People with uncontrolled type 2 diabetes (T2DM) often delay initiating and titrating basal insulin. Patient‐managed titration may reduce such deferral. The Italian Titration Approach Study (ITAS) compared the efficacy and safety of insulin glargine 300 U/mL (Gla‐300) initiation and titration using patient‐ (nurse‐supported) or physician‐management in insulin‐naïve patients with uncontrolled T2DM.1c. Secondary outcomes included hypoglycaemia incidence and rate, change in fasting self‐monitored plasma glucose, patient‐reported outcomes (PROs), and adverse events.ITAS was a multicentre, phase IV, 24‐week, open‐label, randomized (1:1), parallel‐group study. Insulin‐naïve adults with T2DM for ≥1 year with poor metabolic control initiated Gla‐300 after discontinuation of SU/glinides, and were randomized to self‐titrate insulin dose (nurse‐assisted) or have it done by the physician. The primary endpoint was change in HbA1c reduction from baseline was non‐inferior in patient‐ vs physician‐managed arms . The incidence and rates of hypoglycaemia were similarly low in both arms: relative risk of confirmed and/or severe nocturnal (00:00‐05:59 hours) hypoglycaemia was 0.77 (95% CI: 0.27 to 2.18). No differences were observed for improvement in PROs. No safety concerns were reported.Three hundred and fifty five participants were included in the intention‐to‐treat population. At Week 24, HbAIn the T2DM insulin‐naïve, SU/glinides discontinued population, patient‐managed (nurse‐assisted) titration of Gla‐300 may be a suitable option as it provides improved glycaemic control with low risk of hypoglycaemia, similar to physician‐managed titration. Participants were insulin‐naïve adults (≥18 years of age) with T2DM for ≥1 year with poor glycaemic control (HbA1c ≥7.5 to ≤10%) on oral antihyperglycaemic drugs (OADs) and/or non‐insulin injectables. After withdrawal of SU/glinides, if any, to evaluate the hypoglycaemia risk solely due to Gla‐300, all participants were treated with Gla‐300 and were randomized to either self‐adjust their BI dose, or have their dose adjusted by a physician during visits or by telephone. The study consisted of a 2‐week screening period and a 24‐week treatment period . In each arm, visits/contacts were weekly until week 12, and then every 2 weeks until week 24. Additional unscheduled contacts  were made available if clinically required. Full details of the methodology have recently been reported.ITAS was a national, multicentre, phase IV, 24‐week, open‐label, randomized (1:1), parallel‐group study, conducted in Italy (EudraCT Number: 2015‐001167‐39). The primary aim of this study was to assess non‐inferiority of change in HbAAll participants provided informed, written consent. The clinical trial protocol was approved by the appropriate local Ethical Committees and IRB/IEC. The study was conducted in accordance with the Declaration of Helsinki and the ICH guidelines for good clinical practice.2.2All patients were instructed to self‐administer a daily subcutaneous injection of Gla‐300  in the evening, anytime from dinner to bedtime. Gla‐300 was administered at a starting dose of 0.2 U/kg and then adjusted at each visit in the physician‐managed arm, and weekly or even more frequently (but no more often than every 3‐4 days) in the patient (nurse‐assisted) managed arm to achieve a fasting self‐monitored plasma glucose (SMPG) of 80‐110 mg/dL. In both groups, changes in the insulin dose were based on the median of the fasting SMPG values measured on three consecutive days, of which the last was the day when titration was scheduled. Patients randomized to self‐managing insulin titration received a specific educational session regarding self‐adjustment of insulin dose from the study nurse, who monitored algorithm application without however exerting any influence on titration. The patient‐ and physician‐managed titration algorithm is shown in Table Rescue therapy, if needed, either by adding a new OAD and/or by increasing the dose of an existing antihyperglycaemic non‐study drug, was based on the Investigator's judgement, considering primarily the patient's individual clinical needs, but also local guidelines and Gla‐300 labelling.At randomization, investigators provided patients with a blood glucometer  and diary to assess and record daily fasting SMPG until it was stable at the target. A seven‐point SMPG profile was measured at Week 12 and Week 24. The investigator explained the need to measure glucose at the times requested by the study and to record the values correctly in the diary that the patients brought along with the glucometer at each office visit.Patients were instructed to measure capillary plasma glucose whenever they experienced symptoms of hypoglycaemia. All hypoglycaemia episodes were recorded in the patient's diary or documented in the “hypoglycaemia screen/page” of the electronic case report form (e‐CRF). Nocturnal hypoglycaemia was any event that occurred between 00:00 and 05:59. Severe hypoglycaemia was defined as an event requiring assistance of another person to actively administer carbohydrate or glucagon or to perform other resuscitative actions.2.31c from baseline to Week 24 (assessed using 0.3% as non‐inferiority margin). The main secondary endpoint was the percentage of participants with ≥1 confirmed [≤70 mg/dL (≤3.9 mmol/L)] and/or severe nocturnal (00:00‐05:59 hours) hypoglycaemic event from baseline to Week 24. Other efficacy endpoints included percentage of participants with ≥1 confirmed [≤70 mg/dL (≤3.9 mmol/L)] and/or severe hypoglycaemic event during any time of day (24 hours) or between 00:00 hour and pre‐breakfast , and the number of hypoglycaemic events per patient‐year during the study treatment; the cut‐off <54 mg/dL was also investigated for each category of hypoglycaemia. Other secondary endpoints included change from baseline to Week 24 in fasting plasma glucose (FPG), fasting SMPG (a seven‐point SMPG profile was performed at Week 12 and Week 24), insulin dose, body weight, and percentage of patients with HbA1c target achievement .The primary endpoint of the study was change in HbAOther assessments included patient‐reported outcomes (PROs), which were collected using the following: Problem Areas in Diabetes‐5 (PAID‐5) questionnaire, Diabetes Empowerment Scale – short form (DES‐SF) questionnaire, and Diabetes Treatment Satisfaction Questionnaire (DTSQ). Safety and tolerability analyses were based on all hypoglycaemic events, skin reaction at injection site, hypersensitivity reactions, and any other adverse events (AE) or serious adverse events (SAE).2.4Full details of the statistical analyses have previously been reported.1c baseline value as the covariate. To assess the non‐inferiority of patient‐managed vs physician‐managed titration, the upper bound of the 95% confidence interval (CI) for the estimated difference in the mean change of HbA1c from baseline to endpoint at Week 24 between the two titration approaches was compared with the predefined non‐inferiority margin of 0.3% HbA1c. Non‐inferiority was shown if the upper bound of the two‐sided 95% CI of the estimated difference for the ITT population was <0.3%.The primary endpoint was analysed using a linear mixed‐effect model (LMEM) with titration approach and centre as the fixed effect and the HbAThe risk of hypoglycaemic events in the two titration groups was compared in terms of relative risk, whereas a rate ratio was computed to compare the annual incidence rates of same type of events in the two‐titration arms. All statistical comparisons between the two titration groups were based on 95% CI. The cumulative number of confirmed and/or severe hypoglycaemic events per patient was provided by titration approach and described through descriptive statistics for continuous variables. Moreover, the cumulative mean functions of anytime (24 hours) or nocturnal confirmed [≤70 mg/dL (≤3.9 mmol/L)] and/or severe hypoglycaemia were estimated and graphically presented.1c targets were summarized descriptively by titration group, and chi‐square tests were applied for comparison.FPG, fasting SMPG and body weight were analysed using the same LMEM described above using the baseline value as a covariate. The frequency and percentage of patients at different HbAChanges in PAID‐5 and DES‐SF total scores were computed and analysed with a LMEM using the effect of the titration approach and the baseline total score as covariates. DTSQ at the end of the treatment is reported through descriptive statistics in each study arm. Safety endpoints were analysed descriptively.33.11c criteria  [72.33 mmol/mol (SD: 7.32)] and 8.82% (SD: 0.64) [72.88 (SD: 7.00) mmol/mol] in the patient‐ and physician‐managed titration arms, respectively. For the primary endpoint, the HbA1c reduction from baseline to Week 24 was non‐inferior in the patient‐ vs physician‐managed group. The least squares mean (LSM) change in HbA1c was −1.60% (SE: 0.06) [−17.49 mmol/mol (0.66)] vs −1.49% (SE: 0.06) [−16.29 mmol/mol (SE: 0.66)] in the patient‐ and physician‐managed arms, respectively; LSM difference: −0.11% (95% CI: −0.26 to 0.04) [−1.20 mmol/mol (95% CI: −2.84 to 0.44)] (P = .1683) in the ITT population  and physician‐managed (35.2%) groups at Week 24 (P = .30).At baseline, the mean HbAn Figure . The pro3.4P = .987]. Mean FPG at 24 weeks was 109 ± 29 mg/dL and 109 ± 28 mg/dL in the patient‐ and physician‐managed groups, respectively. There was no difference in the profiles of progressive reduction in fasting SMPG in the patient‐ and physician‐managed groups  ; 3.53.5.1] and/or severe nocturnal (00:00‐05:59 hours) event  .9 mmol/LFigure . AnnualiFigure . IncidenFigure .3.5.2No differences were seen in the incidence or annualized rates of confirmed [≤70 mg/dL ≤3.9 mmol/L)] hypoglycaemic events at any time of day (24 hours) between the patient‐ and the physician‐managed groups  or nocturnal confirmed [≤70 mg/dL (≤3.9 mmol/L)] and/or severe hypoglycaemia was not different between the two treatment groups : −0.34 (0.31) kg vs 0.10 (0.30) kg, respectively; estimated difference between groups: −0.43 kg (95% CI: −1.21 to 0.34); 3.81c <7.0% without severe and/or confirmed hypoglycaemia were also not different between the groups  (P = .50). There was also no significant difference between the patient‐ and physician‐managed groups among patients who achieved HbA1c <7.0% without severe and/or confirmed hypoglycaemic events and no weight increase  (P = .21).The proportion of patients achieving HbA3.9P = .70]. Total LSM scores (SE) in the PAID‐5 questionnaire were reduced (improved) by −1.32 (0.42) points in the patient‐managed group and −1.51 (0.41) points in the physician‐managed group .There was no difference between study groups for improvement in DTSQ treatment satisfaction between baseline and Week 24 Figure . For the3.10A summary of treatment‐emergent adverse events (TEAEs) can be found in Table 41c close to 7.0%. Taken together, the results of ITAS demonstrate not only the efficacy of Gla‐300 using a simpler patient‐ vs a physician‐managed titration process but also the safety of such an approach. Of note, the results of ITAS were obtained in the context of identical numbers of visits/contacts in the two arms of the study, that is, with nurses and physicians, respectively. These positive results of ITAS using Gla‐300 may help in reducing the barriers to initiation and titration of BI in uncontrolled T2DM people who need to initiate BI. Several previous studies have compared patient‐ and physician‐managed titration using BI Gla‐100 [AT.LANTUS (insulin‐naïve and pre‐treated participants)1c with patient‐ vs physician‐managed titration. However, overall incidence of hypoglycaemia was higher in the patient‐ vs physician‐managed titration arms in AT.LANTUS (P < .01) and ATLAS . In ITAS, there is a lower risk of hypoglycaemia (any definition) in both patient‐ and physician‐managed BI titration groups, likely because the insulin‐naïve population studied had lower hypoglycaemia risk, and also because Gla‐300 carries a lower risk of hypoglycaemia