypertension  and smoked  .Based on a large cohort of older adults in the UK, our analyses identified three distinct types of frailty trajectories over the ages of 60 to 85 years. Our results suggested that participants with VD deficiency had a greater likelihood of following the “Pre-frail to frail” and the “Frail to severely frail” trajectories relative to the “Non-frail” trajectory. In the causal mediation analysis, PA mediated approximately 48.4% of the association between VD deficiency and the “Pre-frail to frail” trajectory, but not the “Frail to severely frail” trajectory. These findings suggest that VD deficiency may accelerate the transition from pre-frailty to frailty through reduced physical activity. Therefore, it is important to promote physical activity for the primary prevention of frailty among older adults suffering from VD deficiency.Limited studies have explored heterogeneous developmental frailty trajectories. Our study identified three distinct types of frailty trajectories: “Non-frail”, “Pre-frail to frail” and “Frail to severely frail”. Similar to our findings, Howrey et al. identified three groups of frailty: “non-frail”, “moderate progressive frailty” and “progressive high frailty” among older Mexican Americans . HoweverAlthough previous studies have investigated whether VD is associated with frailty, few have examined its association with the developmental trajectories of frailty. A recent systematic review  of 26 stOur study has several advantages over previous studies. First, we gridded the longitudinal frailty assessment data by age; that is, we used age as the underlying time scale, which allowed us to better understand how frailty changes with age in older adults. Second, we took baseline PA into account to minimize reverse causation. However, our study has several limitations. First, we only examined VD at baseline, which ignores changes in VD over time. Second, some components of the FI were long-term or irreversible, which made it difficult to observe any improvement in frailty. Finally, most variables of the FI were self-reported, which makes them subject to recall bias and may lead to inaccurate estimates of the frailty trajectories.Based on a nationally representative sample of older adults in the UK, we identified three distinct frailty trajectories: “Non-frail”, “Pre-frail to frail” and “Frail to severely frail.” Older adults with insufficient and deficient VD were at significantly higher risk of following the “Pre-frail to frail” and the “Frail to severely frail” trajectories. PA mediated approximately half of the association between VD deficiency and the “Pre-frail to frail” trajectory. Our results highlight the importance of developing strategies to promote physical activities to prevent the onset of frailty in older adults with low levels of VD. Given the observational nature of this analysis, interventional studies are needed to investigate the protective effect of PA in reducing frailty among older adults with VD deficiency.


Stress can increase ethanol drinking, and evidence confirms an association between post-traumatic stress disorder (PTSD) and the development of alcohol use disorder (AUD). Exposure to predator odor is considered a traumatic stressor, and predator stress (PS) has been used extensively as an animal model of PTSD. Our prior work determined that repeated exposure to intermittent PS significantly increased anxiety-related behavior, corticosterone levels, and neuronal activation in the hippocampus and prefrontal cortex in naïve male and female C57BL/6J mice. Intermittent PS exposure also increased subsequent ethanol drinking in a subgroup of animals, with heterogeneity of responses as seen with comorbid PTSD and AUD. The present studies built upon this prior work and began to characterize “sensitivity” and “resilience” to PS-enhanced drinking. Ethanol drinking was measured during baseline, intermittent PS exposure, and post-stress; mice were euthanized after 24-h abstinence. Calculation of median and interquartile ranges identified “sensitive” (>20% increase in drinking over baseline) and “resilient” (no change or decrease in drinking from baseline) subgroups. Intermittent PS significantly increased subsequent ethanol intake in 24% of male (↑60%) and in 20% of female (↑71%) C57BL/6J mice in the “sensitive” subgroup. Plasma corticosterone levels were increased significantly after PS in both sexes, but levels were lower in the “sensitive” vs. “resilient” subgroups. In representative mice from “sensitive” and “resilient” subgroups, prefrontal cortex and hippocampus were analyzed by Western Blotting for levels of corticotropin releasing factor (CRF) receptor 1, CRF receptor 2, CRF binding protein, and glucocorticoid receptor, vs. separate naïve age-matched mice. In prefrontal cortex, CRF receptor 1, CRF receptor 2, CRF binding protein, and glucocorticoid receptor levels were significantly higher in “sensitive” vs. naïve and “resilient” mice only in females. In hippocampus, CRF receptor 1, CRF receptor 2 and glucocorticoid receptor levels were significantly lower in “resilient” vs. naïve and “sensitive” mice across both sexes. These results indicate that sex strongly influences the effects of ethanol drinking and stress on proteins regulating stress and anxiety responses. They further suggest that targeting the CRF system and glucocorticoid receptors in AUD needs to consider the comorbidity of PTSD with AUD and sex of treated individuals. Alcohol use disorder (AUD) encompasses both alcohol abuse and dependence, and it is considered a chronic brain disorder that is characterized by compulsive drinking, an inability to stop or to control alcohol use, and the presence of negative emotions when not drinking ,b. Some Post-traumatic stress disorder (PTSD) has been classified as a trauma- or stressor-related disorder . IndividSeveral studies document an association between PTSD and the development of an AUD and suggest that PTSD may precede the onset of AUD , and no interaction. The similar and significant