Background: Genetic risk scores (GRSs) have partially improved the understanding of the etiology of moderate hypertriglyceridemia (HTG), which until recently was mainly assessed by secondary predisposing causes. The main objective of this study was to assess whether this variability is due to the interaction between clinical variables and GRS. Methods: We analyzed 276 patients with suspected polygenic HTG. An unweighted GRS was developed with the following variants: c.724C > G (ZPR1 gene), c.56C > G (APOA5 gene), c.1337T > C (GCKR gene), g.19986711A > G (LPL gene), c.107 + 1647T > C (BAZ1B gene) and g.125478730A > T (TRIB gene). Interactions between the GRS and clinical variables (body mass index (BMI), diabetes mellitus, diet, physical activity, alcohol consumption, age and gender) were evaluated. Results: The GRS was associated with triglyceride (TG) concentrations. There was a significant interaction between BMI and GRS, with the intensity of the relationship between the number of alleles and the TG concentration being greater in individuals with a higher BMI. Conclusions: GRS is associated with plasma TG concentrations and is markedly influenced by BMI. This finding could improve the stratification of patients with a high genetic risk for HTG who could benefit from more intensive healthcare interventions.  Hypertriglyceridemia (HTG) is a frequent disorder in the general population and is related to the risk of cardiovascular disease and pancreatitis . MultiplNonetheless, the accumulation of these allelic variants can only explain 10–20% of the susceptibility to these disorders, and most of the genetic susceptibility in HTG patients remains to be established. Other factors such as epigenetics, gene–gene and gene–environment ,12 may cAn unbalanced diet, sedentary lifestyle, obesity, metabolic syndrome and type II diabetes are secondary causes of HTG ,14, and Interactions between genetic risk factors and clinical phenotypes may account for some of the unexplained heritability of plasma lipid traits. In the present study, we calculated one GRS based on single nucleotide polymorphisms (SNPs) related to TG  and clinical variables including diet, physical activity and body mass index (BMI) in a group of patients followed in our hospital. We studied the influence of clinical variables and the relationship between each GRS and the blood lipid profile based on the hypothesis that these clinical variables modify the polygenic risk associated with high TG levels.2, and 61.2% were current or former smokers, 34.1% had hypertension and 17.8% had DM. The mean score on the diet questionnaire was 7.5. The mean of the highest TG levels recorded from the clinical records of the patients was 7.84 : 7.0 to 8.6) mmol/L. The mean TC, non-HDL-c and HDL-c values corresponding to the sample with the highest TG concentrations were 6.71 (95% CI 6.5 to 6.9) mmol/L, 5.74 (95% CI 5.5 to 6.0) mmol/L and 0.98 (95% CI 0.94 to 1.01) mmol/L, respectively.Patients with DM exhibited good metabolic control as they presented a mean glycated hemoglobin (HbA1c) of 6.8% (SD of 0.7), DM  of the variability in TG concentrations (results not shown).The GRS explained 7%  implies a greater increase in TG levels. For genetic loads of greater than six SNPs in the GRS, a higher increase in TG concentrations was observed for each increment of one allele compared with those observed for genetic loads of less than six SNPs, which were almost zero. These results are similar to those already shown in that the association between GRS and TG levels was statistically stronger ,21 when Visceral obesity, overweight and global obesity ,23, as wIn this study, BMI status significantly influenced the effect of these genetic variants on plasma TG concentrations. Thus, obesity or overweight amplified the effect of GRS by further increasing TG levels, while normal weight attenuated this effect. In this regard, similar results have been described in studies evaluating the effect of abdominal obesity ,24,25 orPolygenic HTG is the result of an imbalance between TG production (with fatty acids from the diet) and TG clearance (affected by the accumulation of SNPs) . In thisThe interaction between BMI and GRS can be explained by different factors. These genes encode proteins that alter both hepatic TG synthesis and peripheral lipolysis, and obesity is not only related to a greater contribution of fatty acids to the liver but also to impaired clearance of TG-rich lipoprotein particles. GCKR gene product [ZPR1 gene corresponds to an intergenic zone located near the APOA5-A4-C3 gene cluster and was related to the development of clinical HTG by decreasing apolipoprotein A5 (apoA5) concentrations [APOA5 [ZPR1 and APOA5 genetic variants are related to a decrease in the lipolytic function of lipoprotein lipase. LPL encodes lipoprotein lipase, a major determinant of peripheral lipolysis of TG-rich lipoproteins. The  product , glucokitrations . The APOs [APOA5 ,29 gene s [APOA5 . Both ZPIn obese individuals, overnutrition is related to a greater contribution of fatty acids (FA) to the liver , a fundaOur results suggest that reducing BMI may also lessen the effects of the GRS on TG levels. Other studies reinforce this concept by finding that lifestyle interventions appeared to partially mitigate the effect of genetic contribution ,35. FurtSome studies carried out in children reported that the GRS was a good predictor of HTG in adulthood . Thus, tSome studies suggest that older people respond less efficiently to lifestyle interventions , thus faSome limitations of our study merit consideration. First, our sample size was relatively small, which might have led to a type 2 error; however, the patients were duly selected by medical specialists. Second, during our literature search, we found differing numbers of candidate SNPs to create the GRS, and, finally, we selected those that showed the greatest effect on plasma TG concentrations and risk of HTG in a GWAS. However, the population presented a high prevalence of some of these SNPs, making it difficult to assess their effect on TG levels.n = 276) with primary HTG, aged 18 to 80 years, visited in the Lipid and Vascular Risk Unit of our tertiary hospital were included in this study.Unrelated patients  or hypothyroidism (with thyroid-stimulating hormone >8 mU/L); alcohol abuse (defined as intake >40 g—4 standard drink units (SDUs)—a day in males and >20 g—2 SDUs—in females) ; and treHbA1c >10% was chosen as this is the value at which insulinopenia should be considered.2 (kg/m2). Data about hypertension, diabetes mellitus (DM) and hypercholesterolemia were also registered.All patients provided information regarding their age, sex, profession, drug therapy, smoking habit and alcohol consumption. Cigarette smoking was categorized as “none”, “past” or “current” and alcohol consumption as the number of SDUs per week. Diet assessment was performed using a validated and standardized diet questionnaire , and phyAll blood tests were performed after eight hours of fasting, in outpatients a few days before each of the follow-up visits in our Vascular Risk Unit. During the follow-up visits, active diseases were ruled out. Fasting lipid profile results corresponding to the highest plasma TG concentrations and the respective drug treatments were recorded for each patient. The results of plasma TG analysis were collected from the clinical records of the patients via the institutional electronic information system. Fasting lipid profiles included plasma concentrations of total cholesterol (TC); low-density lipoprotein cholesterol (LDL-c), calculated using the Friedewald equation only when TG concentrations were lower than 2.3 mmol/L (200 mg/dL); high-density lipoprotein cholesterol (HDL-c); non-high-density lipoprotein cholesterol (non-HDL-c); TG; apolipoprotein B; and apolipoprotein A1. Serum concentrations of HbA1c were also recorded. LDL-c was calculated using the Friedewald equation. This was only used when TG concentrations were lower than 2.3 mmol/L (200 mg/dL); when TG concentrations were higher, only non-HDL-c was calculated.A history of atherothrombotic cardiovascular disease, including coronary artery disease, cerebrovascular disease and peripheral arterial disease, was recorded through anamnesis and a review of medical records. Coronary artery disease was defined as acute myocardial infarction, stable or unstable angina, coronary artery bypass graft or percutaneous transluminal coronary angiography with evidence of significant atherosclerotic disease. Cerebrovascular disease included both past stroke and a transitory ischemic attack, and peripheral arterial disease was defined as the presence of a pathological ankle–brachial index (<1), or clinical manifestations of intermittent claudication.A history of pancreatitis was also recorded from the clinical records of the patients via the institutional electronic information system, or through self-reports when the episode occurred in other hospitals.®). HbA1c was measured in whole blood using the HA-AutoA1C 8180 autoanalyzer from Menarini® .All biochemical analyses were performed in plasma using a COBAS 800 automated analyzer  to form a red quinonimine dye.LDL-c: LDL-c was estimated using the Friedewald equation if TG concentrations were below 2.3 mmol/L. If not, non-HDL-c was calculated instead.TG: Reagent: TRIGL. COBAS 501/502 for COBAS 8000. The present method is based on the work of Wahlefeld and uses a lipoprotein lipase derived from microorganisms to rapidly and completely hydrolyze TG to glycerol, with subsequent oxidation to dihydroxyacetonephosphate and hydrogen peroxide. The resulting hydrogen peroxide reacts under the catalytic action of peroxidase with 4-aminophenazone and 4-chlorophenol to form a red dye in a Trinder endpoint reaction. The color intensity of this red dye is directly proportional to the concentration of TG and can be measured photometrically.HDL-c: Reagent: HDLC4 COBAS 501/502 for COBAS 8000. Test principle: homogeneous enzymatic colorimetric test. Non-HDL lipoproteins including LDL, VLDL and chylomicrons combine with polyanions and detergent to form a complex water soluble. In this complex, the enzymatic reaction of CHE and CHOD versus non-HDL lipoproteins occurs so that only the HDL particles can react with CHER and CHOD. The concentration of HDL-c is determined enzymatically by CHER and CHOD. Cholesterol esters are quantitatively degraded to free cholesterol and fatty acids by CHER.ZPR1 gene rs964184 (NM_003904.4:c.*724C>G), APOA5 gene rs3135506 (NM_001166598.1:c.56G>C), GCKR gene rs1260326 (NM_001486.3:c.1337T>C), LPL gene rs12678919 (NC_000008.11:g.19986711A>G), BAZ1B gene rs7811265 (NM_032408.3: c.107+1647T>C) and TRIB gene rs2954029 (NC_000008.11: g.125478730A>T).The following allelic variants were selected according to the greatest effect on plasma TG concentrations and the risk of HTG shown in a GWAS : ZPR1 geThe characteristics of these genetic variants are shown in ® RSC Instruments, Promega, Madison, WI, USA) and stored at −80 °C.DNA was extracted from peripheral blood using an automated DNA purification system  in 96-well plates including positive and negative controls. Real-time polymerase chain reaction (PCR) was carried out with the 7500 Real-time PCR System, Applied Biosystems , following standard recommendations. Briefly, 1 µL of Assay Mix was mixed with 10 µL Supermix SsoAdvanced , 2 µL genomic DNA (20 ng/µL) and purified water up to 20 µL. The resulting mixture was heated to 50 °C for 2 min and 95 °C for 10 min in the thermal cycler. This was then followed by 40 cycles of denaturing at 95 °C for 15 s and annealing/extending at 60 °C for 1 min.Genotyping was carried out using the TaqMan SNP Genotyping Assay  and “2” if two alleles were present (homozygous). As six allelic variants were selected, the score could range between 0 and 12.Qualitative variables were described as absolute frequencies, quantitative variables were described as means with standard deviations (SDs) and non-normal variables were described as means and interquartile ranges.A GRS was created by adding the number of mutated alleles present in each individual for each of the six allelic variants; for the statistical analysis, the three individuals with scores of 11 were grouped with individuals with scores of 10. The study population was divided into two groups depending on whether they presented GRS > 6 or GRS < 6. Both groups were comparable.A bivariate analysis was carried out to evaluate the clinical variables related to the highest TG concentrations: qualitative variables were compared by analysis of variance (ANOVA) and quantitative variables with the Pearson correlation test (data not shown). Variables that were statistically significant together with age and sex  were considered to be included in the multivariable regression model. Interactions between GRS and different clinical variables  were evaluated; only the interaction between BMI and GRS was significant. Finally, a multiple linear regression model with GRS and BMI (their interaction), including DM, diet, age and gender, was created. Using a backward stepwise procedure, the variables age and sex were excluded, as they did not contribute significant information to the model.There was a positive association between the GRS and TG concentrations, and these scores may be useful for predicting HTG, even before it appears as a phenotypic trait. The main finding of our study is that the polygenetic contribution to HTG was significantly modified by BMI, with a higher BMI being associated with a stronger influence of the GRS on TG concentrations. This finding could improve the stratification of patients with a high genetic risk for HTG, who could benefit from more intensive healthcare interventions to lower their BMI even before the phenotypic trait is present.This section is not mandatory but may be added if there are patents resulting from the work reported in this manuscript.