vs Gla‐100.ITAS is the first randomized controlled trial specifically comparing the efficacy and safety of initiation and titration of Gla‐300 managed by either the patient (nurse‐assisted) or physician, in an insulin‐naïve population with uncontrolled T2DM, after withdrawal of SU/glinides. The results of ITAS show that patient‐managed titration of Gla‐300 achieves non‐inferior glucose control with similarly low risk of hypoglycaemia compared with physician‐managed titration, with a successful end‐of‐study HbA1c reduction. However, this study was made in a population heterogeneous for antecedent insulin use since the majority of subjects at baseline were on long‐term insulin and therefore familiar with this treatment. Incidence and rates of hypoglycaemia were higher in TAKE CONTROL compared to ITAS, likely because of the greater hypoglycaemia risk . Although continuation vs withdrawal of SU at time of BI initiation is still a debated question, evidence indicates that continuation of SU upon initiating BI increases the risk for hypoglycaemia,Recently, the TAKE CONTROL studyInterestingly, in ITAS, there is a lower risk of hypoglycaemia also when the comparison is done vs another study, EDITION 3, in which BI was initiated in insulin‐naïve subjects after withdrawal of SU/glinides.Regardless of the mechanism, reducing hypoglycaemia on BI treatment in T2DM is an important goal to improve long‐term adherence of patients to insulin treatment and titration to target, as demonstrated by the high retention of patients in ITAS in both groups for the entire duration of the study. Minimizing the risk of even non‐severe hypoglycaemia, as in ITAS, is important to prevent long‐term hypoglycaemia unawareness,Interestingly, there was no increase in body weight with Gla‐300 neither in patient‐ nor in physician‐managed BI titration, in line with previous observations.As in TAKE CONTROL, ITAS reported no difference in total PRO scores in the patient‐ and physician‐managed titration arms, demonstrating that patients felt comfortable with self‐titration. In addition, incidence of TEAEs was not different in the patient‐ and physician‐managed groups, as shown in TAKE CONTROL, AT.LANTUS, and ATLAS.ITAS tried to mimic certain features of pragmatism  comparing physician managed titration with patient (nurse‐assisted) self‐titration. Both approaches are similarly successful and safe in ITAS, thus proving the feasibility and convenience of the simpler patient (nurse‐assisted) approach.The limitations of this study include the open‐label design. It should also be noted that the good results of ITAS have been obtained with a number of visits/contacts, which do not necessarily reflect the lower level of interaction that patients would often receive in routine clinical practice in diabetes clinics. Finally, hypoglycaemia events were pragmatically reported and analysed by patient diaries or e‐CRF and not by independent glucose monitoring tools.In conclusion, ITAS provides additional evidence that initiation and titration of BI Gla‐300 are not only efficacious as several previous studies have shown but also safe with low risk for any hypoglycaemia, especially when SU/glinides, a likely confounder, are suspended. Interestingly, these positive results, which include neutrality of BI Gla‐300 on body weight, are achieved similarly when either patients (nurse assisted) or physicians manage BI titration. This encourages development of programmes for wider use of the patient‐managed (nurse‐assisted) approach for BI titration, shifting the locus of control to fight clinical inertia.R.C.B. has received honoraria or consulting fees from Sanofi, Merck Sharp & Dohme, Eli Lilly, Johnson & Johnson, Bristol‐Myers Squibb, AstraZeneca and Janssen. A.G. has received honoraria or consulting fees from Amgen, AstraZeneca, Boehringer Ingelheim, Eli Lilly, MSD, Mundipharma, Sanofi and research funding from AstraZeneca. R.B. has received honoraria or consulting fees from Sanofi, Eli Lilly, Abbott and Novo Nordisk. G.P. has received honoraria or consulting fees from Novo Nordisk, Sanofi, Eli Lilly, AstraZeneca, Boehringer Ingelheim, Takeda, Abbott, Janssen and Merck Sharp & Dohme. D.C. has received honoraria or consulting fees from Eli Lilly, Novo Nordisk, Roche Diagnostics and Sanofi. A.A. has received honoraria or consulting fees from Novo Nordisk, Sanofi, Eli Lilly, AstraZeneca, Boehringer Ingelheim, Takeda and Servier and research funding from AstraZeneca and Vifor Pharma. G.A. has received honoraria or consulting fees from Sanofi, AstraZeneca, IBSA Institut Biochimique and Novartis and research funding from Sanofi, AstraZeneca and Novo Nordisk. M.L. is an employee of Sanofi and holds stocks/shares in Sanofi. C.F. has received honoraria or consulting fees from Sanofi and non‐financial support from Menarini. G.B.B. has received honoraria or consulting fees from Sanofi and Menarini and research funding and speakers' bureau fees from Sanofi.All authors have read and approved the final manuscript. R.C.B. participated as an investigator, provided comments and input to all drafts of the manuscript and interpretation of the results. A.G. participated as an investigator, provided comments and input to all drafts of the manuscript and interpretation of the results. R.B. participated as an investigator, provided comments and input to all drafts of the manuscript and interpretation of the results. G.P. participated as an investigator, provided comments and input to all drafts of the manuscript and interpretation of the results. D.C. participated as an investigator, provided comments and input to all drafts of the manuscript and interpretation of the results. A.A. participated as an investigator, provided comments and input to all drafts of the manuscript and interpretation of the results. G.A. participated as an investigator, provided comments and input to all drafts of the manuscript and interpretation of the results. M.L. provided comments and input to all drafts of the manuscript and interpretation of the results. C.F. participated as an investigator, provided comments and input to all drafts of the manuscript and interpretation of the results. G.B.B. participated as an investigator, provided comments and input to all drafts of the manuscript and interpretation of the results.Table S1 Titration algorithm used by patients and physicians.Table S2. Glucose lowering medications at baseline (ITT population)Table S3. Relative risk and annualized rate of nocturnal, 00:00 hour to pre‐breakfast and anytime (24 hours) hypoglycaemia from baseline to Week 24 (ITT population)Table S4. Summary of patients with treatment‐emergent adverse eventsFigure S1. Study flowFigure S2. HbA1c reduction between baseline and Week 24 (PP population)Figure S3. Hypoglycaemia in an expanded nocturnal window or at any time of day (ITT population)Figure S4. DTSQ at baseline and Week 24 (ITT population)Click here for additional data file.


Anaemia is highly prevalent in gastric cancer (GC) patients. The role of initial haemoglobin levels in predicting the prognosis of GC patients treated by chemotherapy has not been well determined. Our present study aims to evaluate the relationship between the degree of anaemia and the overall survival (OS) and progression-free survival (PFS) of patients with GC.Our retrospective study enrolled 598 patients who were treated with chemotherapy when the recurrent or metastatic GCs were unsuitable for surgical resection. Univariate and multivariate analyses were performed to identify risk factors that had the potential to affect patient prognosis. Additionally, the relationship between clinicopathological characteristics, including treatment method, and degree of cancer-related reduction in haemoglobin was further analysed.ini level ≤ 80 g/L had a trend toward a shortened median OS and PFS . Interestingly, we also found that HBdec ≥ 30 g/L was associated with a significantly shortened median OS and PFS . Multivariate analysis showed that HBini levels ≤80 g/L could be used as an independent prognostic factor for recurrent and metastatic GC. More importantly, HBdec ≥ 30 g/L and treatment response were also significantly associated with OS and PFS. Furthermore, the degree of haemoglobin decrease was associated with chemotherapy including platinum and the number of chemotherapy cycles.Our results revealed that patients with HBOur study concludes that the initial degree of anaemia and a decrease in haemoglobin of ≥30 g/L can serve as biomarkers to predict prognosis in recurrent or metastatic GC patients, while chemotherapy treatment rather than red blood cell (RBC) transfusion can improve their prognosis. Additionally, platinum should not be recommended for treating severely anaemic GC patients. Gastric cancer (GC) is the fifth most common malignant tumour and the third leading cause of death worldwide . RecurreCancer-related anaemia (CRA) is considered to be associated with multiple pathological and clinical factors, such as bleeding, nutritional deficiency, and bone marrow suppression . Bone maOur study aimed to determine the role of initial degree of anaemia and cancer-related haemoglobin reduction in the prognosis of recurrent or metastatic GC patients. The relationships between clinicopathological characteristics, including treatment regimens, and cancer-related haemoglobin reduction degree were further analysed. Our study will contribute to the determination of treatment approaches for recurrent or metastatic GC-related anaemia patients.All procedures followed were in accordance with the ethical standards of the ethical committee of Shandong Provincial Hospital regarding human experimentation and with the 1964 Helsinki Declaration and later versions. Informed consent for inclusion in the study was obtained from all patients.Our retrospective study analysed the data collected from patients diagnosed with metastatic GC or recurrent GC at Shandong Provincial Hospital in China from January 1, 2010, to December 31, 2014. The entry criteria included the following: 1) metastatic GC or recurrent GC after radical surgical treatment was histologically confirmed as gastric adenocarcinoma — radical gastric resection was defined as negative margins, en bloc resection of the greater and lesser omentum, and D2 lymph node dissection, and standard lymphadenectomy was defined as when the number of retrieved lymph nodes was ≥15; 2) The Eastern Cooperative Oncology Group performance score (ECOG PS) was used to estimate a life expectancy of more than 3 months ; and 3) ini) was collected at the initial diagnosis of recurrent or metastatic GC. The lowest haemoglobin level was determined as the lowest level obtained from the day of diagnosis to the date of death or the final follow-up visit. The decrease in haemoglobin (HBdec) was defined by subtracting the lowest haemoglobin level from the initial haemoglobin level. Evaluation and grading of anaemia were performed according to National Comprehensive Cancer Network (NCCN) guidelines for cancer- and chemotherapy-induced anaemia [The initial haemoglobin level ; XP or modifications (capecitabine 1000 mg/m2 twice daily (BID) on days 1–14, cisplatin 75 mg/m2 or oxaliplatin 130 mg/m2 on day 1); FOLFIRI ; paclitaxel liposome 100 mg/m2 (q2w) or 135–150 mg/m2 (q3w) on day 1, combine with capecitabine or S-1; and single agents such as docetaxel 75–100 mg/m2 on day 1, capecitabine 1000–1250 mg/m2 BID on days 1–14, or S-1 80–120 mg on days 1–14, cycled every 21 days. The first line chemotherapy regimens include DCF, Paclitaxel liposome + Capecitabine / S-1 or XP. The second line chemotherapy regimens include FOLFIRI or single agent. The treatment effect of chemotherapy was estimated after 2 cycles of the chemotherapy regimen with 3 weeks or 3 cycles of the chemotherapy regimen with 2 weeks.The regimens used to treat the patients included the combination chemotherapy of docetaxel, cisplatin, and 5-fluorouracil (DCF) and related modifications  1.1 guidelines . The numOverall survival (OS) was calculated as the time