subgroup differences in drinking in males and females reflected our use of identical criteria for defining “sensitive” and “resilient” animals across males and females.By analyzing a large number of animals for each sex, median and interquartile ranges were calculated on the individual data (% change in average PS2-4 intake from BL). A scatterplot of these data, combined across sex, is depicted in F = 38.36, p < 0.001; Subgroup: F = 3.44, p < 0.07; PS: F = 8.32, p = 0.006]. The interaction between subgroup and PS exposure also was significant , but no interactions with sex were statistically significant. When collapsed across sex, there was a trend for an effect of subgroup , significant effect of PS  and significant subgroup by PS interaction . In the “sensitive” subgroup, PS exposure significantly influenced ethanol intake , with post-hoc tests confirming that ethanol intake was increased significantly over BL following PS1-4 . However, in the “resilient” subgroup, PS exposure significantly altered ethanol intake , with post-hoc tests confirming a significant decrease in ethanol intake following PS1-4 vs. BL  = 31.47, p < 0.001] and also differed across subgroups . There was no significant interaction between sex and subgroup. When collapsed across sex, a follow-up t-test indicated that BL intake in the “sensitive” subgroup was significantly lower than intake in the “resilient” subgroup . However, repeated exposure to PS reversed this relationship, such that after PS4, ethanol intake was significantly higher in the “sensitive” vs. “resilient” subgroup .Baseline ethanol intake see  was highF = 3.66, p < 0.06; Subgroup: F = 4.14, p < 0.05; PS: F = 13.00, p < 0.001]. The interaction between subgroup and PS exposure also was significant , but no interactions with sex were statistically significant. When collapsed across sex, ANOVA revealed a trend for an effect of subgroup , a significant effect of PS  and significant subgroup by PS interaction . In the “sensitive” subgroup, PS exposure significantly influenced ethanol preference , with post-hoc tests confirming that ethanol preference was increased significantly over BL following PS2–4 . In the “resilient” subgroup  = 8.17, p < 0.001], with post-hoc tests indicating that preference was decreased significantly from BL following PS1-4. Thus, the effects of PS on ethanol preference were similar to the effects on ethanol intake.To test whether PS specifically affected ethanol intake, or non-specifically affected consummatory behaviors, similar analyses were performed for ethanol preference . The ining PS2–4 . Ethanolsubgroup , PS expoF = 49.46, p < 0.001]. There was no significant interaction between sex and subgroup. When collapsed across sex, a follow-up t-test indicated that BL preference in the “sensitive” subgroup was significantly lower than in the “resilient” subgroup . However, repeated exposure to PS eliminated this difference, such that preference tended to be higher at PS4 (p = 0.08) in the “sensitive” vs. “resilient” subgroups.Baseline ethanol preference see  did not F = 13.42, p < 0.001; PS: F = 18.58, p < 0.001], but the interaction between sex and PS was not significant. When data were collapsed across sex, including the naïve group, and collapsed across PS1 and PS4, a one way ANOVA and post-hoc tests confirmed that plasma CORT levels were significantly increased by PS  vs. naïve and were significantly higher in the “resilient” vs. the “sensitive” subgroup  = 47.56, p < 0.001; Subgroup: F = 15.65, p < 0.001]. The interaction between sex and subgroup also was significant . In male mice, final CORT levels were not significantly altered vs. naïve in the “sensitive” and “resilient” subgroups. In contrast, final CORT levels in females were significantly different , with post-hoc tests confirming that CORT levels were significantly elevated vs. naïve in both the “sensitive” and “resilient” subgroups (ps < 0.001).Plasma CORT levels following a 24 h abstinence after the final drinking session in the “sensitive” and “resilient” subgroups  are shown in Western blots were conducted on tissue from representative animals in the “sensitive” and “resilient” subgroups that are depicted in post-hoc test showed a significant 17% increase in levels of this protein in “sensitive” vs. naïve mice and 22% higher levels in “sensitive” vs. “resilient” subgroups.The Western blot results are depicted in While significant effects of subgroup were found across all four investigated proteins in the hippocampus, interactions between sex and subgroup reached statistical significance only for CRF-BP . TherefoDue to the significantly higher CORT levels upon euthanasia in the female vs. male experimental mice, correlations between final CORT levels and stress-related protein levels were conducted . In the The purpose of the present studies was to begin to characterize “sensitivity” and “resilience” to PS-enhanced drinking in male and female C57BL/6J mice. These studies build upon our prior work, where we determined that repeated exposure to intermittent PS (30 min dirty rat bedding) significantly increased anxiety-related behavior and plasma CORT levels  as well The analysis of drinking following each PS exposure revealed that ethanol intake was increased significantly over BL after PS2–4 in both males and females from the “sensitive” subgroup. There also were comparable increases in ethanol drinking over BL in the sexes; ethanol intake increased over BL by 31%  and 44%  after PS2 and by 62%  and 59%  after PS4. The increases in ethanol intake over BL after PS2 in the “sensitive” subgroup, using a 23 h access procedure, are comparable with our earlier work that utilized a 2 h limited access procedure and found that 2 h 10% ethanol intake was increased by 30–35% in males and by 25–36% in females on the second day after PS exposure . One othIn the current and prior study that measured 23 h drinking, we found that the significant escalation in ethanol intake began after exposure to the second PS. This timing corresponded to the