Global lifetime prevalence of anxiety disorders has been estimated atapproximately 16.6%, with subclinical prevalence likely much higher. Herbalapproaches to reduce anxiety may be as effective as pharmacologicaltreatments and are less likely to be associated with adverse side effects.The herbal species, namely, valerian, passionflower, hawthorn and ballota,have a long history of use as anxiolytics in traditional medicine, furthersupported by recent pre-clinical and clinical trials.®) on psychological state andpsychological and physiological stress responses during a laboratorystressor.To assess the effects of chronic (14 days) supplementation with a multi-herbextract preparation  receiveda MHEP and placebo for 14 days with a 28-day washout. Anxiety (State-TraitAnxiety Inventory), mood and physiological measures of stress  weremeasured before and after an Observed Multitasking Stressor. Cognitiveperformance was also assessed.p = 0.038), with participants showing an attenuatedresponse to the observed multitasking psychosocial stressor following MHEP,evidenced by lower salivary α-amylase (p = 0.041) andgalvanic skin response (p = 0.004).MHEP was associated with reduced tension-anxiety. The global lifetime prevalence of anxiety disorders has been estimated atapproximately 16.6% , with 8.Valerianaofficinalis , Passiflora incarnata L.(passionflower) and Ballota nigra L.  have a long historyof use as anxiolytics in traditional medicine , led tosignificantly lower levels of anxiety during laboratory-induced stress in humans.Here, individuals given a 600-mg dose reported significantly lower levels of anxietythan those given placebo or a higher 1800 mg dose  is an herbal substance that has beenseen to provide similar anxiolytic properties as the commonly prescribedbenzodiazepine midazolam within dental patients, at a dose of 260 mg (Passiflora caerulea) acts asa partial agonist on benzodiazepine receptors  contains several phenylpropanoids,precursors to flavonoids, which are compounds able to bind to benzodiazepine,dopaminergic and opioid receptors in rodents, possibly explaining the neuro-sedativeproperties of the plant (Crataegussp. (hawthorn) are a species rich in polyphenols including flavonoids andprocyanidins. Hawthorn preparations are effective in the treatment of cardiovascularand ischemic heart disease, with hypotensive effects often reported (N = 36) assessing the effects of 10 weeks’administration of 500 mg hawthorn extract alone or in combination with magnesium inmildly hypertensive adults has provided initial evidence of the anxiolytic effectsof this extract. Trends for reduced blood pressure and reduced anxiety in thoseadministered the hawthorn extract were observed, both with hawthorn extract aloneand in combination with magnesium , 40 mg P. incarnate L., 10 mg Crataegus sp. (from the leaf and flower) and10 mg B. nigra L. (from the flowering tops)). Evidence has shownthat this MHEP combination is able to interact with benzodiazepine receptors, whichmay underpin the anxiolytic effects , Euphytose effects . In outpreatment .Currentreatment , thepotreatment .Therefore, the aim of the present study was to assess the effects of chronic(14 days) supplementation with a MHEP (Euphytose) on psychological state withregards to perceived stress and overall mood as well as psychological andphysiological stress responses during a laboratory stressor in a sample of healthy,sub-clinical participants.A randomised, placebo-controlled, double-blind, crossover design was utilised.Participants attended the Brain, Performance and Nutrition Research Centrelaboratory at Northumbria University and were assessed after 14 dayssupplementation with MHEP and a matched placebo. The study was performed inaccordance with the ethical principles that have their origin in the Declarationof Helsinki (1996). The trial was conducted in compliance withprotocol/GCP/applicable regulatory requirements and commenced only when afavourable ethical opinion was obtained from the University of NorthumbriaDepartment of Psychology Ethics Committee, United Kingdom, approval number13339.d = 0.56) reported in the study by The power calculation was made with reference to the medium effect size . The remaining 27 participants , aged19–58 years , self-reported being in good health andwere free from any relevant medical condition or disease including psychiatricand neurodevelopmental disorders. Blood pressure was taken at screening, andparticipants were enrolled into the study if it measured <159 mmHg systolicand <99 mmHg diastolic. Participants confirmed they were not currently takingany relevant pharmaceuticals and had not taken any antibiotics within 4 weeks ofscreening. They also confirmed they had not taken part in another clinical trialwithin 30 days and had not experienced an event likely to have impacted their emotional and/or psychological state within theweek prior to starting the study and that they did not have an event planned likely to affect their emotional, psychological orhormonal state during the course of the study. A full list of the inclusion andexclusion criteria can be found in V.officinalis L., 40 mg P. incarnate L., 10 mgCrataegus sp. and 10 mg B. nigra L.) and amatched placebo in a counterbalanced order. The full composition of the activetreatment and placebo is listed in Supplemental File 2. Treatments were delivered from themanufacturer  in boxes labelled as placeboand verum. The bottles for each treatment arm were identical. An independentthird party who had no further involvement with the trial procedures created afully counterbalanced computer-generated randomisation schedule (www.randomization.com) and assigned the treatment codes A and Bto the treatments. Bottles were labelled with a randomisation number accordingto the counterbalancing schedule by the lead researcher; randomisation numberswere issued to participants sequentially at visit 1.Participants received MHEP  each with a four-point Likert-type scale. Participants rate howmuch they feel like each statement at the time of making the response.Scores on the STAI range from 20 to 80, with higher scores representinghigher levels of anxiety. The Trait subscale also consists of 20 statementsbut refers to how participants generally feel . STAIStThe GHQ-12 is a screening instrument used for assessing general psychologicalhealth in both clinical settings and non-clinical research settingsrequiring repeated measurements over time. The GHQ-12 consists of 12 items,each assessing the severity of a mental problem over the past few weeksusing a four-point scale (0–3) with higher scores indicating worseconditions .The PSS is a 10-item questionnaire that assesses the degree to whichsituations in one’s life are appraised as stressful using a five-point scale(0–4). It is a widely used research instrument, and its validity has beenestablished within a number of populations .The POMS is a well-established, factor-analytically derived measure ofpsychological distress for which high levels of reliability and validityhave been documented . The POMParticipants completed a series of VAMS anchored by 27 antonyms relating tomood and psychological state. Participants moved a marker along the line todescribe how they currently feel. Each line was scored as % along the linetowards the more positive antonym. Factor analysis of the original 27 itemsrevealed three factors incorporating 18 items (unpublished data). Thefactors were labelled Alertness , Stress  andTranquillity .Supplemental File 3.The OMS incorporates two elements that have previously been shown to engendera stress response in laboratory studies; extended multitasking and socialevaluation. The OMS has previously been shown to provoke a psychologicalstress response across repeated administrations . BrieflyHR and GSR was measured throughout performance of the OMS. GSR and HR weremeasured on testing visits using the Vilistus Digital Sampling Unit . The GSR sensors, which measuredrelative changes in skin conductance, were attached to the middle and fourthfingertips on the participant’s non-dominant hand using Velcro straps. TheHR sensor clip, which measured blood volume pulse (BVP), was placed on thetip of the index finger or thumb on the non-dominant hand. These sensorswere attached at least 1 min prior to the commencement of recording to allowfor stabilisation of the readings. The unit measured 32 and 128 samples persecond for GSR and HR, respectively.g for 2 min.Samples were transferred into Eppendorfs and frozen at −80°C. Beforeassaying, the samples were thawed and the cortisol and α-amylase levels inthe saliva samples were measured using enzyme-linked immunoassay.Saliva samples were obtained throughout the protocol at various time points usingsalivettes to measure salivary cortisol response  and saliapp (www.cognimapp.com) allowsfor at home assessment of participants on a range of cognitive and mood measuresthroughout the course of the intervention period. To capture response totreatment for both morning sleep inertia and ‘post lunch dip’ periods of theday, as well as ongoing effects of treatment on subjective stress and anypotential sedative effects of the intervention, the Cognimappassessment  was completed before breakfast and after lunch. Apretreatment Cognimapp assessment took place on days 7 and 36 andthen again on days 7 and 14 of each treatment period  on five separate occasions. The firstwas an introductory visit where informed written consent was obtained. Followingthe introductory visit, participants attended the laboratory at a prearrangedtime in the afternoon on four separate occasions (visits 1–4). The first andthird visits comprised the baseline assessments. Visits 2 and 4 were chronicassessments and occurred 15 days (±3 days) after visits 1 and 3, respectively.Each visit was identical, except for the intervention consumed between visits 1and 2 and visits 3 and 4  break, participants were taken to an ‘interview’ roomwhere they underwent the OMS for 15 min in front of a panel of two observerswhile also being video recorded and having their GSR and HR readings measuredthroughout. The STAI-State and VAMS were completed in the laboratory immediatelyprior to and after the OMS and at 30, 60 and 90 min post-OMS. Seven salivasamples were collected in total (see app assessment battery just before breakfast and afterlunch on days 7 and 14 in each treatment period following their baselineCognimapp assessments on days 7 and 36 (see app assessments). Afull description of the procedure is provided in Supplemental File 5.Before leaving on testing visits 1 and 3, participants were provided with theirtreatment. Participants were also instructed to complete theCognimFor the data collected during the study visits, the general statistical approachcomprised the analysis of data collected following each treatment period (i.e.visits 2 and 4), including data collected at the pre-intervention assessment(i.e. visits 1 and 3) as a covariate. The MIXED procedure in SPSS  was used for all analyses. For each model,restricted maximum likelihood estimation methods were used and covariance matrixstructure was chosen based on the structure that produced the lowest Schwarz’sBayesian criterion, an indication of the best fitting model . SubjectTo investigate the effect of treatment on the direct psychological andphysiological response to the OMS, data collected at all other time pointsduring the testing visit were analysed in a separate