from the date of initial diagnosis of metastatic GC or the date of recurrence after GC resection to the date of either death or the final follow-up. Progression-free survival (PFS) was calculated as the date of either disease progression, confirmed by magnetic resonance imaging or computed tomography using a contrast medium if possible, or death from any cause.Clinical variables for risk assessment consisted of patient demographics, surgical and pathological factors, chemotherapy regimens, and packed red cell transfusion. Data regarding recurrence, defined as disease recurrence at any site, and survival outcomes were also collected.Peritoneal metastasis is a frequent type of metastasis of gastric cancer and is a definitive determinant for prognosis. Peritoneal metastasis was diagnosed by histological diagnosis of peritoneal metastasis and/or by peritoneal lavage cytology positive for cancer cells.dec as the outcomes, with a significance level of p < 0.05. Multivariate analysis was carried out with a Cox proportional hazards model to evaluate prognostic factors with respect to PFS, OS and HBdec. The factors which were potential risk factors for GC patient’s prognosis or having statistical significance from the univariate analysis data were performed in Cox multivariate model. Hazard ratios (HRs) and 95% confidence intervals (CIs) were calculated. A value of p < 0.05 was considered statistically significant. All statistical analyses were conducted using SPSS statistical software .Survival analyses were performed by Kaplan-Meier curves with log-rank tests for significance. Statistical analysis included univariate analysis and multivariate analysis. Univariable Cox regression analyses were performed using PFS, OS and HBBased on the inclusion and exclusion criteria, 598 patients were included in our study. Our study included 170 recurrent GC patients and 428 metastatic GC patients. The general characteristics including the kinds of chemotherapy regimen of all enrolled patients are listed in Table There were 312 patients treated with the first line chemotherapy regimens, yet the GC in 188 patients remained in a development condition, and then those patients were treated with the second or/and third line chemotherapy regimens including FOLFIRI and docetaxel single agent. The cycles of chemotherapy used for our GC patients was 4.4 ± 3.705 . Two hunOf the 598 GC patients, the median follow-up time was 11.60 months (range 0–76), and the median OS after chemotherapy was 12 months (95% CI 11.221–12.779), with 1-, 3-, and 5-year OS rates of 45.40, 3.80, and 0.90%, respectively.ini ≤ 80 g/L cohort and the HBini level > 80 g/L cohort. Our study included 40 patients in the HBini ≤ 80 g/L cohort and 558 patients in the HBini level > 80 g/L cohort. The clinical features which have potential effects on GC patient OS and PFS were well matched between our two groups  transfusion is an important treatment modality, while chemotherapy is beneficial for improving the prognosis of recurrent and metastatic GC patients. We analysed the different treatment modalities and clinicopathological parameters for the OS and PFS in our patients.ini level, HBini ≤ 80 g/L, metastatic sites ≥3, liver metastases, paclitaxel-based combination of three regimens, the number of chemotherapy cycles, treatment response, and HBdec ≥ 30 g/L (p < 0.05). Additionally, HBini level, the lowest haemoglobin level, metastatic sites ≥3, liver metastases, bone metastases, number of chemotherapy cycles, chemotherapy including paclitaxel, treatment response and HBdec ≥ 30 g/L were significantly associated with PFS (p < 0.05)  Table .Table 4ini level ≤ 80 g/L , liver metastases , chemotherapy including paclitaxel , treatment response , and HBdec ≥ 30 g/L  were significant adverse prognosis factors of OS. More importantly, the number of chemotherapy cycles was also significantly correlated with improved OS  , chemotherapy including paclitaxel , treatment response , the number of chemotherapy cycles , and HBdec ≥ 30 g/L  were independent prognostic factors  , and was not relevant with chemotherapy included paclitaxel  Table . Multivael Table .Table 6Chemotherapy drugs can not only kill cancer cells, but also damage healthy cells, which causes side effects. Our results revealed that the most common side effects of chemotherapy were myelosuppression, diarrhea and vomiting, yet which could not influence the OS and PFS in our cohort Table .Table 8CRA occurs as a result of multiple aetiologies, including blood loss, functional iron deficiency, erythropoietin deficiency due to renal disease, chemotherapy-induced myelosuppression, marrow involvement with tumours and other factors. The relationship between anaemia and the prognosis of GC patients is rarely reported. Zhang et al. reported that patients with less than ≤65 g/L haemoglobin had a significantly shorter median OS than patients with 65 g/L to normal haemoglobin or patients with normal haemoglobin and demonstrated that a lower haemoglobin level might predict poorer OS in advanced GC patients . There iAccording to the NCCN guidelines for cancer- and chemotherapy-induced anaemia, a haemoglobin level ≤ 80 g/L is used to define severe-grade anaemia. Our present study also chose a haemoglobin level of 80 g/L as the cut-off value for severe anaemia. Our results revealed that pretreatment of severe anaemia could serve as a prognostic factor in metastatic GC or recurrent GC patients who underwent radical resection and were then treated with chemotherapy. Multivariate analysis also showed that an initial haemoglobin level ≤ 80 g/L was an independent adverse prognostic factor for our patients. In addition, the degree of haemoglobin decrease (haemoglobin level ≥ 30 g/L) during chemotherapy or the follow-up period was also an important risk factor for the prognosis of recurrent or metastatic GC.The cause of anaemia in patients with cancer is often multifactorial. The malignancy itself can lead to or exacerbate anaemia, and underlying comorbidities may also contribute to anaemia. Cancer cells can directly suppress haematopoiesis through bone marrow infiltration and produce cytokines, leading to iron sequestration. Chronic blood loss, nutritional deficiencies, myelosuppressive effects of chemotherapy, and radiation therapy to the skeleton can further exacerbate anaemia in patients with cancer –10. Due dec. Our results were also different with previous research which showed that docetaxel, a newly taxoid anticancer drug, can cause a progression in anaemia from grade III to IV in 9% of patients [Previous researches recognized paclitaxel as first- or second-line chemotherapy, in which median overall survival was several months , 17. Ourpatients . The reaTo the contrary, our results showed that chemotherapy including platinum was associated with a decrease in haemoglobin in recurrent or metastatic GC patients, which are similar to the findings of previous reports. Groopman et al. reported that platinum-based regimens are well known to induce anaemia due to the combined bone marrow and kidney toxicity, and the use of chemotherapy regimens including paclitaxel is an adverse prognostic factor for decreased haemoglobin, although this effect is not significant . TherefoThe most common treatment options for CRA include erythropoietic-stimulating agents, RBC transfusion and nutritional therapy, such as iron intake. Previous studies have reported that the lowest postoperative haemoglobin level and postoperative transfusion were the most significant risk factors for postoperative complications in GC surgery . SquiresHowever, our study also has several limitations. First, our study is a retrospective analysis, so our results should be confirmed by multicenter-randomized trials. Second, the inequality in the number of patients enrolled in our different cohorts can also generate potential bias. Third, high proportion of patients  only received 1–2 cycles of chemotherapy treatment, which can also influence the treatment effect of chemotherapy.Our study demonstrated that the initial degree of anaemia can serve as a biomarker for predicting the prognosis of recurrent or metastatic GC patients, while chemotherapy treatment rather than RBC transfusion can improve OS and PFS. In addition, platinum should not be recommended to treat severely anaemic GC patients.Additional file 1 Table S1. Median OS and PFS


The dual contrast technique enabled simultaneous determination of CA4+  and gadoteridol  partitions within cartilage. Cartilage proteoglycan content and biomechanical properties correlated significantly  with CA4+ partition in superficial and middle zones at both diffusion time points. Normalization of the CA4+ partition with gadoteridol partition within the cartilage significantly (p < 0.05) improved the detection sensitivity for human osteoarthritic cartilage proteoglycan content, biomechanical properties, and overall condition . The dual energy technique combined with the dual contrast agent enables assessment of human articular cartilage proteoglycan content and biomechanical properties based on CA4+ partition determined using synchrotron microCT. Additionally, the dual contrast technique is not limited by the beam hardening artifact of conventional CT systems. © 2019 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 38:563–573, 2020Dual contrast micro computed tomography (CT) shows potential for detecting articular cartilage degeneration. However, the performance of conventional CT systems is limited by beam hardening, low image resolution (full‐body CT), and long acquisition times . Therefore, to reveal the full potential of the dual contrast technique for imaging cartilage composition we employ the technique using synchrotron microCT. We hypothesize that the above‐mentioned limitations are overcome with synchrotron microCT utilizing monochromatic X‐ray beam and fast image acquisition. Human osteochondral samples ( Cartilage injury can initiate the development of post‐traumatic osteoarthritis (PTOA) if not detected and treated early after injury. Therefore, early diagnosis is crucial for the initiation of surgical or pharmaceutical treatment to slow down the lesion progression to PTOA.q = +4, and iodine k‐edge 33.2 keV) and non‐ionic, gadolinium‐based  contrast agents. The diffusion of non‐ionic contrast agent reflects the water content and permeability when the contrast agent partition is determined prior to diffusion equilibrium. Further, by normalizing the cationic contrast agent distribution with that of non‐ionic agent distribution, the PG content within cartilage is estimated more accurately.k‐edges .Contrast‐enhanced computed tomography (CECT) enables detection of changes in cartilage composition along with quantitative evaluation of subchondral bone density and structure.k‐edge of the chosen contrast agent. In addition, beam hardening artifacts can occur. These factors might limit the accuracy of the dual contrast technique. Moreover, with conventional microCT scanners, high‐resolution images require long acquisition time. When imaging during active diffusion , the image acquisitions with both energies should, in theory, be conducted simultaneously. To achieve very high resolution with relatively short imaging time (~ 2 min per acquisition), synchrotron‐based microCTThe main limitation of conventional microCT and clinical CT scanners is the relatively wide energy spectrum. With one tube voltage, the photon energies can spread on both sides of the Herein, for the first time, we employ two different monochromatic X‐ray energies in high‐resolution synchrotron‐based microCT to image the diffusion of dual contrast agent in human articular cartilage. The aim of this study is to assess the full potential of the dual contrast technique to detect human articular cartilage degeneration at a clinically relevant diffusion time point (2 h) and at diffusion equilibrium (72 h). We hypothesize that synchrotron‐based microCT will provide sensitive dual contrast diagnosis of spontaneous arthritic human