significant increase in anxiety-related behavior that was observed after exposure to the second PS in naïve male and female mice, measured on the elevated plus maze . We founOur retrospective determination of PS sensitivity subgroups revealed that BL ethanol intake was significantly lower in animals in the “sensitive” vs. “resilient” subgroup. This finding is similar to our earlier observation in females, where there were two separable BL subgroups, and PS-enhanced drinking was evident only in the subgroup of female mice with lower BLs . For thePlasma CORT levels were measured as a bioassay for HPA axis responsivity after intermittent PS exposure and a 24 h abstinence after the final drinking session. There were several interesting findings. First, PS exposure significantly increased CORT levels vs. naïve, consistent with data from our and other laboratories, where various models of PS exposure significantly increased CORT levels . DespiteOur study analyzed levels of proteins involved in regulation of stress responses at 24 h of abstinence following continuous ethanol drinking. We acknowledge that distinct changes in these protein levels could occur at different time points of the procedure due to plasticity associated with the effects of ethanol and PS e.g., . Our priTaking into consideration the potential treatments for individuals with PTSD-AUD comorbidity, it is notable that CRF-R1 was the least affected among stress-related proteins in the “sensitive” mice. Thus, the only increase in CRF-R1 in the “sensitive” mice in the present study was observed in the mPFC of female mice as compared to “resilient” females. This increase was not statistically significant when compared to naïve mice. On one hand, this observation suggests that the previously observed increase in CRF-R1 levels in PS-exposed alcohol-exposed mice in the hippocampus  could beIt needs to be specially noted that changes in CRF-BP appeared the most consistent between the subgroups. Specifically, CRF-BP showed increased levels in “sensitive” mice of both sexes in mPFC when compared to both “resilient” and naïve animals, and showed increased levels in the hippocampus of male “sensitive” mice vs. both “resilient” and naïve, while showing no changes in the hippocampus of female “sensitive” mice vs. the other groups. It is possible that the higher CRF-BP levels in the “sensitive” subgroup contribute to the significantly lower CORT concentrations after PS in the “sensitive” vs. “resilient” subgroup, as one role of CRF-BP is to bind and sequester circulating CRF and the urocortins e.g., . Both stIn conclusion, we developed an animal model where repeated exposure to predator odor as a traumatic stress significantly increased later ethanol drinking in a proportion of PS-sensitive male and female mice. The heterogeneity of responses is consistent with results from studies utilizing other predator odors as models of PTSD in rodents. Our initial characterization of “sensitivity” and “resilience” to PS-enhanced drinking revealed that “sensitivity” was associated with a blunted CORT response to PS in both males and females, but also with sex-dependent differences in proteins related to stress-regulating systems in mPFC and hippocampus. Despite large differences in sample size between males and females in the full dataset , similar results in drinking measures and CORT responses were obtained with the dataset of animals utilized for the Western blot study when group size was similar in males and females. Future studies will use this model to examine sex differences in biological and molecular mechanisms that confer susceptibility for increased drinking following traumatic stress so that the information can guide the development of new interventions for the treatment of PTSD-induced AUD, which likely differ in males and females.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Institutional Animal Care and Use Committee at VA Portland Health Care System.DF, AR, and LD contributed to conception and study design. DF, MH, and MN conducted the drinking studies. MH and MN dissected prefrontal cortex and hippocampal tissue. MA and LD isolated protein and prepared samples for Western Blot analysis. MA conducted the Western Blots and MA and LD quantified the protein levels. MH conducted preliminary analysis of drinking data and DF, MH, and MN identified animals for the Western Blot analysis. DF, MN, MH, and AR participated in final analysis of drinking data. MH, DF, and AR contributed to analysis of the Western Blot data. LD and AR assisted in the interpretation of the Western Blot analyses. DF and AR wrote the first drafts of the manuscript, but MA wrote sections of the Methods and Results. All authors contributed, read, and approved the final version of manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The CON rested for 30 min while the EXP performed the downhill running. Before, 10 min, 24 h, and 48 h after the interventions, dependent variables  were measured. Knee extensor muscle soreness was significantly greater in the EXP than the CON group following the intervention throughout the entire 48 h. This was accompanied by the greater decline in the knee extension strength in the EXP than the CON . The elbow flexion strength, VA, and EMG amplitude were not affected by the exercise. However, the decline of the elbow flexion resting twitch was greater in the EXP than the CON . Therefore, the downhill running impaired the remote elbow flexor muscles at a peripheral level.The purpose of this study was to examine the effects of a 1-h downhill running exercise on the elbow flexor muscles’ neuromuscular functions. Seventeen adults (Control [CON]:  Different from running on a flat surface, running downhill imposes unique biomechanical demands to some lower limb muscles, such as knee extensors, ankle flexors, and hip extensors.,Skeletal muscle damage is referred to as a state of prolonged impaired muscle functions resulting from physical damage to muscle tissues.,,In addition to influencing the intervened muscle