analysis. Outcomes includedSTAI-State, VAMS, GSR, BVP and salivary cortisol and salivary α-amylase. Thesewere analysed as above, including treatment and assessment as fixed factors andpre-intervention values as a covariate. For the dual tasking performanceoutcomes, task was included as an additional factor.app data were analysed as above, including the fixed factorstreatment, visit and time of day.The CognimIn order to assess the stress response elicited by the OMS procedure itself, theVAMS mood, STAI-State and saliva analyte outcomes collected at visits 1 and 3 inthe absence of treatment were analysed as above including assessment and visitas fixed factors.Missing data were left as empty cells as the linear mixed model approach that wasapplied to the data uses maximum likelihood to estimate the missing values.Supplemental Tables for data from all measures.Thirty-one participants were randomised to receive treatment see . One parOne participant only completed the first phase of the trial including visits 1and 2. However, these data were included in the analysis; therefore, 27 datasets were eligible for analysis.Participant demographics and baseline characteristics are summarised in 2(1) = 0.619, p = 0.431).Compliance was at 97.2% during the placebo phase and 98.3% during the MHEP phaseof the study. Compliance was based on (returned) treatment counts. Participantsresponses to the treatment guess questionnaire, completed on the final visit,were analysed via a chi-square test and revealed that there was no significantdifference between the ability to correctly detect the active treatment and theplacebo (χPre-intervention visit data (i.e. visits 1 and 3) were analysed for treatmentgroup effects and treatment group × visit interactions to confirm an absence ofbaseline differences between the groups, or carryover effects from the firsttreatment period.app outcomes.No baseline differences were observed for any of the outcomes included in thechronic effects analysis or any of the CognimF = 8.43, p = 0.004). Participants assigned to MHEPreported lower anxiety (30.81) than placebo (32.97) before treatment commenced.A significant effect of treatment was also observed for the OMS dual task speed = 7.30, p = 0.007) andaccuracy  = 14.79, p < 0.001)measures (z scores). Participants assigned to MHEP were faster(0.14) and more accurate (0.18) than those assigned to placebo  before treatment commenced.With regards to the analysis of OMS-associated effects for data that werecollected between −15 min pre-OMS until 90 min post-OMS, a significant effect oftreatment group was observed for state anxiety  = 38.71, p < 0.001),stress  = 13.99, p < 0.001)and tranquillity  = 8.14,p < 0.001). Post hoc comparisons revealed that theassessment completed immediately after the OMS was significantly higher  or lower (tranquillity) compared to all the other assessments = 9.26, p = 0.004), with lower anxiety reported at visit3 (30.24) compared to visit 1 (33.40). Similarly, an effect of visit was alsoobserved for alertness  = 9.15,p = 0.003), with higher alertness reported at visit 3 (65.08)compared to visit 1 (62.64).An effect of visit was detected for state anxiety  = 3.83, p = 0.002). Post hoccomparisons revealed that the value of the sample collected immediatelyfollowing the OMS (240.69) was significantly higher than the sample collectedimmediately prior to the OMS . A significanteffect of assessment was also observed for salivary cortisol = 10.29, p < 0.001). However,the pattern of response here was more anticipatory; post hoc comparisonsrevealed that cortisol concentration was elevated from −15 min pre-OMS and onlybegan to decline 60 min post OMS  = 4.84,p = 0.038), with post hoc pairwise comparisons revealing MHEPresulted in significantly lower tension-anxiety (6.33) than placebo (7.75)(A significant main effect of treatment was identified for tension-anxiety on thePOMS questionnaire (o (7.75).F = 4.20,p = 0.041), with participants having lower salivaryα-amylase following MHEP (209.51) compared to placebo (232.21) overallduring the OMS assessment  = 8.63, p = 0.004),with participants having a lower GSR following MHEP (7.59) than placebo(8.43) overall during the OMS assessment  = 4.13,p = 0.044). However, post hoc pairwise comparisonsrevealed no significant differences between the groups.A significant interaction between treatment × time of day was identifiedfor digit vigilance false alarms  = 4.27, p = 0.041), with post hoc pairwisecomparisons revealing that MHEP made significantly less false alarms(2.07) than placebo (2.67)  false alarms (o (2.67) .F = 3.42, p = 0.036), with post hoc pairwisecomparisons revealing that placebo had significantly faster reactiontimes (494.16 ms) than MHEP (509.80 ms) but only in the +7 day morningassessment (p = 0.026) (A significant interaction between treatment × visit × time of day wasidentified for digit vigilance reaction time (= 0.026) .app,MHEP led to significantly fewer false alarms on the RVIP task compared with placebo.A significant reduction in speed of performance on the digit vigilance taskfollowing MHEP was also observed. However, this isolated negative effect was onlyobserved during the morning assessment on day 7 and appears to contradict thepattern of response for the other assessments where performance was numericallyfaster following MHEP.In the current study, 14 days’ supplementation with MHEP was associated with reducedtension-anxiety. In addition, participants showed an attenuated response to the OMSpsychosocial stressor following MHEP, evidenced by lower salivary α-amylase and GSR.With regards to cognitive performance assessed at home via CognimV.officinalis species as in MHEP) has been shown to increase central GABAlevels. This leads to a reduction in central nervous system activity  isrecognised as a good indicator of activation of the sympathetic nervous system. The obsAlthough it could be expected that the active treatment would have a beneficialeffect across all the physiological parameters, it should be noted thatinconsistencies in these measures are also found in the literature. Cortisol, asteroid hormone, is a reliable measure of the response to acute stress .A-amylaapp cognitive performance outcomes, MHEP led tosignificantly fewer false alarms on the RVIP task compared with placebo. However,the findings here do not appear to represent a consistent pattern of effects foreither treatment, rendering interpretation difficult. Specifically, digit vigilancereaction time was significantly slower following MHEP compared to placebo in the+7 day morning assessment. The number of dependent variables should also beacknowledged; the small effects seen here may not have been detected if the numberof analyses conducted were adjusted for. Importantly, despite these minimal andcontradictory effects, the null findings overall provide evidence of an absence ofconsistent adverse effects on performance observed either during the study visit oron the Cognimapp assessments as a result of the active treatment.Furthermore, we also observed no effect of MHEP on subjective alertness or on theKSS, a reliable measure of subjective drowsiness. Of the extracts contained withinMHEP, those understood to have sedating properties include valerian and ballota. Theability of valerian to bind to adenosine receptors has been reported within animalstudies and proposed as one of the mechanisms by which the sedating effects mayoccur  have been used traditionally for their sedativeproperties, among others , it is recommendedthat chronic assessments of mood should be completed in a more neutral setting, on adifferent day to the OMS in order to capture any potentially subtle effects oftreatment.app is a valuable assessment tool with the ability to capturecognitive performance and mood measures in any setting, but inevitably this comeswith some practical limitations. A laboratory setting provides a quiet environment,free from daily distractions where engagement can be monitored by a study team.Although guidance is provided to the participant to complete theCognimapp assessments with these principles in mind, it is not alwayspracticable when fitting the assessments into their daily lives. Without the abilityto monitor participants, there is also the possibility that assessments will not becompleted within the appropriate timeframe. In order to monitor time of day effects,including the impact of morning sleep inertia and the post-lunch dip, participantswere required to complete the assessments before breakfast and 1 h (2 h maximum)after finishing their lunch. It was evident from the raw data that not allparticipants adhered to this period and/or consumed breakfast and lunch at irregulartimes of the day. However, it could be argued that ‘real life’ environments providethe ideal setting within which to assess cognitive performance since any findingsdetermined as a result, either positive or negative, would potentially be even morevalid. It is likely that a larger data set with this measure would tease out many ofthese nuances and individual differences to reveal a clearer pattern of effects.CognimThe findings of the present study demonstrate that 14 days’ supplementation with acombination of the herbal extracts valerian, passionflower, ballota and hawthornreduces subjective anxiety in a healthy population and lowers electrodermal skinconductance and concentration of salivary α-amylase in response to a psychosocialstressor, compared to placebo. Future studies may benefit from conducting mood andwell-being assessments in the absence of the OMS to remove any anticipatory effectsof this measure and/or assessing all physiological and mood outcomes over a longerpre- and post-OMS time frame in order to ascertain what the extent of the effect ofthe preparatory response is in this environment.Click here for additional data file.Supplemental material, sj-doc-1-jop-10.1177_02698811221112933 for The chroniceffects of a combination of herbal extracts (Euphytose®) on psychological moodstate and response to a laboratory stressor: A randomised, placebo-controlled,double blind study in healthy humans by Fiona Dodd, David Kennedy, EmmaWightman, Julie Khan, Michael Patan, Rian Elcoate and Philippa Jackson inJournal of PsychopharmacologyClick here for additional data file.Supplemental material, sj-docx-2-jop-10.1177_02698811221112933 for The chroniceffects of a combination of herbal extracts (Euphytose®) on psychological moodstate and response to a laboratory stressor: A randomised, placebo-controlled,double blind study in healthy humans by Fiona Dodd, David Kennedy, EmmaWightman, Julie Khan, Michael Patan, Rian Elcoate and Philippa Jackson inJournal of PsychopharmacologyClick here for additional data file.Supplemental material, sj-docx-3-jop-10.1177_02698811221112933 for The chroniceffects of a combination of herbal extracts (Euphytose®) on psychological moodstate and response to a laboratory stressor: A randomised, placebo-controlled,double blind study in healthy humans by Fiona Dodd, David Kennedy, EmmaWightman, Julie Khan, Michael Patan, Rian Elcoate and Philippa Jackson inJournal of PsychopharmacologyClick here for additional data file.Supplemental material, sj-docx-4-jop-10.1177_02698811221112933 for The chroniceffects of a combination of herbal extracts (Euphytose®) on psychological moodstate and response to a laboratory stressor: A randomised, placebo-controlled,double blind study in healthy humans by Fiona Dodd, David Kennedy, EmmaWightman, Julie Khan, Michael Patan, Rian Elcoate and Philippa Jackson inJournal of PsychopharmacologyClick here for additional data file.Supplemental material, sj-docx-5-jop-10.1177_02698811221112933 for The chroniceffects of a combination of herbal extracts (Euphytose®) on psychological moodstate and response to a laboratory stressor: A randomised, placebo-controlled,double blind study in healthy humans by Fiona Dodd, David Kennedy, EmmaWightman, Julie Khan, Michael Patan, Rian Elcoate and Philippa Jackson inJournal of PsychopharmacologyClick here for additional data file.Supplemental material, sj-docx-6-jop-10.1177_02698811221112933 for The chroniceffects of a combination of herbal extracts (Euphytose®) on psychological moodstate and response to a laboratory stressor: A randomised, placebo-controlled,double blind study in healthy humans by Fiona Dodd, David Kennedy, EmmaWightman, Julie Khan, Michael Patan, Rian Elcoate and Philippa Jackson inJournal of PsychopharmacologyClick here for additional data file.Supplemental material, sj-xlsx-7-jop-10.1177_02698811221112933 for The chroniceffects of a combination of herbal extracts (Euphytose®) on psychological moodstate and response to a laboratory stressor: A randomised, placebo-controlled,double blind study in healthy humans by Fiona Dodd, David Kennedy, EmmaWightman, Julie Khan, Michael Patan, Rian Elcoate and Philippa Jackson inJournal of Psychopharmacology