articular cartilage and that limitations of conventional CT systems are overcome with synchrotron microCT employing monochromatic X‐ray beam and fast image acquisition.1,N3‐bis(2‐aminoethyl)‐2,4,6‐triiodoisophthalamide, q =  +4, M = 1,499.88 g/mol) and gadoteridol  diluted with phosphate‐buffered saline (PBS). Two dual contrast agent immersion baths (for 2 and 72 h immersions) containing 5 mg I/ml and 10 mg Gd/ml were prepared. Inhibitors of proteolytic enzymes  were added to both immersion baths. Furthermore, penicillin–streptomycin–amphotericin B  was added to the 72‐h immersion bath to minimize the sample degeneration. The osmolalities of the contrast agent mixtures were ~308 and ~297 mOsm/kg for the 2‐ and 72‐h immersion baths, respectively. The osmolalities of the contrast agents and PBS were measured with a freeze‐point osmometer .The dual contrast agent was composed of CA4 + )bis were extracted from human cadavers’  left and right proximal tibia (N = 8) and distal femur (N = 8). The Research Committee of the Northern Savo Hospital District  approved the sample collection. A detailed flowchart describing sampling and experiments is shown in Fig. A total of 41 osteochondral samples  were determined by indentation loading using a custom‐made material tester equipped with an actuator having displacement resolution of 0.1 µm  and a load cell with 5 mN force resolution .Biomechanical properties of cartilage . After reaching mechanical equilibrium at pre‐stress of 12.5 kPa,ν = 0.2)ν = 0.5).First, the average cartilage thickness was calculated from four measurements around the cylindrical osteochondral sample using a vernier caliper (resolution = 0.01 mm). Subsequently, samples (from bone) were glued to the bottom of a sample holder, which was then filled with PBS to fully immerse the samples. The container was adjusted to set the cartilage surface in perpendicular contact with cylindrical, custom‐made, flat‐ended, metallic indenter (k‐edge (33.2 keV). The image acquisition was conducted by combining 1:1 magnifying visible light optics microscope , a 300‐µm thick scintillator , and an sCMOS detector . A field of view (FOV) of 16.640 × 3.497 mm2 and an isometric voxel size of 6.5 × 6.5 × 6.5 µm3 were applied. To minimize the radiation exposure of the samples, an off‐beam alignment system was used.The microCT imaging was conducted at X02DA TOMCAT beamline . Two monochromatic X‐ray energies (32 and 34 keV) were selected by utilizing a double‐multilayer monochromator  based on iodine 10 photons/mm2/s for the 32 keV and 1.0 × 1011 photons/mm2/s for the 34 keV, respectively. The cartilage samples were then modeled as soft tissue (ICRU‐44) by considering the X‐ray mass energy‐absorption coefficient from the NIST database,In order to estimate the dose deposited on each sample, the X‐ray flux was measured with calibrated passivated implanted planar silicon diodesFirst, a set of contrast agent phantoms with varying CA4+ and gadoteridol concentrations in distilled water were imaged using both energies. In the phantoms, the iodine (CA4+) concentrations were 3, 6, 12, 18, 24, 30, 36, and 42 mg I/ml and the gadolinium (gadoteridol) concentrations were 6, 9, 12, 15, and 18 mg Gd/ml  or gadolinium (Gd) in the mixture. The contrast agent concentrations in the mixtures and within cartilage are solved using Equation The concentrations in the contrast agent mixture phantoms and the contrast agent partitions within cartilage were solved using Beer–Lambert law and Bragg's additive rule for mixtures,The articulating surface and bone‐cartilage interface were manually determined using a segmentation software  for every sample at both time points. Due to the limited vertical FOV of approximately 3.5 mm, the cartilage–bone interface was not visible for 9/41 samples, and therefore, for those samples the interface was determined to be at the image border, resulting in the loss of the calcified cartilage (<200 µm). The volume of interest  was selected from the center of the osteochondral sample by using a custom‐made MATLAB  code.First, a non‐contrast‐enhanced X‐ray attenuation profile , middle (10–40%), deep (40–100%) zones, and into full cartilage thickness (0–100%) starting from articulating surface (0%) to cartilage–bone interface (100%). The decrease in bath concentration (due to the contrast agent diffusion into cartilage) was taken into account when calculating the partition profiles. The masses of gadolinium and iodine were calculated based on their contents within cartilage and sample volume and then deduced from the masses of gadolinium and iodine in the bath. With this information, the bath concentrations of CA4+ and gadoteridol at 2‐ and 72‐h time points were determined. Finally, the partitions of CA4+ and gadoteridol were calculated using Equation The sample halves were decalcified in EDTA, cut into 3 µm thick sections, and stained with Safranin‐O. Safranin‐O is a cationic dye, and it is stoichiometrically attracted to PGs, thus quantitatively revealing the PG distribution within cartilage.The severity of OA was evaluated along three histological grading systems: Mankin score,The relations between the measured and the true contrast agent concentrations in the mixture phantoms were evaluated with Pearson's correlation analysis. The statistical dependency between contrast agent partitions within cartilage and biomechanical data (equilibrium and instant modules), and PG content (OD) were assessed with Pearson's correlation, and dependency between contrast agent partitions and histological score and gradings  were assessed with Spearman's correlation. The improvement in correlation by the normalization of the CA4+ partition with that of gadoteridol was tested according to SteigerR2 = 1.000, p < 0.0001). The difference between the true and measured concentrations were 1.46 ± 0.37% for iodine and 2.62 ± 1.11% for gadolinium  and instantaneous modulus  and instantaneous modulus . Further, 2‐h gadoteridol partition in all zones and in full cartilage correlated significantly with Mankin score , OARSI score , and ICRS score . At diffusion equilibrium CA4+ partition correlated significantly  with PG content in full cartilage and all zones. Importantly, at 2‐h time point, the Spearman's correlation coefficients were significantly (p < 0.05) higher between the normalized CA4+ partitions in the superficial and middle zones, and in full cartilage thickness and histological score and grades  than for non‐normalized CA4+ partitions (Table p < 0.05) higher between the normalized CA4+ partitions in the middle zone and in full cartilage thickness and optical density, equilibrium, and instantaneous modules than for non‐normalized CA4+ partitions  correlations were found between normalized CA4+ partition and reference parameters  in the superficial and middle layers, and in full thickness cartilage. The normalization significantly improved the correlation between CA4+ partition and the reference parameters in superficial , middle , deep (equilibrium modulus) zones, and full thickness cartilage (equilibrium and instant moduli).The contrast agent partition profiles within cartilage for CA4+ and gadoteridol at 2‐ and 72‐h diffusion time points are presented in Fig. p < 0.05) improved the correlations with the reference parameters at an early diffusion time point and at diffusion equilibrium.As compared with anionic contrast agents, cationic agents provide better contrast and are more sensitive to variation in PG content.R2 = 1.000, p < 0.0001). This correlation along with minor error between the true and measured concentrations, reflects that the determination of the concentrations is accurate and linear within whole range of concentrations with the present dual contrast technique. A similar correlation between measured and true concentrations in the mixture phantoms was observed in our recent synchrotron microCT study with different X‐ray energies (25 and 37 keV).k‐edge (33.2 keV) to minimize the time difference between the measurements due to energy change and to maximize the image contrast.The measured CA4+ and gadoteridol concentrations in the mixture phantoms correlate linearly with the true concentrations (CA4+ distributions within cartilage were as expected (at 2 h decreasing and at 72 h increasing trends toward cartilage–bone interface) and in line with the previous studies at both diffusion time points measured in humanp < 0.05) at 2 h after contrast agent immersion. In general, the correlation coefficients between CA4+ partition and all the reference parameters  greater when the CA4+ partition was normalized with that of gadoteridol in the superficial and middle zones, and in full cartilage thickness at both diffusion time points. At the 2‐hour time point, the diffusion profiles show minimal CA4+ and gadoteridol accumulation in the deep cartilage . As from two to eight osteochondral samples were extracted from the same knee joint, there is a dependency between the samples. In addition, the samples were extracted from tibia and femur as well as both medial and lateral sides of the joint. This causes degeneration independent variation in cartilage composition, which cannot be differentiated with scoring and grading systems and therefore, might have led to weaker correlations. Furthermore, the samples were frozen and thawed three times before the synchrotron microCT experiment, which may have caused some degeneration. However, this degeneration can be assumed to be similar between the samples and with no major effect on biomechanical properties.Several limitations are present in this synchrotron microCT study with human osteochondral samples. First, the theory behind the dual contrast technique assumes that the images are acquired simultaneously with two separate energies. This was not possible in the present study. However, the time difference between the 32 and 34 keV acquisitions was approximately only 5 min 31 s. Furthermore, the 34 keV acquisitions conducted at the beginning and end of the imaging sequence were averaged to more closely match the 32 and 34 keV acquisitions. This was especially necessary for the 2‐h time point when the diffusion is very fast. After averaging, the time difference between the 32 and 34 keV acquisitions was minimal and the effect to the results is assumed to be negligible. The FOV height (~3.5 mm) was close to the thickness of the thickest (~3.2 mm) cartilage samples. To ensure the visibility of the cartilage surface, a safety margin was left at the top of the image. Therefore, the cartilage–bone interface was invisible for 9/41 samples, and a region of maximum 200 µm of the deepest cartilage was lost, resulting in a minor shift of the zones and therefore, a minor error in the contrast agent partition in every zone for those samples. Another limitation of this study was the low number of cadavers . This synchrotron microCT study reveals the potential of the dual contrast technique and provides the proof‐of‐concept for assessment of cartilage composition, as imaging‐related uncertainties were minimized. The results are in line with our earlier studies with conventional CT systems and indicate that beam hardening error is minimal with conventional CT scanners when imaging in vitro samples. The dual contrast technique offers significant diagnostic potential for detection of articular cartilage degeneration.M.K.M.H., A.E.A.S., M.J.T., G.L., and J.T. designed the study. H.K. and A.J. applied for ethical approval and participated in sample collection. CA4 +  contrast agent was synthesized in the laboratory of M.W.G. Synchrotron microCT measurements were conducted by M.K.M.H., A.E.A.S., M.J.T., G.L., and J.T. M.K.M.H., A.E.A.S., M.J.T., and J.T. participated in the data interpretation. Biomechanical measurements were conducted by M.K.M.H. and M.P. Digital densitometry measurements and analysis were done by R.S. M.K.M.H. wrote the manuscript with critical revision and contribution by all co‐authors. Finally, all the authors have approved the submitted version of the manuscript.