groups, high-intensity exercise interventions can also affect remote non-exercised or less active muscle groups. These phenomena have been reported, and they were generally described as the term non-local muscle fatigue.Even though non-local effect research has been emerging over the last decade, very few non-local research studies have used eccentric contractions as the exercise intervention, which can induce prolonged neuromuscular functions and performance declines. Recently, evidence has shown that high intensity unilateral eccentric knee extension exercise can contribute to the reduced electromyographic (EMG) amplitude and physical work capacity of the non-exercised contralateral muscle.Therefore, in this study, we utilized the 1-h downhill running protocol to induce lower limb muscle damage, and the main purpose was to determine if this damaging protocol would cause prolonged declines in the remote heterologous upper limb muscles (elbow flexors) neuromuscular functions and performance. In addition, surface EMG was used to examine the muscle excitation levels of the biceps brachii when performing submaximal contractions , as examined in Hedayatpour et al.To examine whether a 1-h downhill running exercise would cause prolonged declines in the neuromuscular parameters  of the non-local upper limb elbow flexor muscles, a between-group  repeated-measures design was utilized . Subjectn = 8, Age = 20.5 ± 1.4 years, Height = 175.1 ± 6.2 cm, Weight = 67.6 ± 3.2 kg; CON group: n = 9, Age = 21.0 ± 1.1 years, Height = 180.6 ± 6.3 cm, Weight = 78.2 ± 10.9 kg). On average, the subjects engaged in aerobic exercises (mostly jogging and running) 4 h per week prior to the study. None of the subjects had previous experience with downhill running. Before participation, written consent was obtained from each subject via the consent form. All subjects also completed a pre-exercise questionnaire, which indicated that they had no current or previous neuromuscular and musculoskeletal disorders, and they were healthy to engage in exercise. In addition, all subjects were instructed to refrain from any exercises or running activities during the entire study period and to maintain all their normal routines, such as dietary intake, hydration, and sleep for the duration of their participation. This study was approved by the University of Mississippi Institutional Review Board . We estimated the sample size based on the data from a previous study with a similar design,f = 0.40, the alpha level of 0.05, and power (1−β) of 0.80 24 by G∗Power .A total of 17 healthy and physically-active adults  participated in and completed this study (EXP group: The first visit to the laboratory was to familiarize the subjects with the testing procedures and the downhill running exercise (only EXP group). During this visit, the subjects' standing height and body mass were measured first, followed by the familiarization to the Visual Analog Scale (VAS) muscle soreness measurement. Then the subjects were familiarized with the elbow flexion isometric strength testing. Specifically, they practiced contracting their dominant elbow flexor muscles against an immovable apparatus a severalmax = 220 - age. Then the subjects ran with an HR monitor  for a duration of 5–10 min on a treadmill set at 0% grade at a pace corresponding to 85% of their HRmax. After the pace was determined, the subjects were told to take a rest, during which the treadmill was adjusted to the downhill setup at −10% grade. The subjects were instructed to run at the determined pace for a few minutes to ensure they would be comfortable with this exercise. Once the familiarization was completed, the subjects were asked to return to the laboratory for the remaining experimental visits for any three consecutive days at roughly the same time each visit.After the stimulation amplitude determination, the subject was familiarized with the submaximal isometric contractions at the 50% MVIC. Once completed, the subjects were asked to rest as the researcher set up the leg extension machine  for the familiarization of the dominant knee extension isometric strength testing. This familiarization followed a similar protocol as used in the elbow flexion isometric strength testing . Finally, as for subjects in the EXP group, they were familiarized with the downhill running protocol. Specifically, the subjects’ maximal heart rate (HR) was estimated by using the Fox formulamean ± SD = 7.7 ± 0.7 miles per hour). During the run, we did not specifically instruct the subjects how to adjust the upper and lower body gait patterns . Instead, every subject adjusted their postures to their comfortable positions throughout the whole running intervention. The Subject's HR was monitored every 5 min throughout the entire downhill running protocol , the subjects returned to the laboratory where Pre-testing dependent variables were measured in the following order: knee extensor muscle soreness, elbow flexion isometric strength with twitch interpolation, surface EMG amplitude during the submaximal elbow flexion isometric contractions, and knee extensor isometric strength. If assigned to the EXP group, following the Pre-tests, the subjects were allowed to warm up for approximately 5 min with the running speed they preferred, before they attempted to complete the 1-h downhill running exercise with the pre-determined pace .For the elbow flexion isometric strength testing, the subjects were seated with an upright position, with the dominant arm resting on a padded table, where the pad was firmly pushing against the subjects' armpit. Meanwhile, the subject's dominant wrist was attached by a strap, which was connected to a load cell . The other end of the load cell was connected to an immovable steel frame. Extra care was taken to ensure the subjects' arm was parallel to the floor, and the elbow joint was at a 90-degree angle a. FollowTo monitor neuromuscular efficiency throughout the entire experiment, the submaximal isometric contraction testing was performed with the same