Circulating tumor cells (CTCs) in peripheral blood have been shown to reflect the prognosis of patients with colorectal cancer, and epithelial and mesenchymal markers further predict the likelihood of cancer dissemination. This study was conducted to identify possible association of clinical features of colorectal cancer with CTC counts, their subtypes, and systemic inflammatory markers.  Blood samples of 316 colorectal cancer patients were used for CTC detection and subtyping with EpCAM, CK8/18/19, vimentin, and twist as biomarkers. The neutrophil/lymphocyte ratio, platelet/lymphocyte ratio, C-reactive protein/albumin ratio, lymphocyte/monocyte ratio, and systemic immune-inflammation index (SII) were also measured. The relationship between clinical data and these markers or parameters was analyzed. P < 0.05). Also, more patients with metastasis tested positive for mesenchymal CTCs (P < 0.05). Of the systemic inflammatory markers, platelet/lymphocyte ratio was positively correlated with CTC counts (P < 0.01), and lymphocyte/monocyte ratio was negatively correlated with CTC counts (P < 0.05).  Total CTC counts were correlated with whether there was lymph node involvement but was not correlated with TNM staging. There was a difference in mesenchymal CTCs between patients with and without lymph node involvement ( Colorectal cancer patients with the mesenchymal markers on their CTCs are more likely to have lymph node involvement or distant metastasis than those without these markers. Colorectal cancer (CRC) is one of the most common malignancies and a leading cause of death. CRC-related mortality is mostly attributable to metastasis to other organs and tissues, especially the liver, lung, and peritoneum , 2. AbouThe detection of circulating tumor cells (CTCs) in peripheral blood has been used to predict the existence of disseminated tumor cells and the risk of cancer recurrence. Studies have shown that CTCs are able to predict distant metastasis and overall survival in several types of cancer , 6. For A recently introduced technology uses epithelial and mesenchymal markers  to classify CTCs into three subtypes, epithelial CTCs, epithelial/mesenchymal CTCs, and mesenchymal CTCs –16. Yet,Subjects were recruited between May 2016 and September 2020. Patients who were histologically diagnosed with colon or rectal cancer and had complete medical records at our hospital were eligible for inclusion. The research protocol was approved by the ethics committee of Henan Provincial People's Hospital. Written informed consent was obtained from participants in accordance with the Declaration of Helsinki. A total of 316 patients entered the study, with general data containing clinical staging, recurrence, sites of metastases, and histological classifications.4Cl, 10 mM KHCO3, and 0.1 mM EDTA)  was added. After 30 min, centrifugation was performed at 1850 rpm for 5 min, and then the supernatant was extracted. The remaining cells were resuspended in a mixture of 4 ml PBS and 1 ml 4% formaldehyde. The suspension was vortexed and placed at room temperature for 8 min. CTCs were isolated with a filtration system consisting of a filtration tube containing a membrane with a pore diameter of 8 μm, a multiwell plate vacuum manifold, and a vacuum pump with the pressure set at ≥0.08 MPa.Immediately following diagnosis with CRC, 5 ml of the patient's peripheral blood was drawn into a centrifuge tube containing EDTA after discarding the first 2 ml to prevent skin cell contamination. The CTC isolation method used in this study was initially described by Wu et al. . To remoμl of a preamplifier solution composed of 30% horse serum, 1.5% sodium dodecyl sulfate, 3 mM Tris-HCl (pH 8.0) , and 0.5 fmol of the preamplifier. After cooling, the membranes were again washed three times with 0.1× SSC and then incubated with 100 μl of the amplifier solution.RNA in situ hybridization (RNA-ISH) was used to detect CTC biomarkers, which included epithelial markers EpCAM and CK8/18/19, mesenchymal markers vimentin and twist, and CD45 as a leukocyte marker. + cells. CD45+ cells were labeled with Alexa Fluor 647 (white fluorescence), epithelial markers (EpCAM and CK8/18/19) were labeled with Alexa Fluor 594 (red fluorescence), and mesenchymal markers (vimentin and twist) were labeled with Alexa Fluor 488 (green fluorescence). Cells possessing epithelial and mesenchymal markers (E + M) showed both red and green fluorescence. Images of white blood cells, epithelial CTCs, mesenchymal CTCs, and epithelial/mesenchymal CTCs are shown in Three fluorescent dyes were used to label the probes, with Alexa Fluor 594 for the EpCAM and CK8/18/19 probes, Alexa Fluor 488 for the vimentin and twist probes, and Alexa Fluor 647 for the CD45 probe. After washing with 0.1× SSC, the cells were stained with 4′,6-diamidino-2-phenylindole (DAPI)  for 5 minutes and examined with an Axio Imager Z2 fluorescence microscope . After filtration, CTC counts were obtained with the subtraction of CD45Patients' blood samples were sent to the hospital's clinical laboratories for the detection of carcinoembryonic antigen (CEA) and cancer antigen 19-9 (CA 19-9). Commercial electrochemical luminescence kits  were used for these markers. Tests were conducted according to the manufacturer's instructions. Laboratory results for the neutrophil/lymphocyte ratio (NLR), platelet/lymphocyte ratio (PLR), C-reactive protein/albumin ratio (CAR), lymphocyte/monocyte ratio (LMR), and systemic immune-inflammation index (SII) were also collected.χ2 test or the t-test, where applicable.All statistical analyses were conducted using SPSS, version 18.0 , and significance was set at .05 for all tests. For laboratory parameters, common reference values were used to categorize patients. Correlations between variables were examined using the two-sided A total of 316 CRC patients were included in this study. Demographic data, clinical features, and histological results are listed in Using CTC counts ≤ 3 as negative detection and >3 as positive detection, we found that total CTC counts were correlated with whether there was lymph node involvement but were not correlated with TNM staging. In addition, there was no relationship between total CTC counts and any of the features such as history of smoking, tumor size, or histological grade . As for P < 0.05) while no difference was found in the epithelial phenotype or the mixed phenotype between these two patient groups (P < 0.05)  .P < 0.01), and LMR was negatively correlated with CTC counts (P < 0.05), whereas none of NLR, CAR, and SII showed any correlation with CTC counts (More CRC patients had CEA and CA 19-9 levels within the reference ranges (≤ 3.5 ng/ml for CEA and ≤30 U/ml for CA 19-9) than those with CEA and CA 19-9 levels exceeding the reference ranges. Also, the levels of these two markers were not associated with CTC counts . Of the C counts .One of the major findings in the current study is that total CTC counts were positively correlated with lymph node involvement but not with TNM staging in CRC patients. We were unable to identify any relationship between CTC subtypes and specific sites of metastasis. A number of studies have demonstrated that CTCs may serve as a prognostic tool for CRC , 6. Arouβ signaling pathways [The demonstration in our study that patients with higher counts of mesenchymal CTCs were more likely to have remote metastasis further illustrates the important role EMT plays in CRC dissemination. During EMT, epithelial tumor cells acquire a mesenchymal phenotype through a highly coordinated program that involves extracellular signals such as multiple growth factors and cytokines, transcription factors of genes responsible for cell adhesion and mesenchymal differentiation, and consequently molecules that confer a distinctive epithelial or mesenchymal identity to the cells , 20. We pathways . Ck8/18/pathways , 25. Vimpathways , 27. As pathways . Its ovepathways , 30. Of In addition to biological processes required for remote metastasis such as EMT, factors that influence the route and site of CRC metastasis remain poorly understood. EMT promotes the development of mesenchymal features in tumor cells. However, CTCs may also undergo mesenchymal-to-epithelial transition (MET), the reverse of EMT, to colonize metastatic sites , 32. TheSince inflammation has been suggested to play a critical role in the development and metastasis of many types of cancer, we included several systemic inflammatory parameters and analyzed their potential association with CTCs in this study. Our results showed that CTC counts were positively correlated with PLR and negatively correlated with LMR but had no correlation with NLR, CAR, or SII. These ratios reflect different aspects of interaction between immune and inflammatory factors in response to tumor development. Platelets suppress lymphocyte proliferation through P-selectin, resulting in decreased proinflammatory cytokine levels and increased anti-inflammatory cytokine levels; so, its positive correlation with CTCs should be expected , 37. In There were some limitations in our study. As a convenient and minimally invasive technology, CTC detection has proved to be a viable alternative biopsy option, but the method we used is based on mRNAs of epithelial and mesenchymal markers and may not reflect protein expression. Moreover, aneuploidy of chromosomes, especially 7 and 8, is often seen in CRC , 44. An In conclusion, available studies, including ours, have demonstrated that CTC detection may provide a convenient tool for the diagnosis and treatment planning of CRC. Subtyping of CTCs is an especially promising approach to the prediction of clinical outcomes because of the increasingly recognized role of EMT in tumor dissemination. For a better understanding of the value of CTC detection and subtyping in CRC management, more rigorous evidence needs to be obtained, preferably through improved research designs, standardized detection protocols, and follow-up of large cohorts.