Escherichia coli compete for a carbon source and exchange resources with a ‘shared mutualist’ strain of Salmonella enterica. In laboratory experiments, competing E. coli strains readily coexist in the presence of S. enterica, despite differences in their competitive abilities. We use ecological modeling to demonstrate that a shared mutualist can create temporary resource niche partitioning by limiting growth rates, even if yield is set by a resource external to a mutualism. This mechanism can extend to maintain multiple competing partner species. Our results improve our understanding of complex mutualistic communities and aid efforts to design stable microbial communities.Although mutualisms are often studied as simple pairwise interactions, they typically involve complex networks of interacting species. How multiple mutualistic partners that provide the same service and compete for resources are maintained in mutualistic networks is an open question. We use a model bacterial community in which multiple ‘partner strains’ of  Mutualists benefit their partners by providing resources that would be difficult to obtain independently. Here, the authors show in a bacterial community and with mathematical modeling how a mutualist can promote coexistence between competitors by providing them with different limiting resources. Traditionally, studies of mutualism have focused on interactions between two species. However, communities often contain many species of mutualists that interact in complex networks4. For example, many flowering plants are pollinated by multiple insect species5, and corals interact with a phylogenetically diverse set of endosymbionts6. In fact, two-partner mutualisms are now thought to be the exception rather than the norm7. Understanding the ecology of multiple mutualist communities is an important goal8. To do so, we need theoretical predictions and experimentally tractable multiple mutualist communities.Mutualisms—bidirectional positive interspecies interactions—are abundant and important8. For example, a guild consisting of two plant species may provide carbon compounds to a shared arbuscular mycorrhizal fungi mutualist that provides both phosphorus, from which one plant species benefits, and pathogen protection, from which the other plant benefits9. Recent studies have suggested that interactions between species within the partner guild can affect the coexistence and stability of the whole community12. Within-guild interactions may be especially important if the partner mutualists within the guild are ecologically similar. If partner species’ resource niches overlap, they may compete for resources that are external to the mutualism. When multiple species compete for the same limiting resource, one species may competitively exclude the others, leading to a loss of diversity within the community13. However, because species-rich communities of multiple mutualists exist in nature, certain mechanisms that maintain coexistence must exist14. Here, we seek to understand the conditions in which multiple partner mutualists are able to coexist despite competition for a common resource.In many multiple mutualist systems, several functionally similar species within a “partner guild” supply resources or services to a “shared mutualist,” which supplies resources in return Fig. 3,8. For Escherichia coli strains that compete with one another for a carbon source and engage in mutualism with a strain of Salmonella enterica, the shared mutualist. The strains engage in mutualism via cross-feeding, with the E. coli strains providing acetate and receiving amino acids from S. enterica. We show that the competing E. coli strains are unable to coexist when they are provided amino acids in the growth media rather than obtaining them from the shared mutualist because one strain has a faster growth rate. However, when the two E. coli strains obtain their amino acids from the shared mutualist, the two E. coli strains coexist, maintaining the diversity of the multiple mutualist communities. Next, we use a resource-explicit ecological model to identify factors that promote coexistence. We show that limitation by the shared mutualist is key—if the shared mutualist sets the growth rate of the community, the two-partner mutualists coexist because they are temporarily limited by different resources. Finally, we demonstrate computationally that this phenomenon can promote the coexistence of more than two-partner mutualists. This work helps us understand how diversity is maintained in multiple mutualist communities and can inform efforts to design stable microbial communities.Using a community of mutualistic bacteria, we explore the potential for the coexistence of multiple partner species. Our system consists of a partner guild of E. coli strain that is a methionine auxotroph (“Em”) and another E. coli strain that is an arginine auxotroph (“Er”)—each strain lacks a gene in the biosynthetic pathway for its respective amino acid, so in order for a strain to grow, its required amino acid must be available in the environment. The two E. coli strains compete for lactose, which we provide in the growth media, and excrete acetate as a byproduct of lactose metabolism. We experimentally evolved a “shared mutualist” strain of Salmonella enterica (“Smr”) that secretes methionine and arginine. Smr was derived from a strain that we had previously evolved to secrete methionine15, and acquired a mutation in argG causing arginine secretion. Smr consumes acetate and is unable to metabolize lactose.We studied competition between two-partner mutualists using a laboratory system of cross-feeding bacteria Fig. . The par16. Therefore, we started by measuring the growth rates of the three strains in monoculture and in pairwise coculture. For all experiments, we used a batch culture setup in which populations grow until resources are depleted. When grown in monoculture in media containing each strain’s required nutrients (with both amino acids in excess in Em and Er monocultures), the three strains have different maximum growth rates  = 9897, P = 9.1e-16; Fig. n = 3, mean = 0.666 h−1, SD = 0.003; Er: n = 3, mean =  0.680 h−1, SD = 0.007; Tukey HSD: P = 0.037), and both E. coli strains grow faster than Smr . When each E. coli strain is grown separately in coculture with Smr in lactose media with no amino acids, the coculture containing Em grows faster  = 15, P = 0.004; Fig. Growth rates are a classic measure of competitive ability in microbial systemsE. coli strains coexist in a lactose environment with excess amino acids and no Smr present. We predicted that the strain with the faster monoculture growth rate, Er, would outcompete Em  = 48, P = 0.0004). Competitive exclusion of Em by Er also occurs in this environment in the presence of Smr : One E. coli strain will outcompete the other. Er may outcompete Em due to its faster monoculture growth rate and greater competitive ability, or Em may outcompete Er due to its faster coculture growth rate when paired with Smr. Hypothesis (2): The two E. coli strains coexist.These findings led to two alternative hypotheses about coexistence in the three-strain multiple mutualist community: E. coli strains in the three-strain community, we again conducted a mutual invasibility test. We inoculated cultures with six different initial frequencies of the E. coli strains, with a constant initial density of Smr. In line with Hypothesis 2, both E. coli strains increased in frequency when initially rare, indicating coexistence  = −58, P = 0.0003), and decreased in frequency when initially common  = 6, P = 0.02). Smr also increased in frequency from rare and decreased when initially common  and the four resources :To understand why the two Em lcts), which are in units of cells/ml. Production terms  are in units of cells/cell. Default values and parameter descriptions can be found in Supplementary Table Em = 1.0, µEr = 1.1, and µSmr = 0.5 with units of 1/timestep.Em, Er, and Smr are the population densities of each strain (cells/ml). Resources  are in units of cell-equivalents/ml (the density of cells that a unit of resource can produce). Growth is governed by Monod saturation rates using Monod constants  until lactose is depleted, and the strain with the faster growth rate, Er, takes over  and amino acid equal for both E. coli strains. As shown elsewhere, unequal Monod constants can allow two competitors to coexist when competing for two essential nutrients20. However, in a system like ours where competitors are limited by equivalent resource concentrations, coexistence is not possible with the complete overlap in benefits provided by the shared mutualist.We explored complete overlap in amino acid consumption by creating a model in which both E. coli amino acid auxotroph  into our model and added production of this amino acid by S. enterica (“Smrx”)  is abundant, allowing it to grow at its maximum growth rate, while the initially common strain(s) is limited by its amino acid  Fig. . We set ist Fig. . Howeverins Fig. . The mec12. We explored the impact of resource competition between partner species that interact with a shared mutualist on coexistence and stability. Laboratory results showed that two E. coli partner mutualist strains that receive different amino acids from a S. enterica shared mutualist can coexist, despite the fact that one E. coli strain is a better competitor for lactose, the resource that ultimately limits growth. Modeling indicated that stability is possible when the E. coli strains are temporarily limited by different resources. While lactose sets the total E. coli carrying capacity—growth ceases when lactose is exhausted—the availability of amino acids during growth determines the instantaneous growth rate of each E. coli strain. When one E. coli strain begins rare, its amino acid is always abundant, so its instantaneous growth rate is faster and it consumes lactose quickly. In contrast, the initially common E. coli strain is amino acid-limited at early timepoints and grows more slowly than the rare strain, which allows the initially rare E. coli strain to increase in frequency. The community is therefore stable through negative-frequency dependence. We found that three key parameters affect the potential for coexistence via temporary amino acid limitation. Coexistence is not possible if S. enterica’s growth rate is high, if S. enterica’s production rate of the initially common E. coli’s amino acid is high, or if the initially common E. coli strain depletes its amino acid at a low rate. In these situations, the initially common E. coli strain is never limited by its amino acid, and the stronger competitor excludes the weaker. In summary, coexistence requires temporary amino acid limitation for one partner strain.In communities of multiple mutualists, competition between species within a partner guild can affect the coexistence and maintenance of diversity22. Theoretical work predicts that multiple species typically do not coexist if they are limited by the same resource20 . However, if the species are limited by different resources, they can more easily coexist24. In our system, the carrying capacity of the E. coli strains is ultimately limited by lactose. However, during growth, the limiting resources are temporarily “partitioned.” One strain’s instantaneous growth rate is limited by its amino acid, while the other achieves its maximum growth rate due to an abundance of lactose and its amino acid. An interesting element of our system is that a biotic factor creates the potential for temporary niche partitioning, rather than an aspect of the environment. The shared mutualist, S. enterica, causes the two-partner species’ instantaneous growth rates to be determined by different resources early on in growth. In addition, the shared mutualist creates the potential for niche partitioning by providing two different resources for the partner strains. Coexistence is not possible if both partner strains receive the same resource from the shared mutualist . Together with this study, our results suggest that understanding the stability of multiple mutualist systems requires consideration of competition for external resources in addition to competition for access to the shared mutualist. We show that even when both competing partners are ultimately limited by a resource external to the mutualism, coexistence can be maintained through temporary niche partitioning. Our work also identifies the importance of the shared mutualist providing different resources to members of the partner mutualist guild.Recent work has explored the importance of competition between partner species in multiple mutualist communities. Several empirical studies have documented competition between species within a partner guild for access to the shared mutualist. For example, flowering plants compete for pollinator services29. An open question in microbial ecology is why natural communities appear to contain several ecologically similar species that consume the same resources and carry out the same functions30. Our work suggests that these communities may be stable despite the potential for competition between strains that provide redundant functions . We also showed that temporary limitation by different resources allows for the coexistence of three partner strains  has a ΔmetB mutation, and the arginine auxotroph (“Er”) has a ΔargA mutation. LacZ was added to both strains using conjugation for Em, and phage transduction for Er38. Em has a cyan fluorescent protein inserted in the lambda attachment site. We also used a Salmonella enterica serovar Typhimurium LT2 strain that secretes methionine and arginine (“Smr”). This strain was derived from a strain containing mutations in metA and metJ that cause overproduction of methionine40. We selected for arginine production by coculturing this strain with the E. coli arginine auxotroph as a lawn on lactose minimal