setup as the elbow flexion isometric strength testing. The subjects contracted their elbow flexor muscle with a “ramp-up, hold, and ramp-down”  manner. A computer monitor was provided to display the target force template, as well as the subject's real-time force output. Specifically, the subjects gradually increased the force output from 0% (rest) to 50% of their Pre-MVIC for 5 s (10% MVIC per second), held it for 10 s, and then gradually decreased it to 0% MVIC for 5 s. Two repetitions of the trapezoid contraction were performed at each measurement point. In addition, the force template (50% of the Pre-MVIC from Visit 2) was always constant throughout all experimental visits. Therefore, if there were any fatiguing effect on the elbow flexor muscles due to the downhill running, then an increased normalized EMG amplitude should be seen.For the knee extension isometric strength testing, the subjects performed three trials, 3-s MVICs with a 60-s rest between trials. The same load cell  was used to measure the isometric force generated by the knee extensor muscles. Specifically, one end of the load cell was connected to the lever of the ankle pad, and the other end was connected to the backside of the knee extension machine through a steel chain b. Prior After the elbow flexion and knee extension isometric strength testing, the offline force signals were digitized with a 16-bit analog-to-digital converter , sampled at 20 kHz with a 16-channel Bagnoli desktop EMG system , and then stored in a laboratory computer  for further analyses. For each 3-s MVIC, the peak 0.5 s window (rather the superimposed twitch area) was chosen and then calculated as the maximal force output. The average of the three maximal force outputs was then calculated as the maximal isometric strength.During the elbow flexion isometric strength testing, the superimposed twitch force and resting twitch force were first selected and calculated for each MVIC c–e. The During the elbow flexion maximal isometric contractions, as well as the submaximal trapezoid contractions, bipolar surface EMG signals were recorded through an EMG sensor  attached to the biceps brachii muscle belly based on the sensor location recommendations from SENIAM.t-tests between groups [CON vs. EXP]. The absolute change values  of the VAS, VA, and the normalized EMG amplitude during the submaximal isometric trapezoid contractions, along with percent change values  of the elbow flexion and knee extension isometric strength, and elbow flexion resting twitch force, were calculated for further statistical analyses. Separate two-way (time [ΔPost-Pre vs. ΔPost24-Pre vs. ΔPost48-Pre] × group [CON vs. EXP]) mixed factorial analyses of variance (ANOVAs) tests were conducted to examine the change scores of all dependent variables across time between groups. When appropriate, the follow-up tests included one-way repeated-measures ANOVA with Bonferroni-adjusted pairwise comparisons, as well as independent t-tests. The partial mean ± standard deviation (SD).Assumptions for normality of distribution for these dependent variables were checked and confirmed using the Shapiro-Wilk test. The baseline  dependent variables were examined via the independent F = 5.873, p = 0.007, partial F = 101.058, p < 0.001, partial F = 4.814, p = 0.026, partial F = 0.672, p = 0.439, partial mean ± SD: 44.3 ± 19.3 mm vs. 63.4 ± 17.5 mm, p = 0.002). In addition, the independent t-tests showed significant differences between the two groups at all time points  = 2.153, p = 0.134, partial F = 2.929, p = 0.044, partial F = 9.011, p = 0.001, partial t-test showed a significant difference between the two groups . With both groups merged, significantly greater percent declines of the knee extension isometric strength were observed at Δ(Post-Pre) than that at Δ(Post24-Pre) , and at Δ(Post-Pre) than that at Δ(Post48-Pre) .For the knee extension isometric strength, the two-way ANOVA showed that there was no significant group × time  = 0.317, p = 0.731, partial F = 0.236, p = 0.634, partial F = 5.423, p = 0.010, partial mean ± SD: -7.0 ± 2.1% vs. −1.8 ± 1.9%, p = 0.003), and at Δ(Post-Pre) than that at Δ(Post48-Pre) .The two-way ANOVA for the elbow flexion isometric strength showed that there was no significant group × time  = 1.113, p = 0.342, partial F = 10.760, p = 0.005, partial F = 5.646, p = 0.008, partial t-test showed a significant difference between the EXP and CON groups , and at Δ(Post-Pre) than that at Δ(Post48-Pre) .For the elbow flexion resting twitch force, the two-way ANOVA showed that there was no significant group × time  = 1.212, p = 0.320, partial F = 1.394, p = 0.268, partial F = 1.214, p = 0.320, partial The two-way ANOVA for the elbow flexor muscle VA showed that there was no significant group × time  = 4.239, p = 0.024, partial F = 3.176, p = 0.073, partial F = 1.278, p = 0.305, partial t-tests showed no significant differences between the two groups at all time points.The two-way ANOVA for the normalized EMG amplitude during the submaximal isometric contraction indicated a significant group × time  of the remote upper limb muscles such as the elbow flexors. The main findings of this study are: 1) Changes in the elbow flexion isometric strength, VA, and biceps brachii muscle excitation levels were not different between the EXP and CON; 2) The reduction in the elbow flexor muscle resting twitch was significantly greater after the downhill running exercise, relative to the CON; 3) The downhill running exercise-induced knee extensor muscle soreness were greater and remained elevated at 24 and 48 h, when compared to CON; and 4) The overall percent decline of the knee extension isometric strength following the downhill running exercise was significantly greater than that of the CON.Lower body muscle DOMS has often been assessed following downhill running exercise interventions.2peak  in the current experiment. In fact, when looking into the current result of the knee extension isometric strength percent decline immediately after the downhill run, our