Bipolar disorder (BD) and metabolic disturbance represent a chronic state of low-grade inflammation and corticostriatal circuitry alterations. Herein, we aimed to investigate whether plasma leptin, an adipokine that plays a key role in the interplay of metabolism and inflammation, is associated with corticostriatal connectivity in patients with BD. Twenty-eight BD I patients, 36 BD II patients and 66 healthy controls were enrolled and completed the Hamilton Depression Rating Scale, the Young Mania Rating Scale, and the Recent Life Change Questionnaire. Fasting plasma leptin and C-reactive protein (CRP) levels were measured, and corticostriatal connectivity was examined using functional magnetic resonance imaging (fMRI). The relationships between leptin, CRP and body mass index (BMI) identified in the controls and BD II patients were absent in the BD I patients. We did not find a significant group difference in the leptin level; nevertheless, the negative correlation between leptin level and corticostriatal connectivity  observed in the healthy controls was absent in the BD patients. The disproportionate increase in leptin level with increasing BMI in BD indicated a potential inflammatory role of white adipose tissue in BD. Furthermore, higher CRP levels in BD I patients might induce leptin resistance. Collectively, our results implied vulnerability to inflammatory and metabolic diseases in patients with BD, especially BD I. Furthermore, evidence has shown that aberrant immune signaling contributes to all stages of BD, and also to metabolic disturbance6. Recent structural and functional studies have directly implicated reward system dysregulation in BD9, and evidence has shown that core network functional abnormalities lead to aberrant reward-processing, and the neural circuitry in BD is highly associated with systemic inflammation11. Such systemic inflammation might drive changes in dopaminergic corticostriatal circuitry connectivity13, and a recent large-scale study indicated that both BD and body mass index (BMI) were associated with similar regional brain volumes, including that of the basal ganglia14.Bipolar disorder (BD), characterized by emotion dysregulation, increases the relative risk of mortality18. In addition, there is compelling evidence implicating leptin as an important modulator of the inflammatory process. Leptin can increase the production of pro-inflammatory cytokines and modulates both innate and adaptive immune responses20. Leptin itself has been found to be able to stimulate C-reactive protein (CRP) synthesis from the liver and endothelial cells21. A positive and independent relationship between peripheral leptin and CRP has been found previously in healthy subjects22. Taken together, leptin could modulate the reward system and plays a key role in the interplay of metabolism and inflammation23.Leptin is secreted by white adipose tissue and regulates energy homeostasis. There is direct brain-adipose crosstalk through leptin, which regulates the energy balance and motivation through action at distinct neural circuits26. However, whether the pathophysiological processes induced by leptin or CRP are enhanced or redundant in BD remains unknown27. We previously reported altered corticostriatal circuitry in BD that was associated with the level of CRP28. In the current study, we aimed to investigate the relationship between peripheral leptin and CRP levels. Regarding the role of the caudate nucleus in both metabolic control and BD29, we also investigated the possible correlation between leptin and caudate-seeded corticostriatal connectivity in patients with BD32. We hypothesized that the alterations in caudate-seeded functional connectivity (FC) in the corticostriatal circuitry may be linked to leptin metabolic feedback dysregulation in BD patients.Both leptin and CRP are linked directly and independently through a number of pathophysiological mechanisms to BDn = 14, 22.6%), valproic acid plus antipsychotics , lithium , and lithium plus antipsychotics . Only one patient received valproic acid plus lithium, and one received valproic acid plus lithium plus antipsychotics; eight patients (12.5%) received only antipsychotics, 3 (4.7%) only antidepressants, and 4 (6.3%) did not take any medication. There were no group differences in the usage of valproic acid (p = 0.182), olanzapine (p = 0.100), quetiapine (p = 0.832), amisulpride (p = 0.466), aripiprazole (p = 0.404), or lurasidone (p = 0.824); however, increased usage of lithium (p = 0.013), risperidone (p = 0.008) and clozapine (p = 0.018) was observed in the BD I patients  and 2.00 ± 3.80 on the 11-item Young Mania Rating Scale (YMRS)  and BD II patients , but this was not the case in the BD I patients . Similarly, BMI was significantly correlated with the CRP level among the healthy controls  and BD II patients , but not in the BD I patients . In contrast, both the BD I and BD II patients exhibited significant correlations between the plasma leptin level and BMI ; however, the healthy controls showed a sub-significant correlation .There were no significant differences in BMI, plasma leptin level or CRP level between groups Table . The plar =  − 0.597, p < 0.001; r =  − 0.532, p < 0.001; r =  − 0.345, p = 0.011) , while a significant positive correlation between plasma leptin level and the DC-inferior temporal gyrus FC was observed in the BD II patients .Among the healthy controls, the dorsal caudate (DC)-ventrolateral prefrontal cortex (vlPFC), DC-inferior temporal gyrus and DC-putamen FC were negatively correlated with the plasma leptin level  as compared with the BD II patients Table . The BD 2. Interestingly, the relationship between leptin and CRP found in the controls and BD II patients was absent in the BD I patients, indicating that higher CRP levels in BD I patients might induce leptin resistance33. Moreover, such dysregulation may arise from obesogenic diets, medications and/or BD per se35. Although we still cannot conclude whether the pathophysiological processes induced by leptin or CRP are enhanced or redundant, their possible additive or even synergistic effects need to be taken into consideration, as both leptin and CRP might increase simultaneously in BD. Our results also implied vulnerability to inflammatory and metabolic diseases in BD I patients.In line with a previous meta-analysis study, our results showed that the leptin level was not altered in the BD patients as compared with the healthy controls10. The results here were in line with a unified model, suggesting that emotional dysregulation in BD arises from dynamic alterations in circuits that involve the perception of interoception36. In the current study, we demonstrated that there were significant correlations between the level of leptin and the corticostriatal circuitry in healthy controls, indicating a leptin-modulated effect on the interoceptive reward circuitry38. The results showed that the negative correlation between leptin and DC-seeded FC was reversed in both the BD I and BD II patients, implying that the aforementioned leptin-modulated corticostriatal circuitry was dysregulated. Furthermore, the dysregulated regions involved the emotion-regulation circuitry, including the vlPFC . A proportion of the subjects  overlapped with those enrolled in our previous published studies58.All patients were recruited from the psychiatric outpatient department at National Cheng Kung University Hospital, while the healthy controls were recruited from the community through advertisement. All participants, who were either Mandarin or Taiwanese speakers, were screened by a psychiatrist using the Chinese version of the Mini International Neuropsychiatry Interview (MINI)The exclusion criteria for all the participants were: (1) any psychiatric (other than BD and tobacco use disorder), neurological, autoimmune, serious surgical, or severe physical illnesses, such as acute coronary syndrome, kidney dialysis, hepatic failure, or transplant; (2) any head injury history with loss of consciousness; (3) any medications that could affect the immune system or use of anti-inflammatory medications; (4) any contraindications in relation to magnetic resonance imaging (MRI), such as having a metal implant, a pacemaker implant, or claustrophobia; (5) plans for pregnancy or a positive pregnancy test.59. The RLCQ, which consists of 39 items querying representative life change events, is the Taiwanese version of the Schedule of Recent Experience60, which consisted originally of 43 life change events, later termed life change units61. Blood samples were taken for the measurement of fasting plasma leptin and CRP levels, and FC was examined during the resting state using functional magnetic resonance imaging (fMRI). For detailed information on image acquisition, image preprocessing, definition of the seeds in the left DC and other areas among the basal ganglia , and seed-based FC maps, please refer to our published papers58.After enrolment in the study, all patients received treatment as usual, and the administration of medications was recorded. There was no significant relapse of BD that necessitated clinical care changes during the study. All participants in the three groups completed the Recent Life Change Questionnaire (RLCQ) to assess the type and magnitude of life events during the previous 12 monthsg for 15 min at 4 °C and immediately stored at − 80 °C. The levels of fasting plasma leptin  and CRP  were measured using ELISA methods. For detailed information on the catalog numbers, minimum detectable levels, and intra- and inter-assay coefficients of the ELISA kits used for the measurement of leptin and CRP, please see Supplemental Table The participants were instructed to fast for at least 9 h prior to each examination. Blood samples for the leptin and CRP assays were collected between 08:00 and 10:00 am in 5-mL EDTA tubes and stored at 4 °C in a fridge. Plasma was isolated by centrifugation at 1800×p < 0.05 (two-tailed). As most of the variables shown in Table post-hoc Mann–Whitney U test was performed whenever the between-group difference was significant. Pearson correlation analyses were performed to test the relationships among the plasma leptin level, CRP level, and BMI.SPSS Statistics 20.0  was used for all analyses. Missing data were excluded from the calculations, and the numbers of participants with missing data are indicated in the footnote of each table. Results were considered significant at https://www.fil.ion.ucl.ac.uk/spm/). Statistical maps were computed to identify changes in the DC-seeded FC for between-group comparisons. Significance was thresholded at the uncorrected voxel-level p = 0.001, followed by the voxel-level family-wise error (FWE) rate-corrected cluster-level at p = 0.05 for whole-brain multiple comparisons.One-way ANOVA was employed to analyze the FC maps using SPM12  values as regressors to identify brain regions exhibiting higher correlations with the DC-seeded FC among the three groups. Significance was thresholded at the uncorrected voxel-level http://www.mccauslandcenter.sc.edu/mricrogl). To show the regression results in scatterplots, we extracted the DC-seeded FC values in each brain region showing higher correlations with the DC-seeded FC among the three groups (peak MNI coordinates are presented in Tables r) and p values were analyzed using SPSS Statistics 20.0.As SPM12 reports peak coordinates as identified within a confluent cluster, there can be multiple peaks that sit on different brain regions/areas . We reported one representative peak for each region/area. To display 3D imaging, we used MRIcroGL for 3D rendering (Department of Psychology, University of South Carolina Supplementary Tables.