media plates containing x-gal (0.05% v/v) for four 7-day growth cycles with 1:6.67 dilutions at each transfer41. The appearance of a blue colony suggested the evolution of arginine production in S. enterica, which we confirmed by isolating S. enterica from the colony on citrate minimal media plates and cross-streaking with Er. We sequenced this strain using Illumina NextSeq and identified mutations using breseq42. We found a T → A point mutation in argG at position 3459818 (reference strain NC_003197).We used two 2HPO4, 16.3 mM NaH2PO4, 0.814 mM MgSO4, 3.78 mM Na2SO4, 3.78 mM [NH4]2SO4, and trace metals 6Mo7O24, 1 uM CuSO4, 2 mM CoCl2, 0.33 µm Na2WO4, 20 µM CaCl2). Monocultures and cocultures of Em and Er were grown in this medium with 250 µM of methionine and 250 µM of arginine added, concentrations that we found to be unlimiting , cultures were diluted in saline (0.85% NaCl) and plated on selective Hypho minimal media plates with 1% agar. Plates for the 600 in a Tecan InfinitePro 200 plate reader at 30 °C, shaking at 432 rpm between readings, which were taken every 20 min. Growth rate estimates were calculated by fitting growth curves to a Baranyi function43 by obtaining nonlinear least-square estimates and using the growth rate parameter estimate. For all experiments , we performed three biological replicates, except for Smr monocultures, where we performed six biological replicates. All measurements were taken from distinct samples.All experiments were performed in 96-well plates with 200 µl of media per well, inoculated with a 1:200 dilution of log-phase monocultures (1 µl of each strain). We measured ODE. coli strain to increase in frequency from rare was our criterion for coexistence18. To measure whether invasion from rare occurred, we set up cultures with different starting frequencies and tracked their changes over one growth cycle. We performed several batches of these experiments on different weeks, with three biological replicates per batch. The initial Smr density was kept constant , and the E. coli total density was kept constant (1.6e6 CFU/ml ± 5.4e5 (SD) for batch A,_1.7e6 CFU/ml ± 5.3e5 (SD) for batch B, 3.6e6 ± 6.7e5 (SD) for batch C) but the frequency of each strain differed across a range of frequencies—0.024, 0.320, and 0.899 Em/(Em + Er) for batch A, 0.006, 0.393, and 0.985 Em/(Em + Er) for batch B, and 0.08 for batch C. (Note that batch C was only performed in lactose + methionine + arginine media with no Smr present.) After growth, the cultures were diluted and plated on strain-specific plates to measure yields as CFU/ml (media described above). The change in Em frequency was calculated as [Em/(Em + Er)]final – [Em/(Er + Er)]initial.The ability of each The ecological model is shown in “Results” and Supplementary Table α = 0.05. The coculture growth rates and mutual invasibility experiments were analyzed using Welch’s two-sided two-sample t test, which is designed for unequal sample distribution variance and assumes normality, with α = 0.05. We used the R package ggtern to make the ternary plots in Fig. Modeling, data visualization, and statistical analyses were done in R version 3.6.0. Monoculture growth rates were compared using a one‐way analysis of variance (ANOVA) and post hoc Tukey HSD tests, with Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Despite guideline recommendations, dual antiplatelet therapy (DAPT) is frequently used for longer than 1 year after an acute coronary syndrome (ACS) event. In Asia, information on antithrombotic management patterns (AMPs), including DAPT post discharge, is sparse. This analysis evaluated real‐world AMPs up to 2 years post discharge for ACS.There is wide variability in AMP use for ACS management in Asia.EPICOR Asia (NCT01361386) is a prospective observational study of patients discharged after hospitalization for an ACS in eight countries/regions in Asia, followed up for 2 years. Here, we describe AMPs used and present an exploratory analysis of characteristics and outcomes in patients who received DAPT for ≤12 months post discharge compared with >12 months.Data were available for 12 922 patients; of 11 639 patients discharged on DAPT, 2364 (20.3%) received DAPT for ≤12 months and 9275 (79.7%) for >12 months, with approximately 60% still on DAPT at 2 years. Patients who received DAPT for >12 months were more likely to be younger, obese, lower Killip class, resident in India (vs China), and to have received invasive reperfusion. Clinical event rates during year 2 of follow‐up were lower in patients with DAPT >12 vs ≤12 months, but no causal association can be implied in this non‐randomized study.Most ACS patients remained on DAPT up to 1 year, in accordance with current guidelines, and over half remained on DAPT at 2 years post discharge. Patients not on DAPT at 12 months are a higher risk group requiring careful monitoring. Among these, 12 922 were eligible for inclusion; 83 patients were excluded . Data on patient demographics, medical history, and management were collected from symptom onset to discharge and AMPs at hospital discharge and post discharge by means of telephone interviews scheduled at 6 weeks and then every 3 months post index event until 2 years.https://astrazenecagrouptrials.pharmacm.com/ST/Submission/Search). All patients provided written informed consent.Study procedures adhered to International Conference on Harmonization (ICH) Good Clinical Practice guidance, the Declaration of Helsinki, and local regulations, and Institutional Review Board/Ethics Committee approval was obtained at each participating center in each country , and anticoagulant  were evaluated at discharge, and at 1.5, 3, 6, 9, 12, 18, and 23 months post discharge. In practice, final interviews were conducted within ±2 weeks of the 24‐month timepoint, hereafter referred to as 24 months or 2 years. Where appropriate, to avoid “loss” of patients whose last interview was 1 or 2 weeks prior to the 24‐month timepoint, medication status at 23 months is reported. For patients on SAPT or DAPT at discharge, the proportions remaining on SAPT or DAPT, or who switched to another antiplatelet pattern, were evaluated. Possible associations between baseline factors, SAPT use at discharge, and DAPT duration >12 months vs ≤12 months were investigated.The aim of this analysis was to characterize oral AMPs (antiplatelet and anticoagulant) at discharge and during a 2‐year follow‐up period. Patterns of antiplatelet therapy  was assessed, including analysis by EPICOR 2‐year risk score, based on previously published data from the EPICORAll outcomes were validated as previously described.2.2P values derived from chi‐square test for categorical variables, two‐sample t‐test for comparison of means, and Wilcoxon test for comparison of medians. Logistic regression models were used to evaluate relationships between baseline characteristics, and (a) SAPT use at discharge, and (b) DAPT use >12 vs ≤12 months, with odds ratio (OR) and two‐sided 95% confidence interval (CI) presented. A stepwise model selection was made with the P value cut‐offs for selection at P < .01 and retention at P < .20.Patient characteristics are described using appropriate descriptive statistics, with P values. The proportion of patients discharged on DAPT who remained on DAPT beyond 12 months is also described by percentile  for each EPICOR 2‐year risk score category.Data are presented in terms of OR and associated two‐sided 95% CIs and An exploratory analysis of time from 12 months after discharge to each clinical event during the second year of follow‐up by DAPT duration (>12 or ≤ 12 months) was made based on Cox proportional hazards models with stepwise model selection with DAPT duration (>12 or ≤12 months), index event diagnosis, age , gender, country group , and in‐hospital management always kept in the model. The data are presented in terms of hazard ratio (HR) and two‐sided 95% CIs.Statistical analysis was performed using SAS version 9.3 or later .33.12) in patients from Malaysia and Singapore. Most had undergone coronary revascularization, except those from India and Malaysia.Details of baseline patient characteristics have been published previously.3.212 receptor antagonist in 88.8% of cases (Baseline demographics by discharge antiplatelet medication and by DAPT duration ≤12 or >12 months (in patients discharged on DAPT) are shown in Table of cases . By 12 mof cases . Patientof cases .Of those patients discharged on SAPT (n = 1121), 90.2% remained on SAPT at 12 months, and 85.4% at 2 years; these patients were more likely to switch to no antiplatelet  than to DAPT . No patient discharged on SAPT received TAPT post discharge.Of the 11 817 patients discharged on DAPT who survived at least 12 months, 1542 received DAPT for ≤12 months (median 7.7 months) and 9275 for more than 12 months (median 23.6 months) Table . They weMultivariable analysis indicated that significant predictors of DAPT duration >12 vs ≤12 months in patients who survived at least 12 months were younger age, obesity, residency in India vs China, any drug‐eluting stent (DES) placement, and in‐hospital percutaneous coronary intervention (PCI)/coronary artery bypass graft (CABG) Table . Other sWhen evaluated in terms of EPICOR 2‐year risk score, the number of patients remaining on continuous DAPT were generally lower as risk percentiles increased, up to the 15‐month visit, although this pattern was less apparent thereafter Figure . There wWhen comparing the groups who were treated with DAPT >12 months vs ≤12 months in the 12‐month survivors, the hazard of composite of death, MI, and stroke was much lower in the group treated with DAPT >12 months (adjusted HR 0.39) , and to have received invasive reperfusion and DES placement, and they were less likely to have had an in‐hospital cardiovascular event. The approximate 60% observed rate of prolonged DAPT use (>12 months post‐discharge) in this study is generally higher than that reported across the APAC region (39%) and Europe (12%), albeit with considerable between‐country variability.Overall, patients in whom DAPT was used for more than 12 months appeared to show lower rates of cardiovascular events and mortality compared with those in whom DAPT cessation occurred earlier, with no apparent increase in risk of major bleeding. However, any observed association between event rates and DAPT duration must be interpreted with caution, due to the possible presence of confounding factors. In particular, treatment allocation bias and confounding occurring 12 months post discharge, that were not explained by the discharge variables, could not be adjusted for.P = .02).A recent randomized, non‐inferiority study evaluating 6 months vs ≥12 months of DAPT following PCI with DES placement in 2712 South Korean patients with ACS  observed a higher rate of MI in the short‐duration DAPT population over an 18‐month follow‐up period , characteristics and outcomes of medically managed patients with NSTE‐ACS and predictors of 1‐ and 2‐year post‐discharge mortality risk were identified.Several potential limitations of the EPICOR Asia study include most patients being enrolled from China, resident in metropolitan areas, and enrolled at well‐equipped centers, thus not being fully representative of the region. Such considerations may partly explain discrepancies in these data and earlier reports for the APAC region; reliance on earlier data must factor in possible greater uptake of evidence‐based guidelines, specifically, the use of both DAPT and PCI, as evidenced in China.In summary, this analysis of results from EPICOR Asia has shown that most patients surviving an ACS in Asia were discharged on DAPT, and more than half remained on DAPT for up to 2 years of follow‐up. Prolonged DAPT was more likely to be employed in younger patients, and those with DES placement or in‐hospital PCI/CABG, and less likely to be used in China compared with India. Patients in whom DAPT treatment was stopped earlier than 12 months post discharge had higher rates of mortality and adverse cardiovascular events. While a causal relationship between outcomes and DAPT duration in this study cannot be inferred, the findings do suggest that patients who receive DAPT for ≤12 months are a high‐risk population and may require greater medical attention after discontinuation of DAPT than patients who receive DAPT for longer.B.Z., Y.H., S.W.L.L., J.P.S.S., H.S.K., A.A.G., J.J., and S.J. have nothing to disclose. R.K. has been a consultant or advisory board member for AstraZeneca and Boehringer Ingelheim. S.J.P. receives research funds from AstraZeneca. V.T.N. has received research grants from AstraZeneca, Servier, Sanofi, and Boston Scientific and has been a consultant or advisory board member for AstraZeneca, Pfizer, Sanofi, Boehringer Ingelheim, Servier, M.S.D., Abbott, Bayer, Novartis, Merck Serono, Biosensor, Biotronic, Boston Scientific, Terumo, and Medtronic. C.T.C. has received research support from Eli Lilly, honoraria from Medtronic, and has been a consultant or advisory board member for AstraZeneca. N.H. is an employee of AstraZeneca, and A.M.V. is a former employee of AstraZeneca. T.K.O. has acted as a consultant or advisory board member for Sanofi‐Aventis, Abbott Vascular, Boston Scientific, Boehringer Ingelheim, Novartis, and AstraZeneca.SUPPLEMENTARY TABLE S1 Demographics, index diagnosis, and general health status of all patients discharged on DAPT who continued on DAPT for ≤12 vs >12 monthsaSUPPLEMENTARY TABLE S2 Demographics and baseline characteristics of patients discharged on SAPT/no antiplatelet or DAPTaSUPPLEMENTARY TABLE S3 Antithrombotic therapy at discharge and during follow‐upSUPPLEMENTARY TABLE S4 Logistic multivariable regression analysis for predictors of SAPT/no antiplatelet vs DAPTa at dischargeSUPPLEMENTARY TABLE S5 Patients on continuous DAPT at each visit according to EPICOR 2‐year risk percentilesaClick here for additional data file.