value (−18%) is close to the upper ceiling of the range (−55% to −14%) from the downhill running literature.With the positive indirect muscle damage markers observed from the knee extensor muscles following the current downhill running protocol, it is our main purpose to examine whether the elbow flexor muscles' neuromuscular functions and performance were altered. To the best of our knowledge, Brandenberger et al.,It is interesting that the prolonged depressions in motor performance were observed in non-exercised and less-exercised muscle groups.The current study did have an interesting finding: the decrements of the elbow flexor muscle twitch force were significantly greater after the downhill running, as compared to those of the CON group. Since the same electrical stimulation was delivered to the biceps brachii muscle belly after the exercise protocol, a decline in the resting twitch indicates the decreased capability of the muscle fibers to generate force. Thus, this finding suggests that our downhill running protocol induced some peripheral impairments at the muscle fiber level in the remote biceps brachii muscle. It was not our intention to examine the exact mechanism(s) leading to this change, but one possibility could be the unaccustomed nature of the downhill running exercise. Specifically, with the altered posture during downhill running, the lumbar spine range of motion has been reported to be greater than that when running on level or uphill.One question remains to be unanswered, is why the different responses of the elbow flexor isometric strength and the biceps brachii muscle resting twitch force were observed, following the downhill running protocol. Our result showed that the downhill running protocol impaired the biceps brachii muscle at the peripheral level. However, it is important to note that other synergistic muscles  and antagonist muscle  that contribute to the elbow flexion force production were not examined in the current experiment. It is unknown if these muscles would have responded similarly to the biceps brachii muscle. Thus, it is possible that the non-local elbow flexor isometric strength responded differently from the biceps brachii muscle resting twitch force. Regarding the biceps brachii surface EMG (to measure neuromuscular efficiency), even though a significant interaction was found for the surface EMG amplitude during the submaximal contractions, we did not observe any further differences between the EXP and CON. However, it is important to point out, that there was a large effect size , which could have influenced the results, as previous studies have reported that the magnitude of muscle damage induced by eccentric exercise differs between men and women.peak2 method, might have provided a less accurate exercise intervention to induce muscle damage. Fifth, we had a fixed order of the testing measurements, which could have led to a potential order effect. Lastly, measurements such as elbow flexor muscle soreness and knee extensor VA, as well as other muscle damage blood markers such as creatine kinase were not conducted, which could potentially add a few insights to the findings of the current experiment.The current study showed some interesting findings regarding the non-local effects following a prolonged downhill running exercise. However, it is important to address some limitations of this study. First and foremost, even though the sample size of the current study was greater than the required estimation number, seventeen  is still a relatively small sample size, specifically under a between-group research design. Second, our study contained a mix of both men and women  of the upper elbow flexor muscles were not altered. These findings suggest that, even though the upper limb muscles are less active during the prolonged downhill run, peripheral changes in these remote muscles can still be observed. Given that the subjects in the current study may slightly be more trained and active than average individuals, future studies should try to explore whether peripheral changes in the remote muscle groups would be magnified, if using less active or sedentary populations.The work described above has not been published previously, that it is not under consideration for publication elsewhere, that its publication is approved by all authors and tacitly or explicitly by the responsible authorities where the work was carried out, and that, if accepted, it will not be published elsewhere including electronically in the same form, in English or in any other language, without the written consent of the copyright-holder.XY and RJB contributed to the design of the work; XY, RJB, WMM, SJ, and JSS contributed to the acquisition, analysis, and interpretation of data; XY, RJB, WMM, SJ, and JSS contributed to the draft of the work and revised it critically for important intellectual content; XY, RJB, WMM, SJ, and JSS approved the version to be submitted, and agreed to be accountable for all aspects of the work in ensuring that the questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.All experimental procedures in this project were in accordance with the ethical standards of 1964 Helsinki declaration and its later amendments or comparable ethical standards and were approved by the University of Mississippi Institutional Review Board (IRB Protocol #: 19-106). After each participant was given an explanation of the study, written informed consent was collected.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. There were no conflicts of interest declared from authors for the completion of this project and manuscript.