Bifidobacterium animalis subsp. lactis  JYBR-190 on the growth performance, intestine health, and gut microbiota of weaning piglets. The results showed that the dietary addition of B. animalis significantly improved growth performance and decreased diarrhea incidence. B. animalis increased villus height in the duodenum and elevated goblet cell numbers and amylase activity in the jejunum. Additionally, B. animalis supplementation markedly increased total antioxidant capacity in jejunal mucosa but declined the malondialdehyde content. B. animalis treatment did not affect the mRNA expressions associated with the intestinal barrier and inflammatory cytokine in various intestinal segments. Microbiota analysis indicated that a diet supplemented with B. animalis significantly increased the relative abundances of health-promoting bacteria in the lumen, such as Streptococcus, Erysipelotrichaceae, Coprococcus, and Oscillibacter. There was a trend for B. animalis fed piglets to have a higher relative abundance of B. animalis in ileal digesta. Moreover, B. animalis-treated pigs decreased the abundance of Helicobacter and Escherichia-Shigella in ileal mucosa-associated microbiota. In summary, this study showed that B. animalis supplementation stimulated growth performance, improved gut development, enriched beneficial bacteria abundances, and declined intestinal pathogens populations, while B. animalis had limited effects on the intestinal barrier and immune function.Probiotics are widely used to promote performance and improve gut health in weaning piglets. Therefore, the objective of this study was to investigate the effects of dietary supplementation with IMPORTANCE In the modern swine industry, weaning is a critical period in the pig’s life cycle. Sudden dietary, social, and environmental changes can easily lead to gut microbiota dysbiosis, diarrhea, and a decrease in growth performance. To stabilize intestinal microbiota and promote animal growth, antibiotics were widely applied in swine diets during the past few decades. However, the side effects of antibiotics posed a great threat to public health and food safety. Therefore, it is urgent to find and develop antibiotic alternatives. The growing evidence suggested that probiotics can be preferable alternatives to antibiotics because they can modulate microbiota composition and resist pathogens colonization. In this study, our results indicated that dietary supplementation with Bifidobacterium animalis promoted growth in weaning piglets by improving gut development, increasing beneficial bacteria abundances, and declining pathogens populations. In the modern swine industry, weaning is a critical period in the pig’s life cycle. Piglets encounter multiple stressors during the postweaning period which cause disruptions of gut microbiota, intestinal inflammation, and diarrhea, consequently reducing their growth performance , 2. To sThe mammalian gastrointestinal tract is inhabited by trillions of bacteria . A stablLactobacillus, Bifidobacterium, Streptococcus, and yeasts  in germfree piglets can prevent Salmonella infection  on day 14 but significantly increased BW on day 28 (P < 0.05). No difference was found in average daily feed intake (ADFI) between the control group (Con) and the B. animalis group (Bif). Average daily gain (ADG) was increased in the group receiving the B. animalis-supplemented diet during days 15 to 28 and days 1 to 28 (P < 0.05). However, the feed conversion ratio (FCR) of piglets fed the B. animalis diet was decreased from day 15 to 28 and day 1 to 28 compared with piglets fed the control diet. Meanwhile, pigs fed the B. animalis diet markedly decreased the incidence of diarrhea  in serum, including IgA, IgG, and IgM, had no differences between the Con and Bif groups . SimilarB. animalis to the diet did not influence the activities of CAT, T-SOD, and GSH-Px in the jejunum. However, B. animalis supplementation increased the activity of T-AOC (P < 0.05) and decreased the content of MDA in the jejunum (P < 0.05).The antioxidant indicators as presented in B. animalis treatment improved the VH and VH:CD ratio in the duodenum compared with the Con group (P < 0.05). In addition, supplementation of B. animalis to the weaning piglet's diet significantly increased the jejunal goblet cell numbers (P < 0.05)   and B. Tzonula occludens-1 [ZO-1], claudin-1, and occludin) in the jejunum, ileum, and colon between the two groups  in the jejunum, ileum, and colon (d-lactate and diamine oxidase (DAO) reflect intestinal barrier integrity. The results showed that the B. animalis treatment also did not affect the contents of serum d-lactate and DAO .In the jejunum, supplementation of d lipase . HoweverIL-1β, IL-6, IL-8, tumor necrosis factor α (TNF-α), and IL-10 in the intestine between the two groups .The inflammatory genes’ relative mRNA expression of different segments in the intestine are shown in o groups  to E. CoP < 0.05, B. animalis-treated piglets displayed an increasing trend . Alpha diversity (describing the diversity of populations within a sample) indices were calculated to assess the richness and diversity of bacterial communities. The richness of the bacterial community was evaluated by Ace, Chao1, and Sobs indices  to C, anStreptococcus and Erysipelotrichaceae_UCG-003 were increased in the ileal digesta of pigs fed the B. animalis diet compared with those fed the control diet . Linear discriminant analysis (LDA) effect size (LEfSe) analysis was used to identify intergroup differential bacteria. Consistently, LEfSe analysis exhibited that the dietary addition of B. animalis also declined the Helicobacter and Escherichia-Shigella abundances of ileal mucosa-associated microbiota (Anaerovibrio was enriched in colonic mucosa samples of pigs from the Con group (P < 0.05)., whereas Streptococcus and Catenisphaera populations were enriched in colonic mucosa samples of pigs from the Bif group . The results showed that neither alpha diversity indices nor beta diversity had differences from ileal and colonic mucosa-associated microbiota between the Con and the Bif groups . Differential bacteria analysis at the genus level found that the relative abundances of crobiota , LDA > 2if group , LDA > 2Lactobacillus and Bifidobacterium species, exhibit various beneficial effects on hosts. Previous studies indicated that supplementation of Lactobacillus or Bifidobacterium improved the growth performance of weaning piglets and decreased diarrhea incidence by regulating the immune function, enhancing the epithelial barrier, increasing the antioxidant capability, and inhibiting the growth of pathogens  on days 21, 28, 35, and 42 but markedly decreased the FCR at related time points in chicken for the Eimeria infection model  for 30 days had remarkably increased final BW on day 60 in comparison with the control group  had increased contents of SOD and GSH-Px in serum, while the concentration of MDA was decreased  and mucin proteins. The serum DAO and d-lactate reflect intestinal permeability, and there were no differences between the Bif and Con groups as well. However, B. animalis-treated pigs markedly increased the number of goblet cells in the jejunum. A recent report demonstrated that the relative mRNA expression of claudin-1 and occludin were not affected in the ileum and colon of B. animalis BB12-treated germfree piglets, respectively, compared to those in the control group  was fed a diet with 1010 CFU Bifidobacterium animalis subsp. lactis per kg. Bifidobacterium animalis subsp. lactis JYBR-190 was provided by ZhongKe-JiaYi Biological Engineering Co., Ltd. . The experimental period included two feeding phases: phase 1, from day 1 to day 14 postweaning; phase 2, from day 15 to day 28 postweaning. The dietary compositions and nutrient concentrations are presented in ad libitum access to feed and water throughout the trial for 28 days.A total of 96 (Duroc × Landrace × Large White) weaning piglets with average initial body weight (BW) of 8.16 ± 0.12 kg were randomly divided into 2 groups based on BW and sex. There were 6 replicate pens per treatment and 8 piglets per pen. The control group (Con) received a control diet without supplementation of probiotics. The Individual pig BW was weighed on days 0, 14, and 28. Pen feed intake was also recorded on days 14 and 28 of the experiment. The average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR) were calculated accordingly. The feces of pigs were scored daily for diarrhea according to the following criteria: 1, firm and well-form feces; 2, soft and form feces; 3, sloppy feces and mild diarrhea; and 4, pasty and liquid diarrhea. The incidence of diarrhea was calculated for the first 7 d after weaning. The incidence of diarrhea was calculated as follows: diarrhea incidence (%) = /(number of pigs per pen × 7 d) × 100%.g for 15 min at 4°C. Fresh fecal samples were collected into 2 mL sterile tubes on day 28 and stored at −80°C for further analysis.On day 28, blood samples were collected from the jugular vein (at least one pig per pen) and serum was separated after centrifugation at 3,000 × On day 29, five pigs from each group were euthanized. The digesta samples from the ileum, cecum, and colon were immediately collected and placed in liquid nitrogen, and then stored at −80°C for further analysis. The different segments of intestinal samples were collected as described previously . Segmentd-lactate, immunoglobulin A (IgA), IgG, IgM, interleukin-1β (IL-1β), IL-6, and IL-10 were detected by commercial ELISA kits  according to manufacturer’s instructions. The serum content of diamine oxidase (DAO) was measured using the biochemical kit following the manufacturer’s protocols . The contents of total superoxide dismutase (T-SOD), catalase (CAT), total antioxidant capacity (T-AOC), malondialdehyde (MDA), and glutathione peroxidase (GSH-Px) capacity in serum were determined using commercial kits , respectively.The contents of serum g and 4°C for 10 min. The protein concentration of the jejunal supernatants was measured using the bicinchoninic acid  method according to the manufacturer's protocols. The jejunum mucosal antioxidant indices were determined as described above. The activities of trypsin, pepsin, lipase, and amylase were determined using commercial kits .Jejunum mucosal tissues (approximately 100 mg) were weighed, homogenized in ice-cold 1 mL PBS (phosphate buffer solution), and centrifuged at 3,000 × The segments of the small intestine were sectioned (5 μm). The sections were stained with the HE method or PAS method. The images were captured under a light microscope . The villus length (VH) and crypt depth (CD) was measured by random measurements of 10 VH and 10 CD per section. The intestinal goblet cell numbers in the field view were counted by a random selection of a field view in each section. The goblet cells are stained red color by PAS.−△△Ct was used to calculate the relative gene expression and glyceraldehyde-3-phosphate dehydrogenase (GADPH) was normalized as the internal control. All samples were measured in duplicate.Total RNA was extracted from the tissue using TRIzol Reagent  according to the manufacturer’s protocol. 