However, variations in the taste quality of Congou black tea manufactured during a single spring season are far from clear. Here, we analyzed the taste quality of HuangJinCha (HJC) Congou black tea using sensory evaluation and found the taste quality of black tea manufactured in the early spring was better than that manufactured in the late spring. Principal component analysis (PCA) and cluster analysis for the data from the electronic tongue confirmed the variation and revealed that April 4 may be the critical time point at which variations in taste quality become apparent. The contents of tea polyphenols (TP), total catechins (TC), total flavones, (‐)‐epigallocatechin gallate (EGCG), (‐)‐epicatechin gallate (ECG), and gallic acid (GA) showed increasing trends, whereas total amino acids (TAA) declined over time. Moreover, the variations in total amino acids (r = 0.846) and total flavones (r = ‐ 0.858) were highly significantly correlated with the average taste quality score , suggesting these two compounds were the primary factors responsible for the fluctuation in taste quality. Fully fermented black tea contains higher levels of oxidation products (including dimeric and polymeric catechins) than unfermented green tea. In recent years, it has been reported that black tea possesses potential protective effects against cancer, cardiovascular diseases, chronic inflammatory diseases, metabolic syndrome, and neurodegenerative diseases , horticultural practices, harvest seasons, and manufacturing methods ‐Catechin (C), (‐)‐epigallocatechin (EGC), (‐)‐gallocatechin (GC), (‐)‐epicatechin (EC), (‐)‐epicatechin gallate (ECG), (‐)‐epigallocatechin gallate (EGCG), (‐)‐gallocatechin gallate (GCG), anhydrous glucose, caffeine (CAF), and gallic acid (GA) were purchased from Shanghai Yuanye Biotechnology Co., Ltd. Company . Alanine , arginine (arg), aspartic acid (asp), cysteine (cys), glutamic acid (glu), glycine (gly), histidine (his), isoleucine (ile), leucine (leu), methionine (met), phenylalanine (phe), proline (pro), serine (ser), threonine (thr), tryptophan (trp), tyrosine (tyr), valine , theanine, the AccQ‐Fluor Reagent Kit, and AccQ‐Tag eluent were purchased from BDH Chemicals Ltd. . Acetonitrile , sodium carbonate, methanol , concentrated sulfuric acid, anthrone, ferrous tartrate , disodium hydrogen phosphate, and potassium dihydrogen phosphate were purchased from Sinopharm Chemical Reagent Co., Ltd. . Pure water from China Resources C'estbon Beverage Co., Ltd.  was used throughout the study.2.2Fresh tea leaves of the HJC variety were harvested from the same tea garden of Dai Linbing Tea Professional Cooperative  in the early spring , mid spring , and late spring  in 2018. Tea leaves were plucked with the same standard of one bud and one leaf in the sunny day  and manufactured using the same method for Congou black tea. Briefly, after approximately 12 hr of withering at 26°C and 70% relative humidity, a rolling machine  rolled the tea leaves for 70 min. Then, fermentation occurred in an environmentally controlled cabinet at 30°C and 90% relative humidity for 4 hr. In the drying stage, tea leaves were initially roasted at 120°C for 15 min and then roasted to a moisture of approximately 7% at 90°C for 60 min in an aroma‐enhancing machine. Black tea samples were ground into powder (100 mesh) using a tube mill and stored at 4°C for future use.2.3Sensory evaluation was performed according to China National Standards outlined in GB/T 23776–2018. Briefly, black tea samples were double‐blinded before 3 g tea was brewed using 150 ml of boiling water for 5 min prior to filtration. Black tea infusions were scored at room temperature (approximately 25°C) by a professional panel composed of seven specialists . Each specialist had a minimum of 150 hr of tea sensory evaluation experience from the Tea Research Institute of Hunan Academy of Agricultural Sciences. The sensory response was evaluated on a 100‐point scale where 90–99 indicated high mellow and fresh, 80–89 indicated neutral mellow and fresh, and 70–79 indicated low mellow and fresh.2.4Experiments were performed using an α‐ASTREE electronic tongue system  equipped with an Ag/AgCl reference electrode and 7 taste sensors . The methods for electronic tongue measurement were based on those described in a previous study , total flavones, soluble sugar, theaflavins, thearubigins, and theabrownins was based on the methods described below  equipped with a Shim‐pack VP‐ODS column  at 278 nm according to the methodology described in a previous study , according to the manufacturer's specifications with slight modifications. Black tea powder (3 g) was infused with 500 ml boiling deionized water and maintained for 45 min prior to filtration with a 0.45 µm membrane. Amino acid standard solutions were dissolved in 0.1 mol/L HCl and filtered through a membrane for further use. Mobile phase A consisted of AccQ‐Tag Eluent A Concentrate in deionized water (1:10 v/v) and mobile phase B consisted of 60% acetonitrile in deionized water. The sample injection volume was 10 µl, and the flow rate was 1.0 ml/min. Column temperature was set at 37°C. Amino acids were detected at 248 nm and identified by comparison of retention times and spectra of standard solutions. Quantification was done via external calibration curves.2.6SD). Principal component analysis was performed using Simca‐p 14 software . Heat map analysis and cluster analysis were performed with Origin software . The significance level was calculated by one‐way analysis of variance (ANOVA) using IBM SPSS statistics software . The statistical significance is indicated in the figures .The data were expressed as mean + standard deviation (33.1p < .05). The results suggested that the taste of HJC Congou black tea during different periods of spring can be divided into several types, and the tea samples from early spring had a relatively better taste quality.Previous studies focused on the taste quality variation of Congou black tea manufactured in different seasons. In this study, the taste quality of black tea manufactured during a single spring season was investigated. The average score of taste quality generally decreased with the progression of time Figure . Black t3.2To verify the sensory evaluation results, we used the data from an electronic tongue to distinguish the taste quality of HJC black tea manufactured throughout the spring. To confirm that the electronic tongue sensors possessed a reliable repeatability for the tea infusion, we calculated the relative standard deviation (RSD) of the sensors' response value and found all levels were less than 1.9% Figure . The PCA3.3p < .05) in black tea samples save for water extract (WE). The TP, TC, total flavones, and GA content exhibited similar increasing trends, whereas the content of TAA showed a decreasing trend . In addition, the content of total flavones was significantly inversely correlated with the taste score . Moreover, the ECG, EGCG, TC, TP, GA, and TR contents were also significantly inversely correlated with the taste quality score (p < .05). Surprisingly, the correlations between the contents of WE, SS, CAF, EC, EGC, TFs, TBs and taste average score were not significant  correlated with taste quality score  correlated with taste quality score, suggesting less importance of catechins in the formation of black tea taste compared with flavones. This result corresponded well to previous report that flavones and their glycosides were the main contributors to black tea taste for the sake of extremely low threshold concentrations (Scharbert, Holzmann, & Hofmann, Many studies have reported a relationship between taste quality and taste compounds. Flavonoids, including catechins, flavones, and their glycosides, are believed to be main taste components in black tea infusion. The catechins in tea polyphenols, especially ester catechins, are the main contributors to the astringent and bitter taste of tea infusion (Chen, Zhao, Guo, & Wang, e Figure . InteresInconsistent results have reported that free amino acids may not be important for the taste formation since the concentration of each amino acid was much lower than their recognized threshold concentration in Darjeeling black tea infusions (Scharbert & Hofmann, CAF was identified as the most important bitter‐tasting compound in tea infusions, while TFs, TRs, and TBs generated during tea fermentation are believed to impart the mouth‐coating, astringent, and long‐lasting oral sensation of black tea infusions (Scharbert, Holzmann, et al., The taste quality of Congou black tea may be influenced by the variety, region, and manufacturing process. In this study, tea leaves of the HJC variety plucked from the same garden were selected as raw materials and manufactured into Congou black tea through the same process in order to investigate the seasonal effect on taste quality. Based on the results of sensory evaluation, electronic tongue analysis, main taste compounds analysis, and correlation analysis, we found that the taste quality of HJC black tea in the early spring was better than that in the late spring and that the critical time point at which taste quality variations begin may be April 4. Furthermore, TAA and total flavones were primarily responsible for the seasonal variations in taste quality of black tea. However, this study only analyzed the variation of highly abundant taste compounds during a single spring season and failed to investigate the flavor characteristics in other potential taste compounds. It is, therefore, necessary to further analyze the taste quality with comprehensive metabolism profiling.In conclusion, the taste quality of HJC black tea manufactured in a single spring season was described and the key compounds of the taste quality variation were investigated in this study. Considering that taste quality is a key factor for tea quality, our observations may serve to provide new insights for the processing of Congou black tea in the spring season.The authors declare that they have no conflicts of interest.This study does not involve any human or animal testing. Written informed consent was obtained from all study participants. Click here for additional data file.