Liquidambar formosana Hance has a highly ornamental value as an important urban greening tree species with bright and beautiful leaf color. To gain insights into the physiological and molecular mechanisms of L. formosana leaf color change, the leaves of three different clones were sampled every ten days from October 13, 2019, five times in total, which are S1, S2, S3, S4 and S5. Transcriptome sequencing was performed at S1 and S4. The chlorophyll content of the three clones decreased significantly, while the anthocyanins content of the three clones increased significantly in the coloring stage. The anthocyanins content of clone 2 was far more than that of the other two clones throughout the period of leaf color change. The transcriptome analysis showed that six DEGs related to anthocyanins biosynthesis, including CHS , CHI , F3′H (flavonoid 3′-hydroxylase), DFR (dihydroflavonol 4-reductase), ANS (anthocyanidin synthase) and FLS (flavonol synthase), were found in three clones. Clone 2 has another three DEGs related to anthocyanins biosynthesis, including PAL , F3′5′H  and UFGT (flavonoid 3-O-glucosyltransferase). We lay a foundation for understanding the molecular regulation mechanism of the formation of leaf color by exploring valuable genes, which is helpful for L. formosana breeding. Liquidambar formosana Hance, a deciduous ornamental tree species, is widely distributed in Southeast Asia [L. formosana has great potential in landscaping and gardening, owing to the fact that its leaf color changes seasonally from green to yellow or red in October [L. formosana is of great importance for breeding individual clones with good ornamental values.ast Asia ,2. This ast Asia . L. form October ; therefoArabidopsis thaliana, the structural enzymes in the anthocyanins biosynthetic pathway include the upstream components chalcone synthase (CHS), chalcone isomerase (CHI) and the downstream components dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS) and others [L. formosana. Many studies on L. formosana have explored the relationship between leaf color change and environmental factors by measuring the content changes of chlorophyll and other related pigments. Studies of Hu and Liu have pointed out that temperature is the main factor affecting the accumulation of pigment in L. formosana leaves [L. formosana.Autumn leaf coloration is one of the most important horticultural characteristics in nature . Variousd others . The strd others ,16. To da leaves ,18. ThisL. formosana development requires the exploration of functional genes. RNA sequencing, based on deep sequencing, has been widely used for gene discovery and analysis of specific genes [L. formosana in autumn, but also aimed to explore the molecular mechanism of its color formation, thereby providing a theoretical basis for L. formosana molecular breeding.Understanding of the metabolic pathways involved in leaf color change during ic genes . The resThe total chlorophyll, carotenoid and anthocyanins in leaves varied significantly at different coloration stages . With inL. formosana were selected, consisting of three clones with three replicates per clone. A total of 936,385,220 raw reads with a total of 141,394,168,220 bp were obtained. After removing the adaptor and low-quality reads, a total of 927,523,280 clean reads with a total of 136,254,863,719 bp were obtained from the eighteen sequencing libraries for further analysis  in at least one of the GO, COG and KEGG databases. In total, there were 82,229 (41.62%), 88,492 (44.79%) and 55,770 (28.23%) unigenes that had annotated to the GO, COG and KEGG databases, respectively.In total, 196,890 unigenes  were identified by BLASTx  in “Function unknown”, followed by “Translation, ribosomal structure and biogenesis”  and “Posttranslational modification, protein turnover, chaperones”  .A total of 55,770 unigenes were assigned to six KEGG categories and 20 sub-categories . “MetaboTo analyze the dynamic expression patterns of the specific genes in leaf color change, the differences in transcriptome profiles between the S1 and S4 stages of different clones were compared. The Transcripts Per Million (TPM) values were statistically analyzed to select different unigenes by using the DESeq method  Table S.We compared the DEGs of the three clones (S1-vs-S4). In clone 1, we identified 20,020 DEGs with 16,005 upregulated and 4015 downregulated a,b. In cIn this study, we carried out an enrichment analysis based on the KEGG database in order to explore the biological functions of these DEGs. A total of 8988 unigenes were assigned to 134 KEGG pathways in clone 1 between S1 and S4 stages. Similarly, a total of 7647 unigenes were assigned to 122 KEGG pathways in the clone 2 (S1-VS-S4) and a total of 6341 unigenes were assigned to 135 KEGG pathways in the clone 3 (S1-VS-S4).Most of the pathways annotated by the three clones were related to “Metabolism”. In the pathways related to leaf color change, the three clones were significantly enriched in “Phenylpropanoid biosynthesis”, “Flavonoids biosynthesis”, “Isoflavonoid biosynthesis”, “Anthocyanin biosynthesis”, “Phenylalanine metabolism” and other pathways .TRINITY_DN11132_c0_g1, TRINITY_DN4277_c0_g1, TRINITY_DN18660_c0_g2, TRINITY_DN11660_c0_g1, TRINITY_DN29005_c0_g1; TRINITY_DN17255_c0_g3 were annotated as CHS, CHI, F3′H, DFR, ANS and FLS, respectively. Another three genes, including PAL(TRINITY_DN17802_c0_g4), F3′5′H(TRINITY_DN28662_c0_g1) and UFGT(TRINITY_DN3115_c0_g1), were significantly upregulated in clone 2  is an enzyme that catalyzes the first step of the phenylpropanoid metabolic pathway [PAL catalyzed the synthesis of anthocyanin [PAL catalyzes the conversion of phenylalanine to cinnamic acid, while C4H catalyzes the conversion of cinnamic acid to 4-coumaric acid. The conversion of 4-coumaric acid to 4-coumaroyl-CoA is catalyzed by 4CL [PAL activity [PAL (TRINITY_DN17802_c0_g4) gene in clone 2 was significantly upregulated at S4 stage, and the expression pattern was confirmed in the qRT-PCR analyses; however, we did not find any DEGs annotated as C4H and 4CL, which encode the enzymes required for the production of 4-coumaroyl-CoA.Phenylalanine ammonia-lyase , a polyketide synthase, mediating the synthesis of naringenin chalcone from 4-coumaroyl-CoA and malonyl-CoA [CHI) to naringenin, the direct precursor of all flavonoid substances [CHS is an important regulatory gene located upstream in the flavonoid biosynthesis pathway, and its