1.0 μg of RNA was used to synthesize the first-strand cDNA by using a reverse transcription kit . The synthesized cDNA was diluted  with ultrapure-water, and then stored at −20°C for future analysis. The quantitative real-time PCR was performed on Roche LightCycler 96 system  with specific primers . The metACTCCTACGGGAGGCAGCAG) and 806R (GGACTACHVGGGTWTCTAAT). The purified PCR products were equimolarly combined and paired-end sequenced on the Illumina Miseq platform .Total microbial DNA of the digesta and mucosa from the ileum, cecum, and colon was isolated using the QIAamp Fast DNA Stool Minikit  following the manufacturer’s protocol. The V3-V4 hypervariable regions of bacteria 16S rRNA genes were amplified with universal primers 338F (https://cloud.majorbio.com/).The raw reads were demultiplexed and quality filtered by QIIME (version 1.9) software with the following criteria (i) the lower-quality sequences with a length of <220 nt or >500 nt, an average quality score of <20, and sequences containing >3 nitrogenous bases were removed; (ii) only overlapping sequences longer than 10 bp and the reads without more than 2 nucleotide mismatches in the primer were assembled. The remaining sequences with 97% similarity were clustered into the same operational taxonomic units (OTUs) using UPARSE (version 7.1) and chimeric sequences were removed. The taxonomy of each OTU representative sequence was analyzed by Ribosomal Database Project (RDP) classifier against the SILVA 16S rRNA gene database using a confidence threshold of 70%. The analysis of the alpha diversity was calculated by the MOTHUR program. For the beta diversity, principal coordinate analysis (PCoA) was performed based on Bray-Curtis distances. The data were analyzed on the Majorbio I-Sanger cloud platform , and Salmonella were presented in Total bacterial DNA was separated from fecal samples as mentioned above. The specific primers of total bacteria, t test. The incidence of diarrhea was analyzed by χ2 tests. A Nonparametric Wilcoxon rank-sum test was performed to compare the microbiota difference between the Con and Bif piglets at different intestinal segments. P values were adjusted with a false discovery rate (FDR). The differential bacteria were identified by linear discriminant analysis (LDA) effect size (LEfSe) analysis. The level of statistical significance was P < 0.05, whereas 0.05 ≤ P ≤ 0.1 was considered a trend of significant difference. Data are presented as means ± standard error of the means (SEM).Data were performed using SAS 9.4 version . The data of the two groups were analyzed by unpaired Student's This work was approved by the Care and Use of Experimental Animal Committee of China Agricultural University (CAU No. AW51211202-1-2).PRJNA828137.All raw FASTQ reads for this study can be found in NCBI Sequence Read Archive (SRA) database


Six species of alfalfa commonly found in northern China were collected in the present study.The chemical composition and epiphytic microbial communities during the ensiling were analyzed; and their effects on fermentation quality and silage bacterial communities were assessed. The effects of physicochemical characteristics of alfalfa on the bacterial community were also investigated in terms of nutritional sources of microbial growth and reproduction.p<0.05). After ensiling, both the fermentation quality and microbial community changed obviously (p<0.05). Specifically, ZM2 had lower pH and ammonia nitrogen (NH3-N) content but higher LA content than other varieties of alfalfa silage. Beneficial bacteria such as Lentilactobacillus and Lactiplantibacillus were predominant in ZM2, which accounted for the higher fermentation quality. Significant correlations between the chemical composition of silage, fermentation quality and bacterial communities composition were observed. Moreover, variations in bacteria community structure during the fermentation of alfalfa were mainly influenced by water-soluble carbohydrates (36.79%) and dry matter (21.77%).The results showed that the chemical composition was significantly different in various alfalfa varieties, yet, the dominant genera attached to each variety of alfalfa was similar, except for pantoea (In conclusion, this study revealed the influence of chemical composition on microbial community and fermentation quality, laying the groundwork for future studies on high-quality silage. Medicago sativa L.) is a leguminous forage with high crude protein (CP) content and high feed value , Zhongmu No. 1 (ZM1), Zhongmu No. 2 (ZM2), Gongnong No. 1 (GN), WL168 (WL) and Xinjiangdaye (XJD) were planted in the Agriculture and Animal Husbandry Interlaced Area Test Base of Institute of grassland research of caas in Shaerqin, Inner Mongolia Autonomous Region (111°45′E、40°34′N), China. Six varieties of squaring stage alfalfa were harvested on June 4, 2021, and wilted in the field for 12 h. The wilted forages from the field were chopped to 10–20 mm lengths using a hay cutter. The silage raw materials were mixed and packed into special vacuum packaging bags  for silage; each bag was about 200 g, which was quickly vacuum sealed by a vacuum packaging machine . Every treatment was repeated three times, and all samples were stored for 60 days at room temperature (20–30°C).3-N was determined using the phenol-hypochlorite reaction method, according to the method described by The chemical composition and fermentation quality of fresh and ensiled alfalfa were analyzed. DM content was determined by oven drying at 65°C for 48 h, according to The DNA extraction was performed according to the kit instructions , and the quality of the extracted DNA was detected by 1% agarose gel electrophoresis. The 16S rRNA gene (V3-V4 region) was amplified using the specific primers 799F (5′-AACMGGATTAGATACCCKG-3′) and 1193R (5′- ACGTCATCCCCACCTTCC-3′) with sample-specific barcodes. The bacterial 16S rDNA gene fragment was amplified by PCR . Each sample was replicated 3 times. The PCR products of the same sample were mixed and detected by 2% agarose gel electrophoresis. The AxyPrep DNA gel recovery kit (AXYGEN Company) was used to cut the gel to recover the PCR products, and Tris HCl was used for elution. Next, the PCR products were detected and quantified by QuantiFluor™-ST blue fluorescence quantitative system (Promega Company) and then mixed in the corresponding proportion according to the requirement of sequencing quantity of each sample. Finally, the PCR products were denatured with sodium hydroxide to generate single-stranded DNA fragments sequenced on the Illumina platform 250PE.Trimmomatic software was used to filter the quality of reads and filter out reads below 50 bp. FLASH software was used to merge the paired reads into one sequence, and the minimum overlap length is 10 bp. Chimera removal was conducted using Usearch software and the Gold database. All sequences were divided into OTUs using Usearch software (Version 10) according to the similarity level of different sequences, and bioinformatics statistical analysis was usually performed on OTUs at the 97% similarity level. The species abundance index was calculated by Mothur software (Version 1.30.1) for sequences with an OTU similarity level of more than 97% . PCA anap < 0.05 was statistically significant, and a value of p < 0.01 was highly statistically significant.The raw data were sorted by Excel 2010 software and statistically analyzed by SPSS 20.0 software. Data from different experimental groups were analyzed using one-way ANOVA and differences were compared using the least significant difference test. A value of p < 0.05). The highest DM content was found in XJD and the lowest DM in ZC. The CP content of WL and the WSC content of XJD were significantly higher than the others (p < 0.05). The NDF content of alfalfa ranged from 364 to 419 g/kg DM, with ZM2 and WL having the highest and lowest NDF content, respectively. The ADF content of alfalfa ranged from 257 to 345 g/kg DM, with ZC and GN having the highest and lowest ADF content, respectively.The chemical composition of different varieties of alfalfa is shown in p < 0.001). After 60 days of ensiling, the DM content of the silage ranged from 481 g/kg FM to 556 g/kg FM, with WL (556.15 g/kg FM) and XJD (545.02 g/kg FM) significantly higher than other alfalfa varieties (p < 0.05). The CP content of WL (204.23 g/kg DM) was significantly higher than the others (p < 0.05). Moreover, XJD exhibited the highest content of WSC, while ZC had the lowest WSC content. Finally, the NDF and ADF contents of XJD were significantly lower.The chemical composition of different alfalfa varieties after fermentation is shown in 3-N, LA, and AA. After 60 days of fermentation, the pH values of each type of alfalfa after ensiling ranged between 4.49 and 5.36, and the pH values of each variety of alfalfa after fermentation were significantly lower before fermentation (p < 0.05). There was a higher NH3-N content in WL and XJD, LA content in ZC, and AA content in XJD compared with other varieties of alfalfa silage (p < 0.05). The LA/AA values of ZC and ZM2 were significantly higher (p < 0.05). Furthermore, no PA or BA was detected in alfalfa silage from either species.The fermentation quality of different varieties of alfalfa silage was described as shown in p = 0.268, p = 0.106, p = 0.223), indicating that each variety of alfalfa had the same bacterial composition before ensiling. After ensiling, the Chao1, Shannon, and ACE indices of alfalfa silage of each variety significantly decreased , which indicated that the composition of alfalfa epiphytic bacterial community was influenced by fermentation during ensiling.High-throughput sequencing of the V3-V4 variable region of 16S rDNA of fresh alfalfa and alfalfa silage epiphytes was performed, and most bacteria were detected in all samples yielding a Good’s Coverage Index of approximately 1. The alpha diversity of bacterial communities was calculated and evaluated . In thisPCA demonstrated the distribution of bacterial communities in each group of samples . The epiProteobacteria and Actinobacteriota dominated at the phylum level for each alfalfa species. After ensiling, the dominant phylum of epiphytic bacteria changed to Firmicutes, and the abundance of Proteobacteria and Actinobacteriota decreased in all varieties of alfalfa.The epiphytic bacterial community composition (phylum level) of fresh alfalfa and alfalfa silage is shown in Bradyrhizobium was the dominant genus for ZC (38.98%), ZM1 (32.95%), ZM2 (36%), GN (36.42%), WL (32.68%) and XJD (47.09%). The abundance of Bradyrhizobium was significantly reduced in all alfalfa species after ensiling. The dominant genera in the ZC group were Fructilactobacillus (47.62%), Lentilactobacillus (31.92%), and Lactiplantibacillus (12.24%). The dominant genera in the ZM1 group were Lactiplantibacillus (85.48%) and Fructilactobacillus (11.60%). The dominant genera in the ZM2 group were Lentilactobacillus (67.10%) and Lactiplantibacillus (31.50%). The dominant genus in the GN group was Lactiplantibacillus (82.25%). Moreover, Enterococcus (43.11%), Pediococcus (23.83%) and Bradyrhizobium (13.67%) were the predominant genera in the WL group. The dominant genera in the XJD group were Fructilactobacillus (37.58%), Enterococcus (31.90%) and Bradyrhizobium (11.81%).A stacked bar chart was generated to visualize the bacterial species composition at the silage genus level for each alfalfa species . Before Pantoea, there was no significant difference in the abundance of dominant epiphytic bacteria in various alfalfa