Severe electrocardiographic (ECG) abnormalities in asymptomatic subjects correlate with cardiovascular risk.The role of minor ECG abnormalities is less well‐known. We evaluated the association between a negative T‐wave and mortality, as a possible marker for prognosis.A prospective, population‐based cohort, examined at 50 years, and followed until death. Time to death (event rates) and predictive role of a negative T‐wave (Cox regression) were analyzed.P = <.0001). A negative T‐wave correlated with elevated risk  for all‐cause and cardiovascular (CV) death (1.59 (1.20‐2.11) P = .0012 vs 1.91 (1.34‐2.73) P = .0004). The association remained after excluding coexisting Q/QS patterns and ST‐junction/segment depression ECG abnormalities . Death from other causes was not associated with a negative T‐wave. A major negative T‐wave carried higher risk than a minor (2.17 [1.25‐3.76] P = .0062 vs 1.78 [1.13‐2.79] P = .012) for CV death.Participants (n = 839) with a negative T‐wave (7.3%) had significantly higher blood pressure (BP) (mean systolic 157.9 mmHg vs 136.8 mmHg without negative T‐wave, A negative T‐wave at 50 years, in asymptomatic individuals, carried an increased risk of all‐cause and CV death during lifetime follow‐up. From the population registry, a sample was drawn in 1963, consisting of all the men born in 1913 on a day of the month divisible by three . These criteria were fulfilled by 973 men born in 1913, of whom 855 (87.9%) participated in a health examination. Participants and nonparticipants have been previously described.2). After an overnight fast, blood samples for serum cholesterol, triglycerides, and blood glucose were drawn from an antecubital vein, with light stasis, and analyzed according to standard laboratory procedures.At baseline, 1963, clinical examination and laboratory analyses were performed. Information on lifestyle, medical history, and smoking habits was collected through a questionnaire. Body weight was measured with a lever balance to the nearest tenth of a kilogram, while body height was measured to the nearest centimeter. Body mass index (BMI) was measured as weight (kg)/height (mBlood pressure (BP) Korotkoff phase 1 and 5 were recorded in the right arm, with the participant in the sitting position after a five‐minute rest with a mercury sphygmomanometer cuff size of 12 x 23 cm. Hypertension was defined as the use of antihypertensive medication or current systolic BP ≥140 mmHg and/or a diastolic BP ≥90 mmHg. Diabetes mellitus was defined as self‐reported in the questionnaire. Smoking habits were classified as: 1 = never smoked, 2 = ex‐smoker since 3 months or 3 = current smoker.Physical activity during leisure time was assessed at a re‐examination in 1967, with the four‐grade Saltin Grimby Physical Activity Scale (SGPALS) and coded as 1 = sedentary, 2 = some light physical activity, such as walking, riding a bicycle, and light gardening for at least 4 hours a week, 3 = regular moderate physical activity for a minimum of 3 hours a week and 4 = regular intense physical training for competition sports.Standard 12‐lead ECGs were recorded with the patients at rest in the supine position. Paper speed was 50 mm/s and calibration was 1 mV: 10 mm. All of the ECGs were evaluated by a physician, who was blinded to the clinical data, and classified according to the Minnesota Code Classification System for Electrocardiographic Findings.2.2Mortality data were obtained from 1 January 1963 until 1 July 2015 from the National Cause of Death Registry, and included date and cause of death. Cause of death was classified according to the International Classification of Diseases (ICD) codes, ICD 8 until 1986, ICD 9 until 1996, and ICD 10 from 1997 and onwards. Only 14 men (1.6%) were lost to follow‐up over time, primarily due to emigration.2.3χ2 test for ordered categorical variables and Mann‐Whitney U test for continuous variables. Event rates per 100 person years were calculated for time to all‐cause death, death due to CV disease and death due to other causes, with 95% confidence interval (CI) estimated applying exact Poisson limits. Univariable Cox proportional hazards models were used when studying the negative T‐wave's predictive ability on time to death. The proportional hazards assumption was examined by adding an interaction term between negative T‐wave and the logarithm of the follow‐up time and was considered fulfilled. Effect size was described by hazard ratios (HR) and 95% CI. Sensitivity analysis was performed excluding individuals with Q and QS pattern or ST‐junction and segment depression. Interaction analyses between negative T‐wave and baseline clinical characteristics  were evaluated applying the same statistical methods. These analyses were performed both unadjusted and adjusted for smoking, physical activity, BMI, systolic BP, BP medication, hypertension, glucose, cholesterol, hematocrit, Q and QS pattern, ST‐junction, and segment depression unless the variable is the main variable studied in the interaction. All tests were two‐tailed and conducted at 0.05 significance level.All data analyses were performed using SAS software version 9.4 . For descriptive purposes, number and percentages were presented for categorical variables and mean, SD, median, minimum, and maximum for continuous variables. For comparison between two groups Fisher's exact test was used for dichotomous variables, Mantel‐Haenszel 33.1At baseline (n = 855), at 50 years, ECG abnormalies were observed in 11.7%, in decreasing frequency: negative T‐wave (7.3%), ST‐junction and segment depression (5.7%), Q/QS patterns (3.0%), and ventricular conduction defects (0.6%). Among these, subjects reporting a prior myocardial infarction (MI) confirmed in medical records (n = 11), or showing ventricular conduction defects  (n = 5) were excluded . The clinical characteristics for remaining participants (n = 839) are presented relative to a negative T‐wave in Table 3.2P = .0012 vs 1.91 [1.34‐2.73] P = .0004). The association remained after excluding coexisting Q/QS patterns and ST‐junction/segment depression ECG abnormalities . A major negative T‐wave carried higher risk than a minor (2.17 [1.25‐3.76] P = .0062 vs 1.78 [1.13‐2.79] P = .012) for CV death. Death from other causes was not significantly associated with a negative T‐wave.The mortality associated with a negative T‐wave in ECG was computed Table : For all3.3We tested the impact on mortality, all‐cause and CV, of all clinical and demographic variables presented in our models, in the presence of a negative T‐wave, while systematically adjusting for smoking, level of physical activity, BMI, systolic BP, BP medication, hypertension, serum levels of glucose, cholesterol, hematocrit, Q/QS pattern, ST‐junction/segment depression unless the variable was specifically studied in the interaction.The HR by presence of T‐wave negativity is documented for all variables, for all‐cause and CV mortality (Supplement 44.1In the present study, a negative T‐wave recorded at 50 years predicted all‐cause and CV deaths during lifetime follow‐up in a male random population‐based cohort. We found the presence of hypertension to be the most important differentiating factor between individuals with a negative T‐wave and those without. No significant differences in other CV risk factors were observed. It is well established that ECG abnormalities are more prevalent in hypertension than in normotension and correlates with its severity.We realize, that our baseline findings are representative of a preceding time period, showing ECG abnormalities in 11.7%, and combined ST‐ and T‐wave changes in 8.9% Table  Cohorts Simultaneous other ECG abnormalities were much more common when a negative T‐wave was present, but limited to ST‐segment/junction depression, most frequently, and also Q/QS pattern. The former would be expected since nondiagnostic ST/T changes are the most common ECG changes overall.4.2The higher event rate for all‐cause and CV mortality associated with a negative T‐wave was consistent with the presence of more hypertension in this group, and of ECG abnormalities, in general, as markers of compromized CV health.Consistent with the above, the HR for death in the presence of a negative T‐wave was also significantly elevated both for all‐cause and CV death, almost doubled for the latter. The increased risk persisted after exclusion of concomitant Q/QS pattern and concomitant ST‐junction/segment depression. This implies that a negative T‐wave marks an elevated risk of death, particularly CV death, and the risk persists, also in the absence of other ECG abnormalities. This is an important finding since the significance of isolated negative T‐waves in an asymptomatic individual has been unclear.Furthermore, although both major and minor T‐wave inversions carried significantly increased risk for all‐cause and CV death, the risk was most expressed for a major negative T‐wave. Although our focus was on analysis of isolated T‐wave inversion, we propose a similar rationale as for general ECG findings: Major ECG abnormalities are more solidly linked to CV disease and prognosis in asymptomatic individuals, than minor.Despite adjustment for numerous risk factors, a negative T‐wave remained an independent lifetime predictor for risk for all‐cause and CV death for lower cholesterol levels. For all‐cause death, the increased risk included also lesser smoking and lower hematocrit. All three conditions per se are associated with lower overall CV risk. Our results indicate that a negative T‐wave in absence of other evident CV risk factors may carry a distinctive message of enhanced risk, not to be ignored. When a negative T‐wave is present in middle age, albeit an otherwise favorable risk factor profile, the enhanced risk appears comparatively even larger, and must always be regarded as a marker of CV risk.However, this study has some limitations to consider. First, the cohort was collected in the 1960s, which makes the results of limited contemporary applicability. Moreover, only men in a limited geographic area were included and the findings cannot be generalized to women and other populations. Second, the size of the cohort was moderate. However, the long‐term follow‐up still gave statistical strength.5A negative T‐wave in ECG registered at 50 years of age in asymptomatic individuals from a randomized observational cohort carried an increased risk of all‐cause and CV death during lifetime follow‐up. The enhanced risk remained after adjustment for other ECG abnormalities and other CV risk factors. A major T‐wave inversion conveyed a higher risk than minor and a negative T‐wave carried enhanced risk also in the absence of other risk factors.The authors declare no potential conflict of interests.Appendix S1: Supplement A. Adjusted Cox proportional hazard models for the effect of T‐wave negativity at 50 years for different patient characteristics, on time to all‐cause death.Supplement B Adjusted Cox proportional hazard models for the effect of T‐wave negativity at 50 years for different patient characteristics, on time to cardiovascular deathClick here for additional data file.