overexpression may positively affect the expression of downstream chalcone isomerase (CHI) genes that affect the production of flavonoids [CHS (TRINITY_DN11132_c0_g1) and CHI (TRINITY_DN4277_c0_g1) gene showed significantly higher expression levels in three clones at the S4 stage. This result indicated that CHS does positively regulate CHI expression during the leaf color change of L. formosana. At the same time, we also found a significantly upregulated flavonol synthase (FLS) gene (TRINITY_DN17255_c0_g3) in three clones, which encodes the enzyme that catalyzes naringenin chalcone to flavonols [F3H), which belongs to the OGD family, converts naringenin into dihydrokaempferol that can be further hydroxylated by flavonoid 3′-hydroxylase (F3′H) or flavonoid 3′,5′-hydroxylase (F3′5′H) into two other dihydroflavonols, dihydroquercitin or dihydromyricetin, respectively [F3′H and F3′5′H are the key enzymes determining the structures of anthocyanins, and therefore, they affect color formation [F3H; however, the DEG TRINITY_DN18660_c0_g2, which was annotated as F3′H, showed a significantly higher expression level at the S4 stage than at the S1 stage in three clones. Another upregulated F3′5′H gene (TRINITY_DN28662_c0_g1) was screened from clone 2. This result was similar to a study that determined the pathway by which red longan (Dimocarpus longan) fruits were produced. Yi et al. revealed that genes related to enzymes leading up to dihydromyricetin were significantly upregulated in red pericarp longan fruits [L. formosana and may also be responsible for the bright red color of clone 2 in autumn.The first committed enzyme in the flavonoid pathway is Chalcone synthase . Anthocyanidin synthase (ANS), which belongs to the OGD family, catalyzes the synthesis of corresponding colored anthocyanidins [ANS gene mutations could cause gentian flowers to turn white [DFR (TRINITY_DN11660_c0_g1) and ANS (TRINITY_DN29005_c0_g1) genes were excavated, and their expression levels increased significantly in all three clones at the S4 stage. In the end, anthocyanidins are decorated and glycosylated by various members of the glycosyltransferase enzyme family, for instance, flavonoid 3-O-glucosyltransferase (UFGT) [UFGT (TRINITY_DN3115_c0_g1) in clone 2 was higher at the S4 stage than at the S1 stage. This result indicated that the biosynthesis of anthocyanin compounds is maintained at high levels in clone 2 at the S4 stage. The higher expression levels of PAL, CHS, CHI, F3′H, F3′5′H, DFR, ANS, UFGT and FLS in red leaf than in green leaf of L. formosana suggested that these genes are responsible for leaf color formation.Next, the three dihydroflavonols are reduced to colorless leucoanthocyanidins by dihydroflavonol 4-reductase . In mid-October, 2019, three clones with the most representative coloration effects were selected for sampling. Beginning October 13th, fresh leaves were collected approximately every ten days. The leaves were immediately frozen in liquid nitrogen and stored at −80 °C until use. The five developmental stages were defined according to the time of collection: S1, the green leaf stage; S2, leaf with red margin stage; S3, leaf with red range-expanding stage; S4, the red leaf stage; S5, the red faded stage .Chlorophyll and carotenoid content of leaves from three clones were measured on the basis of the procedure described by Lichtenthaler and Wellburn . ApproxiThe content of anthocyanins was determined by the hydrochloric acid-ethanol extraction method ,42. In tL. formosana leaves at two stages (S1 and S4) were selected as materials. The leaves of S1 are the green ones before coloration, and the leaves of S4 are the red ones after coloration. There were three clones per stage, and each clone had three biological replicates, resulting in a total of 18 samples. Total RNA was extracted using the Plant RNA Kit  according to the manufacturer’s instructions. The quantity and quality of total RNA were assessed using a 1% agarose gel and a Nanodrop ND 2000 spectrophotometer . Total RNA integrity and concentration were assessed using the Bioanalyzer 2100 RNA 6000 Nano Kit .The Poly(A) mRNA was isolated from total RNA using the Oligotex mRNA Mini Kit  according to the manufacturer’s instructions. The cDNA library was built using methods previously described by Niu et al. . The 18 http://www.geneontology.org, accessed on 3 April 2022), COG and KEGG.Assembled unigenes were aligned to publicly available protein databases, including GO and the DEGs between different stages were identified with padj < 0.05 and |log2 | ≥ 1  Next, GOEight key genes involved in anthocyanin biosynthesis were selected for validation by quantitative real-time PCR (QRT-PCR). The primers were designed by Primer Premier 5.0  and the reference gene was 18S ribosomal RNA . All expt method . The expp-values less than 0.05 were considered to indicate significance between groups. For the elaboration of graphs, Excel 2019  was used.The data analysis included a basic descriptive analysis followed by an analysis of variance (ANOVA). Significant differences were based on Duncan’s test, which were performed using SPSS 23.0 for Windows . The L. formosana was firstly carried out by physiology and RNA-seq. It was found that with increased redness in leaf color, the total chlorophyll levels decreased, while anthocyanin levels increased. The anthocyanins content of clone 2 was far more than that of the other two clones throughout the color-changing period. Six genes, including CHS, CHI, F3′H, DFR, ANS and FLS, play an important role in the anthocyanin’s biosynthesis pathway in three clones. Another three genes, including PAL, F3′5′H and UFGT, were only significantly expressed in clone 2, indicating that there were more DEGs related to anthocyanin biosynthesis in clone 2. Our study will provide molecular information for the selection and breeding of new species of colored-leaf species and provide a reference for the future study of leaf color polymorphisms in L. formosana.Overall, the regulation mechanism of leaf color in