varieties. The abundance of WL epiphytic Pantoea was significantly higher than the others. During the ensiling process, the abundance of alfalfa epiphytic dominant bacteria of all varieties decreased sharply. Accordingly, after ensiling, the dominant genera were Lactiplantibacillus, Lentilactobacillus, Enterococcus, Bradyrhizobium, and Pediococcus.The variation in bacterial communities of each sample is depicted in Bradyrhizobium, Mesorhizobium, Methylovirgula and Mycobacterium and negatively correlated with the abundance of Lentilactobacillus (p < 0.01). The silage DM was significantly correlated with the abundance of Asinibacterium, Bradyrhizobium, Mesorhizobium, Methylovirgula, Mitochondria_norank and Mycobacterium (p < 0.001) and negatively correlated with Lactiplantibacillus (p < 0.05). The silage CP was significantly positively correlated with the abundance of Pediococcus (p < 0.001), Mitochondria_norank (p < 0.01) and Asinibacterium (p < 0.05). The silage WSC showed a significant positive correlation with the abundance of Mitochondria_norank and a negative correlation with Lentilactobacillus. The silage NH3-N was highly significantly positively correlated with the abundance of Asinibacterium, Bradyrhizobium, Enterococcus, Methylovirgula and Mycobacterium (p < 0.001) and negatively correlated with the abundance of Lactiplantibacillus (p < 0.01). The silage NDF was negatively correlated (p < 0.001) with the abundance of Bradyrhizobium, Mesorhizobium, Methylovirgula and Mycobacterium. Moreover, the silage ADF was negatively correlated with the abundance of Mesorhizobium, Methylovirgula and Mycobacterium (p < 0.01). Finally, the silage LA and AA were negatively correlated with Lactiplantibacillus .Pearson correlation analysis was conducted between bacteria and fermentation parameters at the genus level . The silAggregated boosted tree analysis was conducted to assess the relationship between the chemical composition of silage ingredients and the bacterial community of silage . The resIt is well-established that the chemical composition of alfalfa can vary significantly depending on the variety, harvesting period and storage method . In thisClostridium can be inhibited when the DM content exceeds 300 g/kg FM (p < 0.05), which may be attributed to significant protein degradation into soluble proteins under the action of proteolytic enzymes during the ensiling process, which led to decreased non-degradable protein content and significantly increased content of soluble crude protein and non-protein nitrogen . Besides, in this study, the LA/AA values of all alfalfa forages except ZM2 were less than 2, which indicated that the remaining five alfalfa silages were mainly based on acetic acid fermentation. Anisotropic fermenting LAB can convert LA to AA, resulting in lower LA/AA values  repository, accession number SRA412564.JL: methodology, visualization, and data curation. GL: experimental design and implementation of some experiments. LinS: conceptualization, acquisition, reviewing, and editing. SW, XM, and LicS: interpret the data and edit the language. LY and LX: conceive the study and review the manuscript. All authors who contributed to the manuscript have read and approved the final version.This research was received financial support from the Natural Science Foundation of Inner Mongolia (No. 2020ZD08) and Science and Technology project of the Inner Mongolia Autonomous Region (No. 2021GG0157).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 triglyceride glucose (TyG) index has been proposed as a reliable marker of insulin resistance (IR) and an independent predictor of cardiovascular disease risk. However, its prognostic value in patients with acute decompensated heart failure (ADHF) remains unclear.A total of 932 hospitalized patients with ADHF from January 1st, 2018 to February 1st, 2021 were included in this retrospective study. The TyG index was calculated as ln [fasting triglyceride level (mg/dL) × fasting plasma glucose level (mg/dL)/2]. Patients were divided into tertiles according to TyG index values. The primary endpoints were all-cause death, cardiovascular (CV) death and major adverse cardiac and cerebral events (MACCEs) during follow-up. We used multivariate adjusted Cox proportional hazard models and restricted cubic spline analysis to investigate the associations of the TyG index with primary endpoints.During a median follow-up time of 478 days, all-cause death, CV death and MACCEs occurred in 140 (15.0%), 103 (11.1%) and 443 (47.9%) cases, respectively. In multivariate Cox proportional hazard models, the risk of incident primary endpoints was associated with the highest TyG tertile. After adjustment for confounding factors, hazard ratios (HRs) for the highest tertile (TyG index ≥ 9.32) versus the lowest tertile (TyG index < 8.83) were 2.09  for all-cause death, 2.31  for CV death and 1.83  for MACCEs. Restricted cubic spline analysis also showed that the cumulative risk of primary endpoints increased as TyG index increased. When the TyG index was used as a continuous variable, the hazard ratios of the three primary endpoints rapidly increased within the higher range of the TyG index .The elevated TyG index was independently associated with poor prognosis, and thus would be useful in the risk stratification in patients with ADHF. Despite similar clinical presentations, acute decompensated heart failure (ADHF) is a highly heterogeneous syndrome with incompletely understood pathophysiology , 2. In cInsulin resistance (IR), a marker of metabolic disorders and systemic inflammation, is an independent and significant risk factor for HF and cardiovascular (CV) death –13. The However, there are limited clinical studies assessing the TyG index in ADHF. Therefore, we conducted a retrospective cohort study to investigate the relationship between the TyG index and the prognosis in ADHF patients.This is a single-center retrospective analysis of 1376 consecutive ADHF patients admitted to our hospital from January 1st, 2018 to February 1st, 2021. ADHF was defined according to the definitions established in the 2021 ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure . Of the 2). Hypertension was defined as systolic blood pressure (SBP) ≥ 140 mmHg and/or diastolic blood pressure (DBP) ≥ 90 mmHg, any use of the antihypertensive drugs, or a having a history of hypertension. Diabetes mellitus (DM) was defined as fasting plasma glucose (FPG) ≥ 126 mg/dL or hemoglobin A1c (HbA1c) ≥ 6.5%, or a self-reported history of diabetes  for those with normal and skewed distributions, respectively. Categorical variables were expressed as number (percentage).2 test among groups.The patients were divided into tertiles according to their TyG index levels: tertile 1 (T1), TyG index < 8.83; tetile 2 (T2), TyG index ≥ 8.83 and < 9.32; tertile 3 (T3), TyG index ≥ 9.32. Continuous variables were compared by analysis of variance (ANOVA) or the Kruskal–Wallis test among the three groups. Categorical variables were compared by the χ2), and history of diabetes mellitus (no versus yes). Interactions between subgroups were tested by the likelihood ratio test. All data were analyzed with R version 4.1.0 and SPSS for windows version 26, and p < 0.05 was considered to indicate statistical significance.The cumulative event-free survival rates of the three endpoints were analyzed using the Kaplan–Meier plots and the log-rank test. Additionally, to rule out the effects of confounding factors, adjusted Kaplan–Meier survival curves were generated. Adjusted survival curves were obtained using non-parametric estimated weights –32. MultThe baseline clinical characteristics of the included patients by TyG tertile are presented in Table During the follow-up period, all-cause death was found in 140 (15.0%) cases, CV death occurred in 103 (11.1%) cases and MACCEs were detected in 443 (47.9%) cases. The patients who died included 45 (14.5%) in the lowest tertile, 42 (13.6%) in the median tertile and 53 (16.9%) in the highest tertile. The patients who died from cardiovascular events included 34 (11%) in the lowest tertile, 28 (9.1%) in the median tertile and 41 (13.1%) in the highest tertile. Individuals with major adverse cardiac and cerebral events included 156 (51.1%) in the lowest tertile, 130 (42.3%) in the median tertile and 157 (50.2%) in the highest tertile.The unadjusted Kaplan–Meier curves demonstrated no significant differences among tertiles in the three predefined primary endpoints Fig. . HoweverIn Fig. The subgroup analysis showed that the associations of the TyG index tertile with the risk of the three primary outcomes were consistent across the subgroups, including age, sex, BMI, LVEF, and history of diabetes mellitus Figs. , 6, 7. IIn this retrospective research, we first demonstrated the associations of the TyG index with the risk of all-cause death, CV death, and MACCEs  in patients with ADHF. We found that patients with elevated TyG index were at greater risk of adverse endpoints, regardless of their diabetic status. The results indicated that the TyG index was an independent predictor of adverse events in ADHF patients. Most significantly, this research proposed a simple and efficient method for evaluating IR to optimize risk stratification of mortality and cardiovascular disease recurrence in ADHF patients.Epidemiological studies have shown that ADHF is a prevalent and serious condition with significant morbidity and mortality, imposing a growing global public health burden . We urgeIR is defined as a clinical or experimental condition in which glucose uptake and utilization are impaired . AccordiIn recent years, numerous clinical studies have shown that the TyG index is correlated with the risk of incident cardiovascular disease. Two retrospective cohort studies by Sánchez Iñigo et al. and Li et al. showed that healthy participants with elevated TyG index are at higher risk of cardiovascular events , 24. AnoA retrospective cohort study reported the TyG index is correlated with the prognosis of patients with CHF and type 2 DM . AnotherTherefore, for optimizing the risk stratification in patients with heart failure and exploring more potential therapeutic options, it is necessary to examine the link between HF and IR. The TyG index is more convenient for routine screening in clinical practice than HOMA-IR. The TyG index could be monitored in patients with HF, particularly ADHF cases because of difficulty in treatment and poor prognosis.There were several limitations in this study. Firstly, due to the limitations of a retrospective design, we were unable to dynamically measure TyG in patients over the follow-up period. Secondly, as this was a single-center study with a limited sample size, data bias could not be avoided despite correction for multiple confounding factors. Thirdly, because of the limited clinical information available, the differences between TyG and other IR indicators, including HOMA-IR and HIEC, in the prognosis of ADHF were not studied. Furthermore, prospective cohort studies are required to validate our findings.In summary, this study demonstrated that the TyG index was directly correlated with poor prognosis in patients with ADHF regardless of DM status. Elevated TyG index could be a predictor and risk stratification tool for all-cause death, cardiovascular death, and MACCEs in patients with ADHF.